- Email: [email protected]
Chromatographic and biological aspects of inorganic mercury
ChromatograpMc Rev~,ews Elsevier Pubhshmg Company, Amsterdam Printed m The Netherlands
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
LAWRENCE FISHBEIN National Institute of Enwronmental Health Sciences, National Institutes of Health, Public Health Service and Department of Health, Educat,on and Welfare, Research Triangle Park, N.C. 277o 9 (U.S.A.) (Received June Ilth, 1971)
CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 2. Occurrence, utility and ecological aspects . . . . . . . . . . . . . . . . . . . . 196 3. Biotransformation, metabolism and toxicity . . . . . . . . . . . . . . . . . . . 199 4. Paper chromatography and electrophoresis . . . . . . . . . . . . . . . . . . . 2oo 5. Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . 213 6. Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 7- Ion-exchange and column chromatography . . . . . . . . . . . . . . . . . . . 226 8. Analysis of mammalian distribution, transport and excretion of inorganic mercury . . 231 9. Miscellaneous analytical methodology . . . . . . . . . . . . . . . . . . . . . . 233 io. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
I. INTRODUCTION A n earlier review I highlighted the salient chromatographic a n d biological aspects of the organomercurials with p r i m a r y emphasis on m e t h y l m e r c u r y because of its u n i q u e u b i q u i t o u s p o l l u t a n t a n d t o x i c a n t n a t u r e . The objectives of this review are to (I) focus on the diverse chromatographic techniques t h a t have been utilized for the analysis of inorganic forms of m e r c u r y (e.g., elemental, mercurous a n d mercuric salts a n d their complexes) in a d m i x t u r e with other metals a n d cations a n d in diverse biological a n d e n v i r o n m e n t a l sources a n d (2) delineate m a j o r aspects of their adsorption, b i o t r a n s f o r m a t i o n a n d t r a n s p o r t , tissue d i s t r i b u t i o n , retention, excretion, mode of action a n d toxicity. The general considerations of inorganic m e r c u r y t h a t are r e l e v a n t to this review i n c l u d e : sources a n d chemistry 2-8, p h a r m a c o l o g y 9,1° a n d toxicology n-l~. The genetic considerations include: the synergistic effect of mercuric chloride with e t h y l m e t h a n e s u l f o n a t e in increasing chromosome aberrations in H o r d e u m sativurn 18 a n d V i c i a f a b a l S , 19, the cytotoxic effects of mercuric chloride on H e L a cells~° a n d i n t e r a c t i o n of inorganic mercurials with nucleic acids21-2L The carcinogenicity of m e r c u r y in tile rat has also been d e m o n s t r a t e d 28.
Chromatogr. Rev., 15 (1971) 195-238
196
L. FISHBEIN
2. OCCURRENCE, UTILITY AND ECOLOGICALASPECTS Mercury is comparatively a rare element, ranking I6th from the bottom of elements in abundance in the earth. Sufficient concentration of mercury for commercial extraction is found as mercuric sulfide (frequently as the red cinnabar and less often as the black metacinnabar). I m p o r t a n t deposits are located in Spain, Italy, U.S.A., Canada, Mexico, Brazil, Peru, China, Japan, Russia, Hungary, Yugoslavia and Germany. A less common ore is the mercurous chloride found in Texas. The ore found in Spain has the highest mercury content with an average of o.5-1.2 % mercury with values occasionally as high as lO% mercury. I t has been estimated that the outer i6-km-thick layer of the earth's crust contains 2. 7 × IO-a% mercury and the oceans contain 3.0 × lO-9 % mercury or approx. 50 million metric tons. In soils, it generally ranges from o.oi to 0.06 p.p.m., in rocks from o.oi to 0.09 p.p.m., in air from 2 to 5 ng/Ina, and in water and beverage from 0.002 to 0.006 p.p.m. Mercury has been known and utilized since antiquity. Today there are over 3000 recognized applications for mercury and its inorganic and organic derivatives. The world production of mercury amounts to about IO,OOO tons per year of which about 3000 tons are used in the United States. The main areas of utility of mercury include: the electrolytic preparation of chlorine and caustic soda, agricultural chemicals, pharmaceuticals (diuretics, cathartics, antibacterial agents), hair dressings and preservatives in cosmetics, pulp and paper making (slimicides and algicides), paints (anti-fouling, pigment), electrical apparatus, catalysts, dental preparations and amalgamations. Production of chlorine and caustic soda is an electrolytic process where large amounts of mercury are used as a flowing cathode (75,ooo-15o,ooo lbs for a plant with a capacity of ioo tons of chlorine a day) 5. It is estimated that the chlorinealkali industry loses to the environment approx. 0.45 lbs. of mercury per ton of chlorine produced ~9. This loss, based on projected tonage figures, m a y be as much as 3300 lbs. per day or 1,2oo,ooo lbs. per year. Most of the lost mercury finds its way into streams and lakes; traces of mercury are also carried into the atmosphere with hydrogen gas (20-30 mg/m 8) while usually less than 5 p.p.m, of mercury is retained b y the caustic soda. Mercury has also been detected in chlorinated hydrocarbons, glycols, acetic acid, carbon dioxide, fertilizers, sulfuric acid, sulfide ores, industrial catalyst wastes (e.g., in production of acetaldehyde and vinyl chloride) and in bituminous shales and crude oils. Fossil fuels in the United States contain mercury in concentrations ranging from a few parts per billion* to several parts per million. The annual consumption of 500 million tons of coal per year (containing an average concentration of at least I p.p.m.) would contribute one million pounds of mercury per year to the environment, about 45 ° metric tons 2. This value does not include the mercury contributed from the refinement and use of products from crude oil which m a y contain higher concentrations of mercury. The key inorganic mercury compounds used in agriculture (as insecticides, seed disinfectants and fungicides) and in home, garden and lawn are mercuric acetate, chloride and oxide (red and yellow) and mercurous chloride and nitrate. Elemental mercury is one of the most toxic elements used in the control of crop pests. Quick* Throughout this article the American p.p.b. = IO-~ is meant.
Chromatogr. Rev., 15 (I97 I) 195-238
197
C H R O M A T O G R A P H I C A N D B I O L O G I C A L A S P E C T S OF I N O R G A N I C M E R C U R Y
silver (metallic mercury) has itself been employed in India to fumigate grain in closed containers. There is a large variety of miscellaneous sources that present possible mercurial contamination to the environment and include the following: (a) disposal of thermometers, aerometers, barometers, relays, rectifiers, switches, fluorescent tubes, mercury lamps and batteries; (b) refuse from hospitals, laboratories and dental clinics; (c) processing or use of raw materials containing mercury such as carbon, coal, chalk, phosphate and pyrite; (d) use of mercurial compounds to prevent mildewing in commercial laundries; (e) manufacture of and residues from paints and impregnating agents which contain mercury to impart mildew resistance; and (f) refining or redistillation of mercury.
(CH3)2Hg
(C6Hs)2Hg C6HsHg+
~ Hg2
CH30 (CH2)2Hg+ Fig.
I.
Conversion o f m e r c u r y H g ° --+ H g t+ in t h e e n v i r o n m e n t a3.
Normal human tissues contain mercury because there is a daily intake in the food. Bread, flour, milk, pork and beef contain 0.02-0.04 p.p.m, of mercury and certain vegetables a good deal more, depending on soil conditions and sprays used. Ingestion of fish and fowl with high levels of mercury (as methylmercury) via environmental fallout from sources described above introduces yet another dimension to the ever-increasing realization that compounds of mercury present a substantial hazard.
IImport, 22% 1 L
IM,ni.g Re.erve. 3,% I I I
IStockpilo.2.%l I
IRecyc'"' °'oF J
ITo'a'oomoo'oo%l
DISSIPATIVE USES
Paints
26°/° ~ 12% Pulp and
Agriculture
Dental~t Catalysts
RECYCLABLE
4%
I
~ Electrtcal
I
IMa°ufa:t°relTJ ~ ~
Soda
&Control Laboratory
ceutlcals 1% I
2°/° Metallurgy I and I Mining
TO Environment 26%
USES
;33% Chlorine-- I ~
5%
Pharma-
cO
1°o I
L
1 I~ ~
Y
41%
27Olo I
rr
11% 3% [
Addition to
Inventory 10%
To Environment 23%
To Inventory and Environment 23°/o
Fig. 2. D i s s i p a t i v e and recyclable uses of m e r c u r y in t h e U n i t e d States in i9683.
Chromatogr. Rev., 15 {1971) 195-238
198
L. FISHBEIN MEDICAMENTS Hg COMPOUNDS METALLICMERCURY Hg METAL- OTHER USES in INDUSTRY m INDUSTRY LURGY METALLICMERCURY COSMETICS I
AGRICULT, AGENTS
•rrtX~ttngJ F~"Spe~t~4
I/
r WOste$-
~i
iI
fl
I /
.
FS~llage-
.
.
t- ~
.
.=V-rSpillage~
rt-Spillage -
Ti I r~,~'~
T! /
Ti
/:!
T! /
I~ i
I: ~Vapor---~ ~:.Vapor--
-~!,I
TI
.
l-~-,-Sp,llageJ
T:..-Vapor ~
.
.
.
Til
J
l~
:~
.
~i ;i
1 ~,_,_,_,_~z,~_,_,_~_,~_,_,_,_,_,,_,~ ",~
\ ".... \ "'"..
"~
~.~S~FARM
AOq0ATIC ~
\
EXCRETA.I--
/
MAN
Fig. 3. Principal pathways of mercury contamination and environmental movement3~. -w~r ] !
t i
/I~
I
Leakage and..- I lage~
I
rnetallic merc ury in container I i
i
lOrev,ce~ I I~
I
c,orh,ng ~.~a, s 11 amalgams Hands
WORKER
SO d
wastes
Eff~ent I
t- Sett hng tank
Outlet Stream ~1----- - --I~ Bed i I~-Littoral mud
Lak~
Me~hylmer~cury Solution m ~ fake water
Farm animals
Fig. 4. Ecological aspects of mercury distribution 34.
Chromatogr. Rev., 15 (1971) 195-238
~'~Micro- orga nisms
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
199
Of major significance has been the recent realization that certain biotransformations ~°-s3 may take place in the environment (Fig. I) and in the body, changing one form of mercury to another, viz., (a) the changing of inorganic mercury to methylmercury when oxygen is limited or absent by microorganisms, (b) the release of inorganic mercury from phenylmercuric compounds with subsequent conversion to methylmercury, and (c) the conversion of phenylmercuric compounds to inorganic mercury in the body. Figs. 2 and3 illustrate the dissipative and recyclable uses of mercury in the United States in 1968 and the principal pathways of mercury contamination and environmental movement, respectively. Fig. 4 depicts the ecological aspects of mercury distribution.
3" BIOTRANSFORMATION, METABOLISM AND TOXICITY
It is important to distinguish between different forms of mercury (e.g., elemental, ionic, alkyl and aryl) in any evaluation of the potential hazards of mercury residues as well as the development of requisite analytical methodology for their analysis. Inhaled mercury vapor (in many cases a mixture of vapor and aerosols) is mainly an industrial hazard and there is evidence that the vapor is adsorbed through the lungs '~5. The process of adsorption involves oxidation of elemental mercury with the subsequent appearance of ionic mercury in the blood, then distribution in the body in analogous manner to subcutaneous-injected mercuric chloride, with the kidney as the primary site of deposition. The oxidation of elemental mercury occurs partly in the blood (mainly in the erythrocytes) and partly in the tissues 36. The higher uptake of mercury in brain following exposure to vapor of animals as compared to subcutaneous injections of mercuric nitrate has also been reported3L3s. Diffusion of elemental mercury into the tissues and across cell membrane is facilitated by its lipid solubility and its lack of charge. In the plasma divalent mercury is bound to plasma proteins and in the erythrocytes to hemoglobin. Although there is some evidence that mercuric ions can be reduced to elemental mercury in the body 39, the extent of this reduction is not considered of practical importance. Mercurous ions are probably oxidized to mercuric ions in the body. Following acute administration of inorganic salts of mercury to man and animals, the highest level of mercury is found in the kidneys, and the next highest in the liver. Elimination of mercury from brain, thyroid and testis is slow4°, permitting accumulation of mercury in these organs. With chronic exposure to mercuric salts, it is uncertain whether mercury levels in brain or testis reach toxic concentrations before the onset of severe renal damage. Although inorganomercury salts are more acutely toxic than organomercury salts, chronic-oral studies 41 have shown for example that phenylmercuric acetate was more toxic to the rat than mercuric acetate. Inorganic mercury salts are poorly adsorbed, e.g., the adsorption has been estimated to be approx. 2% while for methylmercury the available data (man and rat) indicate an intestinal adsorption of more than 90%. Insoluble inorganic mercurous compounds such as Calomel (Hg2C12) can undergo oxidation to more soluble, adsorbable compounds. Inorganic mercurials, in
Chromatogr. Rev., 15 (i97I) I95-238
200
L. FISHBEIN
suitable vehicles, can be adsorbed through the intact skin on passage into the blood; they become firmly bound to plasma proteins and in the erythrocytes to hemoglobin. Mercury readily redistributes to the tissues and within several hours is found in human and animal tissues (in the approximate order of decreasing concentration): kidney, liver, blood, bone marrow, spleen, upper respiratory and buccal mucosa, intestinal wall, skin, salivary glands, heart, skeletal muscle, brain and lung. Mercury is excreted by the kidney, by the liver via bile, by the intestinal mucosa, sweat glands and salivary glands; urinary and fecal routes are the most important for elimination. Biotransformation has been shown 4~-44 to be important for the excretion of mercury after exposure to methylmercuric chloride. The carbon-mercury bond may slowly undergo cleavage in vivo, resulting in the release of inorganic mercury and its preferential excretion via the gastrointestinal tract (as distinct from the kidney, where a relatively higher amount of the intact organomercurial was found in urine). Biotransformation within the intestinal lumen in the cecum was shown to be the major source of inorganic mercury in feces. CLARKSON45 has suggested that the nephrotoxic action of organomercurials may be related to the rate of biotransformation with enzyme systems participating in the breakdown of organomercurials.
4-
PAPER CHROMATOGRAPHY AND ELECTROPHORESIS
Although paper chromatographic data for inorganic substances have recently been tabulated by LEDERER AND MAJANI4s, a variety of selected methods are included in this review with primary emphasis on specific methods for mercury. The utility of paper chromatography and hydrazine sulfate and hydroxylamine hydrochloride as specific reagents for the separation and detection of mercury and silver ion in toxicological analysis was described by ROMANOWSKI et al. 47. Mercury and silver were separated using ascending paper chromatography in two solvent systems: (a) butyl acetate and (b) acetone-conc, hydrochloric acid-isobutanol-glacial acetic acid-water (58 :I :2o:6.4:14.6); the RF values were 0.86 and 0.4 ° for mercury and silver, respectively. The paper was first sprayed with a 5% ethanolic solution of either hydrazine sulfate or hydroxylamine hydrochloride and then heated. Mercury produced a dark brown spot and silver one of yellow-brown. The presence of other metals did not interfere and the elements could be successfully detected in the presence of biological or mineral materials. The limits of detection of both mercury and silver were o.I and 0.3 #g with hydroxylamine hydrochloride and hydrazine sulfate, respectively. The radial-paper chromatographic separation of mercury and antimony valence states has been reported by JANARDHAN AND PAUL48. Trichloroacetic acid (TCA) was used as a complexing agent for the separation of mercurous from mercuric ions. TCA forms a semicovalent compound (I) with mercurous ion by sp hybridization, which alone moves with the solvent (benzene-TCA) leaving mercuric ions at the origin. On electronic considerations alone, formation of a similar chelate with mercuric ion is not possible. The formation of the chelate structure (I) has not been supported by dipole moment measurements and a callinear configuration (II) was postulated by JANARDHAN AND PAUL. This necessitates the assumption of additional charge on the
Chromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
201
oxygen of the hydroxyl of the COOH in TCA due to three chlorine atoms (this charge prefers only the mercurous ion, which is less positive as compared to Hg*+). Hence, TCA effects a resolution of the Hg2~+ and Hg 2+. The solubilizing effect of benzene on the covalent complex of Hg~2+ was suggested to be due to the association of benzene molecules with TCA (III). In the case of polar solvents such as water and aliphatic alcohols containing TCA, occurrence of double spots due to the disproportionation Hg2e+ ~ H@ + + Hg was observed.
Cl
0
O--C CI -C I I OI ~1
Cl~C_Cx// O"Hg-- Hg-- O;C-CC t3
~g
(~1
7 ( Z)
,o
I
o
Hg--Hg-O-C,~C---CI.---H--~
~ > . . . . H--CI
0~ \ I ('nT)
The radial-paper chromatographic separations were carried out on circles of Whatman No. I filter paper (diameter 15 cm). The spraying reagent for mercury consisted of fleshly-prepared 20% sodium hydroxide, which yields a brown color with mercurous ions, and a 1% ethanolic solution of 1% S-diphenyl carbazone, which yields a violet to blue color with mercuric ion. (Both reagents are sensitive to a microgram of mercury.) Fig. 5 illustrates chromatograms for the separation of mercury oxidation states in different solvent systems. Table I depicts the paper chromato1
2
4
3
5
Fig. 5. Chromatograms for the separation of mercury oxidation states. Elution of Hg2*+ and Hg2+ by o.I N HNO~ (I); EtOH + HNO3 (ioo:i) (2); BuOH-HNO3 (Ioo:i) (3); o.I N HCI (4); and benzene-TCA (ioo:Io, v/w) (5).
Chromatogr, Rev., 15 (1971) 195-238
:20:2
L. FISHBEIN
I I
l
I
l
I I
I I
I
1
1
lq
i I 1
1
I
i
I I
oh
bl
tt~ ~-
I I
1
1
t
f
1
t
1
1 o
I
1
I
I
I
t m
II
I
I
I
I1
I
1
I
I1
1
I
I
It
I
I
1
li
i
o o o o
0
0
0
0
o o
o
0
0
~
~
I
I
o
~
~
I
1
1
1
I
I
~
o
g t
I
l
I
I
I
I
l
I
I
I
I
o
o~ o~ o ~ ~
o~
6°d°d°o~oo
5
o
~oh O~
dd
I I
0
d
I
5
I
5
l
~
1 t
dd
I I
d
I
d
o
I
d
d
d
d ~
d
d
d 0
~d
I i
I
I
I
I t
t I
~
1
~"
d
I
0
±
I
I
I
I
1
I
0
o
~
_.~0 ~
o~
.~ o~
Chromatogr. Rev.
15 (1971) 1 9 5 - 2 3 8
>
2o3
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY TABLE
2
ANALYSIS
OF MULTIPLE
Spot: 4oL2/*g
(so#l,
Chromatogram
SPOTS OBTAINED
FROM MERCUROUS
ION
o.2 M H g , ~+ s o l u t i o n ) .
Solvent system
Found (~,g)
R~, v a l u e a
NO.
Hgb
Hg~a+~
Hg'+
ng='+b,'t Hg~ and Hg=+
i -
o.i N HNO a MeOH-HNO 3
o.o o.o
o.91 0.75
0.95 0.86
400 37 °
8 15
2
EtOH-HNO,
o.o
0.74
0.83
390
15
3
BuOH-HNO~
o.o
o.23
0.46
360
3°
-
o.I M TCA MeOH-TCA
o.o o.o
0.95 o.83
0.98 0.92
380 41o
6 q
-
EtOFI-TCA
o.o
o.64
o.75
39o
5
(1oo:I) (ioo:I) (ioo:i)
(Ioo:5, v/w) (Ioo:5, v/w) a Average of at least three chromatograms. b By disproportionatiou reaction. e By oxidation during chromatography. a Estimated polarographically. e E s t i m a t e d c o l o r i m e t r i c a l l y b y t i l e d i t h i z o n e m e t h o d 49 o n a B e c k m a n model B.
spectrophotometer,
graphic resolution of mercury and antimony valency states using 2o solvent systems and Table 2 shows the analysis of multiple spots obtained during the chromatographic determination of mercurous ion. Dithizone (S-diphenyl thiocarbazone) as well as S-diphenyl carbazone, are extremely useful reagents yielding very sensitive color reactions with many ions (amenable to extraction with immiscible organic solvents). In the case of mercury, the reaction with dithizone results in the colored chelate
(iv). H
Hg~
C=S N=N
The adsorption and development of nineteen mercury compounds were investigated on Amberlite SB-2 anion-exchange paper by using 1.6 N nitric acid as the developing solvent 5°. The RF values were o.19-o.33 for alkylmercuric compounds, o.o9-O.ll for inorganic salts, and O.Ol-O.O3 for phenylmercuric compounds, hence pernfitting the differentiation of organic and inorganic mercury compounds. An ion-exchange paper-X-ray emission procedure for the determination of microgram quantities of mercury has been described by LINK et al. 51. The procedure permitted the determination of I #g of mercury in IOO ml of aqueous acid solution with a precision of ± 0.25/~g. The mercury is first adsorbed by anion-resin-loaded Chromatogr. Rev.,
15 ( 1 9 7 1 ) 1 9 5 - 2 3 8
204
L. FISHBEIN
paper and then determined by X-ray emission spectroscopy. The method which was applied to the determination of mercury in inorganic pigments used in foods and drugs gave 75-125% recoveries of I / , g mercury from acid solutions containing I O g of Na,SO 4, NaC1, Fe20 ~, MgCO s and CaCO s and satisfactory recoveries from the HC1 extract of C, BaS04, chromic oxide, bentanite, kaolin, talc, Ti02 and magnesium stearate. KAO et al. 53 separated mercury from other ions on W h a t m a n No. I paper using methyl acetate-methanol-water (85:5 :IO) or methyl acetate-water (95:5)- The band was first indicated b y spraying with a reagent containing 5% K I - 2 o % Na2SO~ (I :I), then with a reagent of 5% CuSO, in I N HC1 to form orange-red Cu2HgI ,. (Gold interferes, but can be removed with hydroquinone.) For quantitative determination, the mercury band is removed, extracted with I N H~SO4, dithizone and chloroform and determined colorimetrically. TRIPATHI AND TEWARIs4 utilized paper chromatography for the determination of metallic poisons in forensic toxicology. Lead, copper, bismuth, zinc and mercury gave characteristic colors following separations with n-butanol saturated with I N HCI at 20 ° and detection with diphenyl carbazide. NAGAI5s separated mercury(II), copper(II), bismuth(III), cadmium(II) and lead(II) on circular paper impregnated with dithizone and developed with o.I N HNO3-acetone (IO:I). The cations were detected as follows from the center to the periphery: mercury, orange; copper, black; bismuth, orange; cadmium, red; unreacted dithizone and lead, pink. INDOVINA AND RICOTTA5s described a xo-min circular-chromatographic separation of copper, cadmium, bismuth and mercury ions using n-butanol-acetic acid12 N hydrochloric acid-water (45 :IO:I :44) and n-butanol-3 N hydrochloric acid as developers. Detection was achieved using dithizone. Table 3 shows the RF values of copper, bismuth, cadmium and mercury cations on paper discs using both developers above. TABLE RF
3
VALUES
OF COPPER,
BISMUTH,
CADMIUM
AND
MERCURY
IONS ON
PAPER
DISCS
Developing solvents: (I) n-butanol-acetic acid-i2 N HCl-water (45:Io:I:44) and (2) n-butanol3 N HC1. Metal cation
Copper Bismuth Cadmium Mercury
R F value Solvent z
Solvent 2
o. 18 0.46 0.26 o.9o
o.4° 0.80 0.83 o.9o
BURSTALL e[ al. 57 described the paper chromatographic separation of a number of metal cations and acid radicals on W h a t m a n No. I or 3 paper. The best separations were achieved in Group I I A (Pb, Cu, Bi, Cd and Hg) and Group I I I B (Co, Ni, Mn, Zn, the platinum metals and gold). More difficult separations were found in Group I I B (As, Sh and Sn) and Group IV (Ca, Sr and Ba). The separation of Group I I A metals as their chlorides was best achieved using n-butanol saturated with 3 N Chromatogr. Rev.,
15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
205
hydrochloric acid as developer (RF values were: Cu, o.2o; Pb, o.27; Bi, o.6o; Cd, 0.77; and Hg, o.81). A dithizone-in-chloroform spray detected the above metal cations as follows: mercury, pink; cadmium, purple; bismuth, purple; copper, purplish-brown. Lead, which gave a weak color with dithizone, was detected with an aqueous solution of rhodizonic acid as a bright-blue spot. The paper chromatographic separation of mercury, lead, copper, bismuth and cadmium cations has been accomplished using W h a t m a n No. 4 paper and (I) nbutanol-acetic acid-I2 N hydrochloric acid-water (45:1o:1:44) and (2) n-butanolhydrochloric acid (I :I) as developers 5s. Detection was accomplished with dithizone or hydrogen sulfide. Table 4 shows the effect of concentration of hydrochloric acid on the RE values of the metal cations, developed with system 2 (RE values increase with increasing acid concentrations). Table 5 depicts the R F values and spot colors of mercury, lead, bismuth, copper and cadmium cations developed with system I. The separation of lead, mercury, bismuth, copper and cadmium ions as their sulfates, chlorides, nitrates or acetates has been achieved by circular-paper chromatography using either n-butanol saturated with 4 N acetic acid or with 3 N hydrochloric acid at 25-27 °59 (Table 6). An effective separation of Ag +, T1+, Pb *+ and Hg 2+ was achieved 8° in approx. 3 h at 20 ± I ° by ascending chromatography on W h a t m a n No. I paper using a solvent mixture of m e t h a n o l - I I N nitric acid (60:40). The RE values were: Ag ¢, 0-29; T1+, 0.39; Pb 2+, o.51; and Hg 2+, 0.75. The spots were detected by dipping the chronlatogram in a solution of ammonium sulfide; the lower limit of detection was I X 10 -5 g .
RAO61 described the qualitative separation and identification of lead, silver and monovalent-mercury (Group I) cation mixtures on W h a t m a n No. I paper and using 0.2 N and 0. 5 N sulfuric acid and ammonium nitrate-ammonia mixtures for development, as well as a modified RUT~ER'S technique ez. Table 7 lists the RE values of cations of Group I using the above solvents. TEWAR163 described the effects of: (a) various n-butanol-pyridine-water mixtures, (b) temperature, (c) cation concentration, and (d) extent of development on the separation of lead, silver and mercury cations. Tables 8 and 9 list the RF values obtained utilizing varying mixtures of n-butanol-pyridine-water at 3 °0 and nbutanol-pyridine-water (20 :i :4) at temperatures ranging from IO to 4 o°, respectively. Table io depicts the RE values for varying amounts of the cations obtained in an I8-h development at 3 °° using n-butanol-pyridine-water (2o:1:4). POLLARD et al. 64 described the separation and determination of some heavy metal cations in admixture with lead ions by descending chromatography on Whatman No. I paper. Table I I shows the RF values of fourteen ions chromatographed with diethyl ether-methanol-water-nitric acid (50:30:20:2) and the spray reagents employed for their detection. The table indicates that it is possible to separate completely the heavy metal ions of major interest, e.g., Zn(II), Cd(II), Cu(II), Fe(II), Ni(II), Bi(III), S g ( I I ) from Pb(II). The chromatography of ten cations on Macherey-Nagel No. 261 paper developed with 96% ethanol-2 N acetic acid (80:20) and acetone-8 N acetic acid (9 o:Io) and detected with chloranilic acid was described by BARRETO et al. ~5. Table 12 lists the RE values and sensitivity of chloranilic acid toward ten cations separated with the solvent mixtures mentioned above. Chromatogr. Rev., 15 (1971) 195-238
:206
L. FISHBEIN
TABLE 4 R F VALUES OF Hg, Pb, Bi, Cu AND Cd CATIONS D e v e l o p i n g s y s t e m : n - b u t a n o l - H C l (I :I) w i t h v a r y i n g acid c o n c e n t r a t i o n s .
Metal cation
Concentration of H C I
Mercury Lead Bismuth Cop per Cadmium
IN
2N
3 N
0.75 o.ii 0.52 0.08 0.63
0.76 o.16 0.54 o.I I 0.67
0.80 0.28 0.58 o.2o 0.76
TABLE 5
R F VALUES AND SPOT COLORS OF Hg, Pb, Bi, Cu AND Cd CATIONS D e v e l o p i n g s y s t e m : n - b u t a n o l - a c e t i c a c i d - I 2 N H C l - w a t e r (45 : i o : i :44).
Metal cation
R ~ value
Mercury Lead Bismuth Copper Cadmium
Spot color
o. 78 o.o 5 o.4o o. 13 o.2o
H~S
D~thzzone
black -brown brown lemon-green
rose rose violet brown rose-purple
TABLE 6 R F VALUES OF Pb, Hg, Bi, Cu AND Cd WITH DIFFERENT ANIONS S o l v e n t s : (I} n - b u t a n o l s a t u r a t e d w i t h 4 N a c e t i c a c i d ; (2) n - b u t a n o l s a t u r a t e d w i t h 3 N h y d r o chloric acid. T e m p e r a t u r e : 25-27 °. Circular c h r o m a t o g r a p h y ,
Amon
Sulfate Chloride Nitrate Acetate
Solvent z
Solvent 2
pb*+
Hg*+
B~3+ Cu*+
Cd*+
pbu+
Hg~+
Bz3+
Cu2+
Cd~+
o.46 o.51
0.57 0.84 0.69 o.82
o.61 o.62 -
o.21 0.42 0.52 0.54
halo halo
0.82 o.83 o.89 o.82
o.72 0.73 -
0.36 o.36 o.38 o.35
0.79 o.79 0.79 o.79
0.27 0.40 o.54 o.57
TABLE 7 R p VALUES OF CATIONS OF GROUP I S o l v e n t s : (i) N H 4 N O s s o l u t i o n d i l u t e d 8o t i m e s - N H 4 O H (2o:8); (2) 0.05 N H2SO,; (3) o.z N
H2SO4. Catwn
Ag + Hg + P b ~+
Strap
Rutter' s method
Solvent r
Solvent 2
Solvent 3
Solvent 3
o.92 0.94 0.02
0.85 0.95 0.04
0.92 0.98 o. 17
0.82 o.91 o.o5
C h r o m a t o g r . R e v . , 15 (1971) 1 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
207
TABLE 8 R~, VALUES OF
P b ~+, Ag +,
AND
Hg 2~ W I T H
V A R Y I N G n - B U T A N O L - - P Y R I D I N E - - W A T E R M I X T U R E S AT
3 °o Ascending development for 6 h. Mixtures: (I) (2o:1:4), (2) (I5:I:9), (3) (lO:1:14) and (4) (5:I:19) • Cat, on
P b ~+ Ag + Hg 2+
Mixtures
o.o 4 0.22 0.59
2
3
4
0.0 5 0.52 o.92
0.0 5 0.45 o.7o
o.o 3 0.20 0.5o
TABLE 9 E F F E C T O F T E M P E R A T U R E ON W A T E R ( 2 0 : I :4)
R F V A L U E S O F P b 2+, Ag+ A N D Hg 2+ U S I N G n - B U T A N O L - - P Y R I D I N E - -
A 3u-rain circular-paper development. Cation
P b 2+ Ag + Hg ~+
Temperature (°C) IO
20
30
40
o.31 0.59 0.92
0.27 0.55 o.81
o.2I 0.53 0.82
o.i 5 o.5I 0.74
T A B L E 10 E F F E C T O F C A T I O N C O N C E N T R A T I O N ON GRAPHY
R F V A L U E S O B T A I N E D AT 3 0 ° B Y A S C E N D I N G C H R O M A T O -
Solvent system : n-butanol-pyridine-water (20 :I :4). Development time : I8 h. Cation
P b ~+ Ag + Hg 2+
Catzon concentration (12g) o.z
0.2
0.3
0.4
0.5
0.05 0.23 0.4 8
o.o 7 0.24 0.50
o.o8 o.25 0.52
0.09 0.26 0.55
o.Io 0.27 0.58
The separation of silver, mercurous and lead ions by circular-paper chromatog r a p h y w a s r e p o r t e d b y MURTHY et al. 8~ u s i n g n - b u t a n o l - p y r i d i n e - w a t e r (IOO :20:20) ~ i n a 2-tl (9-cm) d e v e l o p m e n t w i t h a m m o n i a c a l h y d r o g e n s u l p h i d e u s e d f o r d e t e c t i o n . T h e R E v a l u e s f o u n d w e r e : Ag+, 0 . 8 6 ; H g +, 0 . 6 2 ; a n d P b 2+, 0.34. Chromatogr. Rev., 15 (1971) 195-238
208
L. FISHBEIN
T A B L E 11 R F VALUES AND SPOT COLORS OF SOME HEAVY METAL CATIONS IN ADMIXTURE WITH LEAD IONS O b t a i n e d w i t h d e s c e n d i n g c h r o m a t o g r a p h y on W h a t m a n No, I paper. S o l v e n t m i x t u r e : die t h y l e t h e r - m e t h a n o l - w a t e r - n i t r i c acid (5 °: 3 ° : 2o :2).
Cation
Spray detector
Spot color
R v value b
TI(I)
0.5% 8 - H y d r o x y q u i n o l i n e in abs. alcohol. H o l d over a m m o n i a a n d u n d e r U V light. 5 % w]v T a n n i c acid in w a r m 60% aqueous methanol, W a r m t h e d a m p strip. 3.0% R h o d i z o n i c acid. H o l d over a m m o n i a . 0.5% 8 - H y d r o x y q u i n o l i n e in abs. alcohol. H o l d over a m m o n i a a n d u n d e r U V light. o . 1 % wlv R u b e a n i c acid in abs. alcohol. H o l d o v e r ammonia. 4 % Salicylaldehyde in 50% a q u e o u s ethanol. H o l d over a m m o n i a a n d u n d e r U V light. o.5% w/v Aqueous potassium ferrocyanide. IO% w / v F r e s h l y p r e p a r e d a q u e o u s s o d i u m dlthionite (warm). 0.5% w / v A q u e o u s p o t a s s i u m ferrocyanide. 0.05% w / v D i t h i z o n e in chloroform,
Yellow-green fluorescence
o.25-o.17
Brown stain
0.36-0.27 e
Red
o.41-0.22
Yellow fluorescence Yellow fluorescence
o.66-o.55 o.66--o.55
Green Yellow-brown Blue Dark spot against a yellow-green fluorescent background Blue Blue Brown
0.66-o.56 0.68-0.55 0.68-0.55 0.70-0.56
Brown
0.78-0.72
Pale p i n k
0.95-0.87
Ag(I) Pb(II) Zn(II) Cd(II) Cu(II) Co(II) Ni(II) Mn(II) Fe(II) Fe(III) Bi(III) a U(VI) Hg(II)
0.70-o.58 0.70-0.58 H y d r o l y z e d M1 d o w n t h e p a p e r to 0.8
a B i s m u t h m a y be c h r o m a t o g r a p h e d successfully o n l y if t h e original solution c o n t a i n e d 5 % v / v nitric acid. T h e n no h y d r o l y s i s p r o d u c t s are formed, a n d t h e R e v a l u e of Bi is 0.78-o.67. b H e a d a n d tail o f spot. e D a r k h y d r o l y s i s p r o d u c t s also p r e s e n t a t s t a r t i n g time. T A B L E 12 R e VALUES AND SENSITIVITY OF CHLORANILIC ACID AS DETECTOR S e p a r a t i o n of IO c a t i o n s on M a c h e r e y - N a g e l No. 261 p a p e r u s i n g d e s c e n d i n g d e v e l o p m e n t w i t h (I) 9 6 % e t h a n o l - 2 N acetic acid (8o:2o) a n d (2) a c e t o n e - 8 N acetic acid (9o:1o). D e t e c t o r : o. i % chloranilic acid in ether.
Cation
Fee+ Ni ~+ Co S+ Cu 2+ Zn *+ Ag+ Cd ~+ H g 2+ P b 2+ U 4+
RF value Solvent x
Solvent 2
o.o8a 0.34 0.42 0.27 0.78 0.37 o.71 o.o o. i o 0.83
I,OO o.07 0.07 o.2o o.31 0.08 o.20 I .oo o.o9 o.86
Sensztivity (ttg)
Color of spot
o.oI o.1o 0.20 o.Io 5.00 4.00 2.00 5.oo o. I o I.OO
brownish greenish yellow light green greenish brownish red brownish brownish brownish brownish
a Tailing.
Chromatogr. R e v . 15 (197 I) 1 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY BLASIUS AND G6TTLING ss d e s c r i b e d t h e c i r c u l a r - p a p e r c h r o m a t o g r a p h y
209 of a
v a r i e t y of cations. T h e following RF values were f o u n d following a 3-h d e v e l o p m e n t on S c h l e i c h e r & S c h t i l l N o . 2045 p a p e r w i t h I O % a q u e o u s a m m o n i u m a c e t a t e : P b *+, o.95; Ag+, 0.80; T1+, 0.75; a n d H g ÷, 0.65. D e t e c t i o n w a s a c c o m p l i s h e d w i t h 5~o a m m o n i u m s u l p h i d e (viz., T1+ a n d P b 2+, b r o w n a n d A g + a n d H g +, g r e e n ) . POLLARD et al. ~9 d e s c r i b e d t h e c h r o m a t o g r a p h y o f t w e n t y - f o u r c a t i o n s u s i n g c o m p l e x - f o r m i n g m i x t u r e s . T a b l e 13 l i s t s t h e R F v a l u e s o f c a t i o n s o b t a i n e d u s i n g n - b u t a n o l - a c e t i c a c i d - a c e t o a c e t i c e s t e r - w a t e r (5 ° :IO : 5:35) for d e v e l o p m e n t . TABLE 13 RF
VALUES OF CATIONS WITH A COMPLEX-FORMING MIXTURE
Solvent system : n-butanol-acetic acid-acetoacetic ester-water (5° :i o : 5 : 35). Cation
R F value
Cat,on
RF value
Ag + Hg + Pb z+ Hg 2+ Bi 3+ Cu *+ Cd 2+ Asa+ Sb 3+ Sn ~+ Sn 4+ AP +
o. I 0.09 o.18 0.84 0.34 o.65 0.29 o. 17 o.i6 o.16 o.18 o. 17
Cr a+ Fe 8+ Zn ~+ Mn ~+ Co s+ Ni *+ Ca 2+ Sr ~+ Ba i+ Mg ~+ K+ Na +
o.64 0.73 0.30 0.23 0.22 0.22 0.20 o. 18 o.16 o.2o 0.25 0,23
T h e s e p a r a t i o n o f n i n e c a t i o n s o n W h a t m a n No. I a n d No. 42 p a p e r s u s i n g cyclohexane-hydrochloric acid and methyl ethyl ketone-hydrochloric acid mixtures w a s d e s c r i b e d b y HALMEROSKI AND SUNDHOLM 70. T a b l e 14 l i s t s t h e R F v a l u e s o f some metal ions with methyl ethyl ketone-hydrochloric acid mixtures. TABLE 14 RF VALUES X IO0 OF SOME METAL IONS W'ITH M E T H Y L E T H Y L KETONE--HC1 Paper: W h a t m a n No. I and No. 4 2. Solvents (mixtures of aqueous HC1 and methyl ethyl ketone) : (I) i ml cone. HC1, and (2) 2 ml cone. HCI in IOO ml of the solvent mixture. Development: descending. Time of run: 7 h with methyl ethyl ketone. Cation
t'Igz+ Cu ~+ Cd 2+ BP + As~+ Sn z+ Sb 3+ FeZ+ UO22+
Whatman No. I
Whatman No. 42
Solvent z
Solvent 2
Solvent z
Solvent 2
74 6 22 3° 13 a 21 38 xo
75 13 25 46 29 a 37 41 22
84 4 16 56 5 79 26 78 7
87 6 28 60 IO 84 49 80 io Chromatogr. Rev., 15 (I97I) I95-238
210
L. F I S H B E I N
BHATNAGER et al. ~1 described the paper chromatography of a number of metal dithizonates. The dithizonates of Ag, Pb, Hg(II), Bi, Cu(II) and Cd were studied in the following solvent systems : isopropanol; acetone; ethyl acetate; methyl acetate; chloroform-acetone (I :I, 1:3 and 1:9) ; chloroform-methyl acetate (I :3 and 1:9) ; chloroform-ethyl acetate (I :3) ; chloroform-ethyl acetate (I :9) ; carbon tetrachloride-acetone (I:I, 1:3 and 1:9); carbon tetrachloride-methyI acetate (I:I, I:3 and 1:9)- The solvent system and the binary pairs which were separated were: isopropanol, Hg-Ag; methyl acetate, Cu-Bi; chloroform-methyl acetate (I:3), Ca-Bi and P b - H g ; chloroform-methyl acetate (I:9), Ag-Hg, Cd-Bi, and P b - H g ; carbon tetrachloride-acetone (1:3 and I:9), Cu-Cd; and carbon tetrachloride-methyl acetate (I :9), Pb-Hg. The chromatographic separation and detection of silver, mercury, copper, cadmium, bismuth, cobalt, nickel and zinc ions with a dithizone-impregnated filter paper was described by NAGAI AND DEGUCHI7~. At the center of a dithizone-impregnated filter paper was placed a drop of an ion mixture of Hg-Cu-Cd-Co-Zn, Ag-CuCd-Co-Zn, Hg-Cu-Cd-Ni-Zn, Ag-Cu-Cd-Ni-Zn, Hg-Cu-Cd-Bi-Zn or Ag-Cu-CdBi-Zn and developed with 3 N acetic acid-acetone (5:6). Each metal ion yielded a circle with a characteristic color and the order of formation of colored circles (precipitated dithizone chelates) agreed with the order of stability of metal chelate. The separation and collection of traces of metals on paper impregnated with a few milligrams of a water-insoluble organic reagent was described ~. The reagent paper was prepared by dipping filter paper, 7 cm in diameter, in 1% dithizone-inchloroform or in o.05% p-dimethylaminobenzylidene rhodamine-in-acetone followed by o.I N nitric acid. Approx. 98% of Au, Ag and Hg in amounts of less than I/~g to 5/~g could be recovered after washing from lO-25o ml of O.l-O. 5 N mineral-acid solution by a single or by repeated filtrations at a rate of 6 ml/min. The method was found applicable for the rapid separation of several p.p.m, of Au, Ag and Hg in Cu and Pb where the concentration factor of Ag with respect to Cu was approx. 200o. The reagent paper could be stored for more than 80 days without any deterioration. AKIYAMA AND SHIOKAWA74 reported the separation of nfixtures of AI(III)Hg(II)-Cu(II), Hg(I])-Co(II)-Zn(II), Fe(III)-Bi(III)-Sb(III), Ug(II)-Ni(II)-Zn(II), Ag(I)-Cd(II)-Pd(II) and Hg(II)-Cu(II)-Cd(II)-Pb(II) by circular chromatography using Toyo filter paper No. 2 or 5B (diameter 9 cm) pretreated with 0.5% dithizonein-acetone solution and developed with o.I N nitric acid-acetone (io:I). QURESHI AND KHAN75 described the precipitation chromatography of various cations on strontium chromate-impregnated paper. Strontium nitrate (0.2 M; pH 7-8) was impregnated on Whatman No. I circles of 7-cm diameter, dried in an oven at 70°, then kept in 0.2 M solution of potassium chromate for about 5 min. After the complete precipitation of strontium chromate on the paper, it was dried, washed with water and redried. For quantitative determinations (20 min) 0.005 ml of the cation solution was applied and 0.2 M strontium nitrate (b) and 10% ammonium hydroxide (a) were used as developers. The RF values were as follows: (a) In ammonium hydroxide solution: Hg22+, Hg 2+, Pb 2+, Cd *+, Bi ~+, As 3+, Sb ~+, Sn ~+, La 3+, V 4+, Be 2+, Au 3+, Ti4+, Fe a+, A13+, Cr~, Mn*+, Co2+, Zn*+, Ca*+, Ba 2+, Mg2+, Ce3+, UO2*+, Th 4+, y3+ and Zr 4+, all were 0.00; Ag + and Ni 2+, 1.0; and Cu 2+, 0.41. (b) In strontium nitrate solution: Ag +, Hg, *+, Pb 2+, Bi 3+, As s+, Sb~+, Sn 2+, V 4+, Be 2+, Au s+, Ti 4+, Fe 3+, A13+, Cr8+, Mn 3+, Zn 2+, Ca ~+, Ba *+, Mg*+, U02 ~+, Th 4+ and Zr 4+, all were 0.00; Cd 2+, La 3+, Chromatogv. Bey., 15 (1971) 195-238
C H R O M A T O G R A P H I C A N D B I O L O G I C A L A S P E C T S OF I N O R G A N I C M E R C U R Y
211
Co2+ and Ni *+, I.OO; Cu ~+, o.41; Ce3+ and ys+ tailed. The main application of this technique is to separate cations with RF values of o.oo from those of RF I.OO. These two RF values can be made to differ by the choice of a l'roper complexing agent for elution. However, to achieve a specific separation, it is necessary to use a paper impregnated with a particular precipitating agent. Thus, using strontium chromate-impregnated paper it is possible to separate silver, mercury and lead cations from a number of different metal ions. The utility of paper precipitation chromatography for the separation of metal ions was described by NAGAI~6, Hg2~+, Ag+ and Pb 2+ were separated as their chlorides on filter paper impregnated with lO% sodium chloride with 4 M aq. ammonium acetate and ammonium hydroxide used as developing agents. Pb ~+ moved with the developing front and was identified with sodium rhodizonate. The Ag+ band which moved next to Pb ~+ was identified by a potassium iodide reaction. Hg~*+ was identified by the appearance of a black spot in the center of the chromatogram. Hg 2+ and Bi a+ were separated on potassium iodide-impregnated filter paper using 3 M propionic acid as developer. SUGAWARA~ described the separation of a number of heavy-metal ions of tile Cu group (Hg ~+, Bi 3+, Cd ~+ and Pb 2+) and Sn group (As3+ and Sb 3÷) on ion-exchange paper, Amberlite SA-2 (Na + type). The groups were developed with I M NH4C1 and I N HC1, respectively, and detected with ammonium sulfide-4-(2-pyridylazo)-resorcinol (for the Cu group) and ammonium sulfide-palladium chloride (for the Sn group), respectively. The RF values were: Hg 2+, 0.04; Bi 3+, o . I I ; Cd 2+, 0.52; Cu ~+, 0.27; Pb ~+, 0.47; As z+, 0.84 and Sb 3+, 0.26. The separation of TI(I), Hg(I), Ag(I) and Cs(I) on ammonium molybdophosphate (AMP)-impregnated papers was described by ALBERTI~s. Table 15 lists the RF values of these cations on AMP-impregnated paper using ammonium nitrate at various concentrations in I N nitric acid as developing solvent. TABLE 15 TI(I), Hg(I), Ag(I) AND Cs(t) SEPARATED ON AMP PAPER Developing solvent" NH4NO3, at various concentrations in I N HNO3.
R F VALUES OF
Cation
TI(I) Hg(I) Ag(I) Cs(I)
Molarity of NH4NO 3 o,z
0.5
z
3
5
7.4
r5
o,o o.15 0.46 o,o
o.o 0.40 0.63 o.o
0.02 o.41 0.65 o.o
o.o6 0.58 0.73 o.io
o.13 0.68 0.74 o,io
0.20 o.75 o.76 o.13
0.4o o.81 0.86 0.23
The separation of the oxidation states of sixteen elements with the use of trin-butyl orthophosphate-5 M acetic acid-acetone solvent system was reported by THAKUR79. Table 16 lists the RF values of ions with separable valency states obtained on Whatman No. I paper developed with tri-n-butyl orthophosphate-5 M acetic acidacetone (I :I :3). The basis on which ions of different valency states can be resolved is that metals form ionic or covalent complexes depending upon their oxidation states Chromatogr. R e w , 15 (1971) 195-238
212
L. F I S H B E I N
a n d t h a t the ionic complexes associate with polar solvents a n d covalent complexes with non-polar ones. The solvent system used in T a b l e 16 is n o n - p o l a r a n d as shown the higher v a l e n c y states exhibit increased migration, a t t r i b u t e d to the formation of their covalent complexes. TABLE 16 RF V A L U E S O F I O N S W I T H S E P A R A B L E V A L E N C Y S T A T E S Values obtained on Whatman No. i paper, developed with tri-n-butyl orthophosphate-5 M acetic acid-acetone (I :I :3) (ascending, 15 cm, 2 h at 19-22°). Ion
R v value
Ion
RF value
As3+ AsS+ Cr3+ Cr6+ Fe 2+ Fe 3+ Hg+ Hg~+ Mn~+ Mna+ Mn7+ TI+ T18+
0.42 0.57 o. I o.26 o.o 0.56, o. 7 o.oa o.92 o.I8 o.o o.o o.05 o. 3
Sba+ Sbs+ P,O: 4PO43PO sFC1C103BrBrO8IIO,-
o.o 0. 5 o.o o.37 o.o7 o.o o.I 3 0.32 o.2 o. 13 o.28 o.o
a Streaking observed. TANAKA et al. 8° described a radioisotope dilution m e t h o d for the determin a t i o n of inorganic a n d organic m e r c u r y b y paper chromatography. Trace a m o u n t s of mercuric chloride a n d p h e n y l m e r c u r i c chloride were first d e t e r m i n e d chelatometrically with penicillamine. T h e chelate was satisfactorily separated b y paper chromatog r a p h y from u n r e a c t e d m e r c u r y a n d the r a d i o a c t i v i t v of the chelate spot on the paper was measured with a well t y p e scintillation counter. A k n o w n a m o u n t of Z°3Hglabeled s t a n d a r d solution a n d substoichiometric a m o u n t of penicitlamine was t h e n added to a n u n k n o w n sample solution, the p H a d j u s t e d to 6-1o a n d the m e r c u r y chelate separated b y paper c h r o m a t o g r a p h y on Toyo filter paper No. 5IA, 2 × 4 ° c a , with isopropanol-conc, a m m o n i u m h y d r o x i d e - w a t e r (7:1:2) as developer. The a m o u n t of m e r c u r y in the u n k n o w n is t r e a t e d in a n analogous m a n n e r a n d the a m o u n t of m e r c u r y in the u n k n o w n calculated from the activities measured. Mercury was detected down to I/~g when lO -6 M of penicillamine a n d lO -8 M of Z°3Hg-labeled mercuric chloride or phenylmercuric chloride were used. Ni, Fe(II), Cd, Z n a n d Co ions did n o t interfere with the d e t e r m i n a t i o n . KURAYUKI AND KUSAMATO81 described the chromatographic separation of inorgano- a n d o r g a n o m e r c u r y compounds on anion-exchange paper, Amberlite SB-2 (R-C1), using 1.6 N nitric acid a n d 2 M a m m o n i u m n i t r a t e as developers a n d dithizone-in-chloroform for detection. The RF values (using 1.6 N HNO3) were: inorganom e r c u r y compounds, o.o9-o.11; alkylmercuriats, o.19-o.33; a n d phenylmercurials, O.Ol-O.O3. The limits of detection were 0.2 #g. I n a n a d d i t i o n a l s t u d y on the determ i n a t i o n of inorganic m e r c u r y c o m p o u n d s b y measuring the area of the colored zone on strips of Amberlite SB-2 (R-C1) the a m o u n t of m e r c u r y (1- 4 rag) was f o u n d to be p r o p o r t i o n a l to the colored area at p H 5.0. Chromatogr. Rev., 15 (1971) i95-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
213
WESTERMARK et al. 8~ described a concentration-electrophoretic method for the separation of charged mercury compounds in aqueous solution. In electrophoresis, the microcomponents concentrate in very narrow bands on the thin-layer strip, the postulated diffusion forces are counteracted or even balanced by the electrical-field strength. This phenomenon was applied to Hg 2+ and CH3Hg+ in an apparatus consisting of an aqueous mercury-containing insert, surrounded by concentration zones and buffer vessels, by using histidine and E D T A ions. The latter move from the cathode and form a negative complex with Hg ~+. This complex moves from the inert zone towards the anode, being partly concentrated there. Under suitable conditions CH3Hg + migrates to the cathode and concentrates behind the insert. At present the method of WESTERMARK and co-workers is limited to 2o #1, but this can apparently be increased. An advantage of this technique compared to gas chromatography is that inorganic mercury and alkylmercury can be measured in the same operation. FUKUDA et al. 83 described the qualitative analysis of metals b y paper electrophoresis following the initial separation of a sample containing nine metal ions into three portions and their precipitation into sulfides with NH4HS, thence treatment of the sulfides with acids into three groups of metal ions. Cu, Zn, Ag, Cd and Pb in the first group were separated b y paper electrophoresis with I N citric acid, 5 N acetic acid and 5 N formic acid as the electrolytes. For the second group (Hg and Bi) o.I M lactic acid or 0. 5 N formic acid were used as the eleetrolvte and for the third group (As and Sb), o.I M lactic acid was used. The paper electrophoresis of Hg(II) and twenty other cations was described b y CATON184. The electrolytes used were : (A) 25 ml 4 N lactic acid and 25 ml 2 M Na2CO~ made up to I 1 with water, (B) 40 ml 0.2 N HC1 and 250 ml 0.2 M potassium hydrogen sulfate made up to I 1 with water; (C) 468 ml o.I M sodium citrate and 532 ml o.I N HC1. 5. THIN-LAYER CHROMATOGRAPHY
SEILER AND SEILER85,86 separated the copper family (Cu 2+, Cd ~+, BP +, Pb ~+ and Hg 2+) on MN-Silica Gel G - H R using n-butanol-I. 5 N HC1-2,5-hexanedione (lOO:2O :0.5) as developer and 2% potassium iodide, ammonia and hydrogen sulfide, reagents for visualization. The addition of Pthe weak comptexing agent hexanedione reduces tailing. The RF values increase in the order of Cu~+
Hg 2+
Cd 2+ - -
[ ] B t3 + ] P b 2+
o,
1
2
3
4
5
6
Fig. 6. S e p a r a t i o n of t h e copper g r o u p on i N - S i l i c a Gel G - H R d e v e l o p e d w i t h • - b u t a n o l - I , 5 N H C I - 2 , 5 - h e x a n e d i o n e (IOO'2O'O.5).
Chromatogr. Rev., 15 (1971) 195-238
214
L. FISHBEIN
3-4-h development. The RF values of the cations increase in the same order as in paper chromatography, e.g., Ag+
Solvent front
@
0
@ 0
0
©
0 0
© StQrt
Bl(~r[) Cd(ZI)Cu(]Z) Pb(]I) Hg(]Z) Mixture
Fig. 7- TLC of metal ions on cellulose MN-3oo developed with tert.-butanol-acetone-water-6 N HNOs-acetylacetone ( 4 : 4 : i . i :o,45:o.45 ). Detection with 2% ethanolic oxine, ammonia vapors and UV inspection.
Chromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY" TABLE TLC
215
17 O F Hg(ll)
AND
METAL
IONS ON
ZIRCONIUM
HYPOPIIOSPHATE
CHROMATOPLATES
Development in (i) o.i N HC1 and (2) 0. 5 N HC1. Cation
R F value Solvent I
Solvent 2
S n z+ P b ~+
o o
o o
F e 3+
0
0
La 8+ Oa a+ Y~+ In s+ Cs+ Cu ~+ Co2+ Cd ~+ Fe 2+ Ca ~+ Zn 2+ Ni 2+ Hg 2+ Ba ~+ Na + Sr 2+
o o o o 0.05 0.05 0.0 7 o.Io o.oi o. io o.15 o.25 o.so o.3 ° 0.35 0.35
o.io o.o o.15 o.2o o.Io 0.55 0.55 0.53 0.56 o.42 0.52 0.50 o.73 o.63 o.55 0.60
Co 2+, Ni 2+, Cu 2+, Z n 2+, C d 2+, H g 2+, S n 2+ a n d P b 2+ w e r e d e t e c t e d w i t h 1 % dit h i z o n e i n c a r b o n t e t r a c h l o r i d e ; F e z+ a n d F e 3+ w i t h i M K3[Fe(CN)6 ] o r i M K a [ F e (CN)~] a n d L a z+ a n d Y ~ w i t h 0 . 1 % I - ( 2 - p y r i d y l a z o ) - 2 - n a p h t h o l i n e t h a n o l . N a +, Cs +, Ca ~+, S r ~+, B a ~+, L a 3+ a n d y a + w e r e i d e n t i f i e d b y u s i n g r a d i o a c t i v e t r a c e r s . T L C w i t h a 0 . 4 - m m l a y e r o f Alz03 a n d silica gel s o r b e n t s w a s a p p l i e d t o t h e d e t e c t i o n o f H g *+ a n d f i f t e e n i o n s o f m e t a l s o f t h e 3 r d a n d 4 t h a n a l y t i c a l g r o u p s 9°. The ions d e t e c t e d , t h e r e a g e n t s used a n d t h e m i n i m u m d e t e c t a b l e a m o u n t s i n / t g are s h o w n in T a b l e 18. TABLE TLC
18 O F Hg(II)
AND
METAL
CATIONS
O F TIIE 3rd A N D
4th A N A L Y T I C A L
GROUPS
Metal catwn
Detect,ng reagent
M i n i m u m detectable amount (,ug)
Cu 2+ Ag + P b ~+ Hg ~+ Bi 3+ Hg, *+ Cd~+ Fe a+ Fe ~+ Co~+ Ni *+ Zn *+ Mn *+ Cr 3+ Ap+
rubeanic acid HCt, SnCI,, NH4OH Na-rhodizonate KI, biphenyl (NH4)~SCN (NH4),SCN Na2S K4Fe(CN), or KSCN NH 3 and dimethylglyoxime a-nitroso-fl-naphthol rubeanic acid (NH4) ~ [Hg(SCN)4] KIO,-HNO 3 2 M Na,HPO 4 NH3_ahzari n
0,24 1.6 o.9 7.5 1.3 6.02 3.4 o.34 0.04 0.038 0.o87 0.30 o. 18 o.18 o.o3
Chromatogr. Rev., 15 (1971) 195-238
216
L. FISHBEIN
SHERWOOD91 described the TLC behavior of thirty cations using Eastman Chromagram No. 6o61 sheets with the following solvent systems: (I) 5% hydrochloric acid-methyl isobutyl ketone-amyl acetate (i :2 :i). Table 19 shows the RF values of the cations as their chlorides arranged in rough order of movement, viz. very fast, fairly fast, slow and very slow--non-movers, following a 2-h development. Visualization of the cations was achieved using nine sprays (the most universal of these being TABLE 19 RF VALUESOF METALtONS ON EASTMAN CHROMAGRAM No. 6061 SHEETS
Developing system : 5% HCl-methyl isobutyl ketone-amyl acetate (I :2 :I). Order of movement
Very fast
Fa,rly fast
Slow
Very slow and non-movers
Cation
RF value
Cat,on
RF value
Cat,on
R~, value
Cation
RF value
Mo Fe
o.91 0.89
Hg As
0.6 o.51
Mn Bi
0.06 0.05
Ni Ti Ce Ca
]
Au
o.89
Cd
o.31
Pb
o.o2
Sr
Sb Te Sn
o.85 o.82 0.78
Zn Cu Pt Co
o.27 o.i8 o.19 o.13
V
o.o2
A1 Zr Li K Na Mg Cr
Ba
All o.o
o.o5% dithizone in carbon tetrachloride) with the following appropriate color reactions: (I)as sprayed: yellow, Fe, Te, As; yellow-orange, Sb; orange, Au, Hg, Cd, Bi, and Pb; red-orange, Mo; cherry red, Sn, Zn, V; yellow-green, Cu; purple, Co; (2) after exposure to ammonia vapors and heating: yellow, Au, Te and As; yellow-orange, Sb and Bi; orange, Fe, Hg, Cd and Pb; red-orange, Mo; cherry red, Sn, Zn and V; pale green, Pt; olive-green, Cu; purple, Co. The detecting reagents used were: (a) 5% ammonium thiocyanate in water-acetone (5:95) ; (b) 5% potassium iodide in wateracetone (lO:9O); (c) 0.5% oxine in ethanol; (d) 5% silver nitrate in water-acetone (30:70); (e) o.5% diphenyl carbazide in ethanol; (f) 1% dimethyl glyoxime in ethanol; (g) o.1% aq. solution of chromotropic acid; and (h) 5% aq. solution of sodium rhodizonate. The limits of detection of the metal ions developed with solvent system I and visualized with dithizone/ammonia vapors/heating were: Hg, 0.5-2 #g/#l; Cd, As and V, 0.2-0.5/~g//,l; Zn, Pb and Pt, O.l-O.2 #g/#l; Cu, Bi, Co, Mn and Ni, all detected down to 0.05 #g//~l. The TLC of a number of metal dithizonates was described by TAKITANI et al. 9~. Pd, Hg, Cu and Ag ions are extracted as dithizonates with o.1% dithizone in chloroform at pH o - i ; then Bi, Cd, Co, Ni, Pb and Zn ions at pH 7-8; and T1, Fe ~+, Mn, and Sn *+ ions at pH 9-1o. The ist- and 2nd-group metals are separated on an MNSilica Gel G-HR layer with carbon tetrachloride-methylene chloride-benzene (i :7:4) and identified by color. The 3rd-group metals are re-extracted with 20% nitric acid and separated with acetone-hydrochloric acid (99 :I) and detected by spraying with appropriate reagents. Identification limits in p.p.m, are: o.2 for Cu, Ni, Co; 0.3 for Hg; 0. 4 for Pd, Ag, Cd; 0.8 for Zn; 3 for Pb; and 5 for Bi. The TLC of metal dithizonates of Hg, Cu, Bi, Zn, Pb, Cd, Co and Ni was deChromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
2I 7
scribed by HRANISAVLJEVICet al. 98. The metals were extracted from aq. solution with o.1% dithizone in chloroform (viz., Hg, Cu and Bi extracted at pH 3; Zn, Pb and Cd at p H 5; Ni and Co at pH 7). The three chloroform solutions obtained were then separated on Silica Gel G. UNTERHALT94 described the TLC of a number of metal dithizonates and cations on Silica Gel G and cellulose MN-3oo F2s4 layers. Fig. 8 illustrates a chromatogram of the dithizonates of Cu(II), Bi(III), Cd(II), Zn(II), Hg(II) and Pb(II) at pH 5 on Silica Gel G developed with benzene-dichloromethane (5 :I) (3° rain, 15 cm). A 0.2%potassium iodide reagent detected Cu(II), Pb(II), Bi(III) and Hg(II) as yellow-brown to red spots, while o.I M sodium sulfite detected Cd(II) and Pb(II).
0 c) o
o
©
Fig. 8. TLC of metal dithizonates at pH 5 on Silica Gel G developed with benzene-dichloromethane (5:I). The utility of TLC in the forensic analysis of metals was described by KUNZI et al. 95. Silica Gel G (Merck) was used with acetone-benzene (75:25) saturated with tartaric acid and 6% nitric acid to effect the separation of the metals in the order of Re: Hg > B i > S b > C d > A s > P b > C u >T1. Both dithizone and ammonium sulfide reagents were used for detection (limit of detection with both reagents was 2 #g). Table 20 illustrates the spot colors of Cu, As, Cd, Sb, Hg, T1, Pb and Bi obtained with the above reagents. SUGIWARA9s reported the separation and identification of metallic poisons (viz., Pb, T1, Cu, Cd, Sb, Bi, As and Hg) on silica gel plates developed with nbutanol-I.5 N hydrochloric acid-acetylacetone (lOO:2O:O.5) and n-butanol-benzene z N nitric acid (70:3:2). The Re values were in the order: H g > T I > B i > S b > C d > A s >Cu>Pb. The determination of Ge(IV), Sn(II), Pb(II) and Zn(II), Cd(II), Hg(II) by ring colorimetry after separation by TLC has been described by JOHRI et al. 9~. Table 2I lists the R~, values and spot colors of the metal ions when developed on Silica Gel G plates with (I) isobutanol-conc, hydrochloric acid-ethyl methyl ketone (Io :8 :I) and (2) isobutanol-acetic acid (3:1) and detected with (a)o.o5% phenylfluorone, (b) o.o5 M aq. potassium thiocarbonate (PTC), and (c) dithizone and ammonia. For the evaluation of different metal ions, the resulting delineated spots as viewed in UV Chromatogr. Rev., 15 (1971) 195-238
218
L. FISHBEIN
TABLE 20 S P O T C O L O R S O F M E T A L S D E T E C T E D W I T H D I T H I Z O N E A N D (NH4),S Developing solvent: acetone-benzene (75:25) saturated with tartaric acid and 6% nitric acid on Silica Gel G.
Metal
Cu As Cd Sb Hg T1 Pb Bi
Dzthizone Acid
A mmoniacal
(NH4) ,S
yellow-green yellow lilac red rose ---purple
yellow-brown -orange bright brown orange-red rose rose orange-red
brown yellow yellow orange black black brown brown
light were r e m o v e d using a micro v a c u u m cleanergS, 99 an d the collected m a t e r i a l t ra n s f err ed to a paper set on a ring oven a n d d e t e r m i n e d b y ring c o l o r i m e t r y utilizing t he t e c h n i q u e of WEISZ 1°°. SEILER103 s e p a r a t e d H g ( I ) a n d H g ( I I ) f r o m a c a t i o n m i x t u r e b y T L C on silica gel using n - b u t a n o l - 4 N nitric acid (80:20) as d e v e l o p i n g solvent. A f t er separation, m e r c u r y was d e t e r m i n e d b y n e u t r o n - a c t i v a t i o n analysis b y y - s p e c t r o m e t r y o f 13o ke V 197Hg(meso). O n e / ~ g o f m e r c u r y was d e t e r m i n e d in t h e presence of I5oktg o f c a t i o n m i x t u r e w i t h a precision of ~ 4 % . TABLE 21 RE × IOO VALUESAND SPOT COLORSOF Ge(IV), Sn(II), Pb(II), Zn([I), Cd(II) AND Hg(II) ON SILICA GEL G Solvent systems: (I) isobutanol-cone, hydrochloric acid-ethyl methyl ketone (lO:8:1); (2) isobutanol-acetic acid (3:I). Detectors: (a) o.o5% phenyl fluoronO°I, (b) o.o 5 M aq. potassium thiocarbonatO °* and (c) dithizone and NH 3. Metal ion
Ge(IV) Sn(II) Pb(II) Zn(II) Cd(II) Hg(II)
RF × ~oo value Solvent i
Solvent 2
io.o 94.4 76.5 ----
---85.7 71.4 50.5
Reagent
Spot color
Limit of identification (ttg)
a b b c b b
pink brown pink-red pink yellow brown-black
6.5 ° I 1.2o 3.9o 3,60 2.20 4. io
K o s s AND BEISENHERZ104 described t h e d e t e r m i n a t i o n o f h e a v y - m e t a l ions as r a d i o a c t i v e sulfides following t h e i r initial separation on Silica Gel G-coated slides, thence t r e a t m e n t w i t h H2ssS. Fig. 9 illustrates the separation of c a d m i u m , copper a n d m e r c u r y salts on a Silica Gel G - c o a t e d microscope slide d e v e l o p e d with n - b u t a n o l 1. 5 N hydrochloric a c i d - a c e t o n y l a c e t o n e (ioo :20:0.5). T h e T L C s e p a r a t i o n of m e t a l - E D T A complexes on microcrystalline cellulose (Avicel) has been described b y MASOOMI AND HAWORTH1°5. T w e n t y - t h r e e m i x t u r e s of Chromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
2I 9
Fig. 9. Separation of cadmium, copper and mercury salts on a coated microscope slide. Layer: Silica Gel G; solvent: n-butanol-I. 5 N HCl-acetonylacetone (lOO:2O:O.5); time of run 2 h; detection with H235S. The chromatogram was evaluated by means of a Geiger-Mfiller counting tube (B) and the autoradiograph evaluated photometrically (A). the metal-EDTA complexes were separated using seven different solvent systems of v a r y i n g c o m p o s i t i o n (Table 22). T a b l e s 23 a n d 24 s h o w t h e R v v a l u e s o f v a r i o u s m i x t u r e s o f m e t a l - E D T A c o m p l e x e s (viz., Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd a n d H g c o m p l e x e s ) a n d t h e d a t a for specific d e t e c t i o n t e c h n i q u e s a n d r e a g e n t s u s e d for e a c h EDTA complex, respectively. T A B L E 22 TLC
S O L V F N T SYSTEMS FOR
Solvent system
SEPARATION OF M E T A L - E D T A
COMPLEXES ON A V I C E L
Compos,tion W'ater-n-butanol-glycol monomethyl ether-conc, ammonia ~ (4.5 : 3.5 : 2.o "o.25) Water-glycol monomethyl ether-methyl ethyl ketone-acetone-n-butanol-conG. ammoniaa (4 .0 : 3-o:1.5 :o.5 :i.o:o.Io) Water-glycol monomethyl ether-methyl ethyl ketone-acetone-conc, ammonia a (4:2:2:2
4 5A 5B 6A 6B 6C 6D 6E 6F 7
:O. I 5 )
Water-n-butanol-acetone-conc. ammonia a (4:4 : 2 :o, Io) \Vater-methyI ethyl ketone-n-butanol-acetone-conc, ammoma a (4.0: 2.5 : x.o: e. 5 :o. i o) Water-methyl ethyl ketone-n-butanol-acetone-cone, ammonia a (4.0 : I.O : I .o :4.° :o. 15) Water-acetone-conc. ammonia • (I.3 : 8.7:0.o5) Water-acetone-conc. ammoma a (2:8:o.o5) Water-acetone--cone. ammoma a (2.5:7.5 :o.05) Water-acetone-conc. ammoma a (3:7:o.o5) Water-acetone-conc. ammoma a (3-5:6.5 :o.o5) Water-acetone-conc. ammoma a (5:5 :o.io) Water-butyronitrile-methanol-HC104a (3 ml : 7 ml : xo drops) a The conG. ammonia and HC104 are on a basis of IOO ml of solvent.
T h e s e p a r a t i o n a n d i d e n t i f i c a t i o n o f P b ~+, A g +, H g 2+ a n d B a 2+, Sr 2+, Ca ~+ a n d Mg 2+ ions has b e e n a c h i e v e d b y T L C o n m a i z e - s t a r c h u s i n g a c e t o n e - 3 N n i t r i c a c i d (I :i) a n d a c e t o n e - 3 N h y d r o c h l o r i c a c i d (2:3) as solventsl°~, 1°~ w i t h t h e d e t e c t i o n o f I × IO-8-5 × IO -9 e q u i v , o f t h e i o n s a c h i e v e d d u r i n g a I 5 - c m 2-h d e v e l o p m e n t . I M a m m o n i u m sulfide s o l u t i o n w a s u s e d for t h e d e t e c t i o n o f t h e g r o u p o f m e t a l s P b ~+, H g 2+ a n d A g + a n d o . 5 % s o l u t i o n o f 8 - h y d r o x y q u i n o t i n e f o l l o w e d b y e x p o s u r e to a m m o n i a v a p o r s a n d d e t e c t i o n u n d e r U V for t h e g r o u p B a ~+, S r ~+, Ca 2+ a n d Mg ~+. T a b l e 25 lists t h e R E v a l u e s a n d s p o t colors for t h e T L C o f e i g h t m e t a l ions on m a i z e s t a r c h c h r o m a t o g r a m s u s i n g a c e t o n e - 3 N n i t r i c a c i d (I :I) as d e v e l o p i n g s o l v e n t a n d I M aq. a m m o n i u m sulfide a n d o . 1 % d i t h i z o n e - i n - c h l o r o f o r m as d e t e c t i n g r e a g e n t s . T h e s e p a r a t i o n o f t h e d i v a l e n t c a t i o n s of m e r c u r y , c o p p e r , c a d m i u m , n i c k e l a n d zinc b y c i r c u l a r - c h r o m a t o g r a p h y w i t h d i t h i z o n e - i m p r e g n a t e d silica gel t h i n l a y e r s w a s d e s c r i b e d b y DEGUCHI 1°8. T h e p l a t e s w e r e p r e p a r e d b y a d d i n g 3o m l o . 3 % di-
Chvomatogr. Rev., 15 (1971) 195-238
220
L. FISHBEIN
T A B L E 23 R F V A L U E S OF VARIOUS M I X T U R E S OF METAL-EDTA COMPLEXES V a l u e s o b t a i n e d w i t h T L C s e p a r a t i o n o n Avicel. S o l v e n t s y s t e m s f r o m T a b l e 22.
Solvent system No.
Metal ion (complexed w,th E D T A ) Cr
Mn
Fe
Co
Ni
Cu
I 2
3 4 4 5A 5B 5B 5B 5B 5B 6A 6B 6B 6B 6C 6C 6D 6E 6F 7 7 TABLE REAGENTS
0.46 0.46
o.54 o.58
0.60 0.67
o.74
o.42 o.42 o.17 o.39 o.65 o.29
0.07
Zn
Cd
Hg
0.70 0.68 0.74
0.65 0.58 0.64
0.62 0.55 0.60
0,59
0.45
0.38
0,II
0.09
0.07
o.12 o.46 o.52
0.09 0.26 o.44
0.54 0.65 0.66 0.78
0.75
o.49
o.57 o.61
o.13
0.07 0.27
0.29 0.54
0.49 0.39
o.13 o.17 0.59 0.60
o.86
0.75 o.i 5 0.22
0.82 0.06 0.09
0.45 0.42
o.58
24 AND
DETECTION
TECHNIQUES
FOR TLC
O F M E T A L - - E D T A COMPLEXES
S e p a r a t i o n o n Avicel. Solvent s y s t e m s f r o m T a b l e 22.
Metal ion (complexed wzth EDTA )
Complex color Reagent and detect,on techmque
Hg Cr
Colorless Violet-blue
Mn
Colorless
Fe Co
Yellow-brown Violet-red
Ni Cu
L i g h t blue Blue
Zn
Colorless
Cd
Colorless
A f t e r s p r a y i n g w i t h 5 % NazS yields a Mack spot. U n d e r U V light c o m p l e x a p p e a r s black. W i t h 0.05% (w/v) dit h i z o n e in CC14 yields a d a r k green color. W i t h a s a t u r a t e d solution o f 8 - h y d r o x y q u i n o l i n e in a m m o n i a yields a yellow color. W i t h 1% K C N S (w/v) yields a r e d color. U n d e r U V light t h e c o m p l e x a p p e a r s black. W i t h dithizone yields a grey-yellow color. A f t e r s p r a y i n g w i t h d i t h i z o n e yields a yellow color. A f t e r s p r a y i n g w i t h 8 - h y d r o x y q u i n o l i n e yields a l i g h t g r e e n color. A f t e r s p r a y i n g w i t h a dilute solution o f a m m o n i a a n d dithizone yields a p i n k color. After s p r a y i n g w i t h 5 % (w/v) solution o f N a , S b yields a yellow color.
a For m u l t i p l e s p r a y i n g , zones colored o n t h e T L C plate were first m a r k e d for t h o s e r e a g e n t s w h i c h were colored ; t h e n followed b y s p r a y i n g w i t h t h e d e t e c t i o n r e a g e n t s in t h e order : K C N S , NayS, d i t h i z o n e a n d 8 - h y d r o x y q u i n o l i n e ; e.g., in t h e d e t e c t i o n o f Hg, Cd a n d Zn s e p a r a t i o n , t h e p l a t e w a s s p r a y e d w i t h NayS to d e t e c t H g a n d Cd, zones were m a r k e d a n d t h e p l a t e s p r a y e d w i t h d i t h i z o n e a n d a m m o n i a to d e t e c t t h e Zn complex. (The H g a n d Cd s p o t s d i s a p p e a r e d in t h e p r e s e n c e o f base.) b Occasionally n e c e s s a r y to r e - s p r a y w i t h a dilute-acid s p r a y to develop t h e color.
Chromatogr. Rev., 15 (1971) 1 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
221
TABLE 25 RF VALUES AND SPOT COLORS FOR SOME METAL IONS ON MAIZE-STARCHCHROMATOPLATES Developing system: acetone-3 N hydrochloric acid (I:I); detecting reagents: (I) I M aq. ammonium sulfide and (2) o.i % dithizone-in-chloroform. Ion
RF value
Spot color Detecting reagent r
Pb 2+ Cu 2+ Bi 3+ Cd 2+ Hg 2+ As 3+ Sb 3+ Sn ~+
0.27 o.4o 0.75 0.80 0.84 0.36 0.74 0.84
brown yellow-brown black-brown yellow black ----
Detecting reagent 2
--yellow-red rose rose
t h i z o n e - i n - c h l o r o f o r m s o l u t i o n to I 0 g silica gel. T h e c a t i o n s w e r e t h u s s e p a r a t e d as m e t a l c h e l a t e s y i e l d i n g c o l o r e d c h r o m a t o g r a m s o f c o n c e n t r i c circles. T h e b e s t s e p a r a t i o n s o f Hg(II), Cu(II), Cd(II), Ni(II) a n d Zn(II) w e r e o b t a i n e d b y u s i n g e i t h e r 2 N a c e t i c a c i d - a c e t o n e (I :I) o r 2 - 4 N a c e t i c a c i d - a c i d (I :0.66) as d e v e l o p e r s . T h e c a t i o n s w e r e d e v e l o p e d in t h e a b o v e order, Z n b e i n g in t h e o u t e r m o s t circle. C i r c u l a r - T L C m e t h o d s for t h e i d e n t i f i c a t i o n of f o r t y c a t i o n s a n d n i n e t e e n a n i o n s f r o m 0.5 m l of u n k n o w n s o l u t i o n s w e r e d e s c r i b e d b y HASHMI et al. 1°9. T h e m e t a l i o n s w e r e i n i t i a l l y s e p a r a t e d i n t o five d e f i n i t e g r o u p s I - V b y s o l v e n t e x t r a c t i o n a c c o r d i n g to t h e p r o c e d u r e of WEST AND MUKHERJ111° (the s u b s e q u e n t d e v e l o p m e n t o f a chromatoplate was complete within two minutes). For cations, aluminum oxide (D-5, c o n t a i n i n g 5 % c a l c i u m s u l f a t e as b i n d e r , C a m a g ) a n d silica gel (D-0, w i t h o u t b i n d e r , C a m a g ) w e r e u s e d w i t h o u t f u r t h e r t r e a t m e n t , for t h e p r e p a r a t i o n o f t h e t h i n layers. F o r anions, A l u m i n u m O x i d e S a n d Silica Gel G ( H o p k i n a n d W i l l i a m s ) w e r e used. C i r c u l a r - t h i n - l a y e r c h r o m a t o g r a p h i c a p p a r a t u s l°s w a s u s e d for t h e d e v e l o p m e n t o f c h r o m a t o p l a t e s . T a b l e 26 lists t h e s o l v e n t s y s t e m s for t h e c h r o m a t o g r a p h i c s e p a r a t i o n o f c a t i o n s o f g r o u p s I - V a n d o f a n i o n s , T a b l e 27 lists t h e s p r a y r e a g e n t s for locaTABLE 26 SOLVENT
SYSTEMS
FOR CIRCULAR-TLC
SEPARATION
OF CATIONS
OF GROUPS
I-V
AND OF ANIONS
Separation on aluminium oxide (3-5 for cations; S for anions) and silica gel (D-0 for cations; S for anions). Solvent system
Composition
Group a
A B C D E Fb
Acetone- 4 N HCl-acetylacetone (45:3 : 2) Acetone-4 N HC1 (47:3) Acetone- 4 N HCl-acetylacetone (48 :i.5 :o.5) Acetone- 4 N HC1 (46:4) Acetoue-conc. HC1 (47:3) n-Butanol-pyridine-water-ammonia (8 :4 : 8 : I)
I, II III IV V V Anions
a Metal ions separated by solvent extraction into five groups: I = chloride group; II = thiocyanate group; I I I = acetylacetone group; IV = diethyl dithiocarbamate group; V = aqueous group. b After shaking, the upper layer is used. Chromatogr. Rev., 15 (1971) 195-238
222
L. FISHBEIN
TABLE 27 SPRAY RI~AGENTS FOR LOCATION ON CIRCULAR-TLC
PLATES OF CATIONS AND OF ANIONS OF GROUPS
I-V
Spray reagent
Solution
Tannic acid (TAw) Tannic acid (TAg) Stannous chloride-potassium iodide (SC-KI) Phenyl fluorone (Pf) Dithizone (DZc) 1Rubeanic acid (IRA) Diphenyl carbazide (DC) Potassium ferrocyanide (PF) Alizarin (AZ) Peroxide-benzidine (PB)
lO% in water lO% in glycerine-water (I :i) Dissolve 5 g SnC12in IO ml conc. HC1 and dilute to ioo ml with addition of 0. 5 g KI. 0.05% in 96% ethanol-conc. HC1 (3:1) 2 % in chloroform o.5% in 96% ethanol i% in 96% ethanol 5% in water saturated solution in 96% ethanol 5% Na,O, in water followed by 1% benzidine in glacial acetic acid Quinalizarin (QAZ) o.o5% in 70% ethanol Sodium sulfide (SS) 2% in water Dlmethylglyoxime (DO) 1% in 96% ethanol 1Rhodizonic acid. sodium salt (SR) 1% in water, freshly prepared Hydrogen peroxide-ammonia (HA) H,O, (3o% wtv)-conc, ammonia (I :I) Sodium cobaltinitrite (SCo) cobaltinitritea-methanol (3: i) AgNO~ 1% in water; after short drying followed by o.1% Silver nitrate-fluorescein (SF) fluorescein in 96% ethanol Silver nitrate (S) saturated solution in water Potassium iodide-HC1 (PH) lO% solution containing lO ml HCl (2 M) Ferric chloride (FC) lO% solution containing io ml HCI (2 M) Potassium dichromate (PD) saturated solution in water, containing io ml H,SO4 (2 M) Sodium nitroprusside (SN) 20% in water Ferrous sulfate (FS) lO% in water, containing 20 ml HzSO4 (2 M)
a 11.4 g cobalt acetate (CHsCOO)2Co.4HzO, 16.2 g lead acetate (CH3COO)zPb.3H~O) , 20 g sodium nitrite and z ml glacial acetic acid are mixed, dissolved and diluted to 15o ml, centrifuged, and filtered. tion of the cations a n d anions, a n d Table 28 the R F values, s e n s i t i v i t y a n d m a x i m u m a m o u n t of cations of groups I-V. The s e n s i t i v i t y in this table indicates the m i n i m u m a m o u n t of the m e t a l ion which m u s t be present before e x t r a c t i o n ; the m a x i m u m a m o u n t indicates the u p p e r limit of each m e t a l ion which does n o t interfere with the succeeding rings when all m e t a l ions of a p a r t i c u l a r group are present in their m a x i m u m a m o u n t . F o r circular-TLC analysis, it was f o u n d essential to apply the m e t a l ions in a m o u n t s which lie between the sensitivity a n d the m a x i m u m a m o u n t shown in T a b l e 28. The s e p a r a t i o n of a n u m b e r of m e t a l diethyl d i t h i o c a r b a m a t e s on Silica Gel G plates was described b y SEI~Fi n using a solvent system c o n t a i n i n g n - h e x a n e - c h l o r o f o r m - d i e t h y l a m i n e (20:2 :I). After the plate was heated, in order to remove the die t h y l a m i n e , the spots were developed b y spraying with 5% copper sulfate a n d o.I # g of each m e t a l was detectable. Interfering metals such as Co, Ni, Fe a n d Mn could be r e m o v e d b y controlling the p H range with c i t r a t e - p h o s p h a t e buffer. T h e R v values of the m e t a l diethyl d i t h i o c a r b a m a t e s were: Hg, o.56; Pb, o.oo; Cu, 0.44; Bi, 0.27; a n d Cd, 0.34. TAKESHITA et al. nz described a reversed-phase TLC m e t h o d for the separation a n d detection of the dithizonates of both inorganic m e r c u r y a n d a series of alkyl-
Chromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHICAND BIOLOGICALASPECTS OF INORGANICMERCURY
223
mercury compounds and found the method applicable to the separation and identification of mercury compounds in foods and sewages. The adsorbents used were cornstarch containing liquid paraffin and Avicel SF (FMC, American Viscose Div.) containing 20% liquid paraffin. The solvent systems consisted of methyl celloso]vewater (75:25) and ethanol and methyl cellosolve treated, respectively, with water in the ratios from o to 30%. The study revealed that the best pattern of separation of all the dithizonates was obtained on Avicet SF-liquid paraffin layers with methyl cellosolve-water (7:3), although the development time was longer than with impregnated cornstarch plates (1-1. 5 h). No interference of other metals and the residual TABLE 29 DETECTION
LIMITS OF MERCURIC
AND ALKYLMERCURIC
DITtlIZONATES
AND CHLORIDES
Layer: Avlcel SF-liquid paraffin. Developer: methyl cellosolve-water (75:25). Compound
Detection limit (fig)
Mercury Methylmercury Ethylmercury n-Propylmercury n-Butylmercury n-Amylmercury n-Octylmercury Stearylmercury
Dithizonate
Chloride
o.oi
o.oo4 o.o53 o.o55 o,o56 0,057 0.029 0.006 o.oo7
o.I o. i o.i o.i 0.05 o,oi o.oi
dithizone was observed. Table 29 lists the detection limits of mercury and alkylmercury compounds (as dithizonates and chlorides) on Avicel SF-liquid paraffin layers with methyl cellosolve-water (75:25) as developer. 6. GAS CHROMATOGRAPHY TADMORna described the gas chromatographic (GC) separation of a number of metal halides, e.g. (a) SnC14, SnBr~, SnI,, (b) AsC13 and GeC14, and (c) FeC13 and HgC12, (2)
(1l
V///////////// ,4,-..
,
*\
.......
0
',;L-'HI (8)
Fig. Io. Home-made gas chromatograph. (i) Electrical furnace; (2) gas preheater; (3) chromatographic column; (4) carrier gas; (5) injection port; (6) heating wire; (7) effluent solvent stream; (8) to radioactivity detector and recorder. Chromatogr. Rev., 15 (1971) I95-238
224
L. FISHBEIN
~
o ~o ~
o
= ~o o
~
~
. ~.~
~: ~b~ ~
~
~.~ ~.
0
o~
~
o
~o~
o
~00°°
~.
~
~
oo
d d d d 5
~
&~d d d
d d
d
d
d d
o Z
%
~,~o
~o
0 ~
"~
~
o
"~
'~
~
o
~o
~oo%%oo~
d
d6~6dd6d6
o
Z 0
~
~
g~
~
~:.~
~ ~
"~ ~ ~
"~"0
o
.,:
.~ ~ "n~ ~ " 0
~
~o®
~
o
m
o
0 0 0
N
++
+
m
Chrom~mg~. Rev., 15 (I97 I) 195-238
~ + +
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS O F INORGANIC MERCURY
225
3g o.
o~
o~
~
~
~ ~ .~ ~ o
,.m
~,
t~
o .o o~
~
0
~
0
~+~ "
~
o ~0 O
~00
~ 0 ~ ~o
oqt~O
~oo
G*
6
. . . .
666666d
O~ O0
6
oooo
6666
~
666dMG o
fi~ o o
o
~,
o ~
~ o o~
o~
~
~
"~
~o
.,~
++++~+~
~+
+ + +
+
"~+ + + Ho
Chromatogr. Rev.,
15 (1971) 1 9 5 - 2 3 8
~ ,1:::~
226
L. FISHBEIN
utilizing a laboratory-constructed apparatus. The apparatus (Fig. IO) was comprised of a chromatographic column preceded by a preheater section for the carrier gas. These consist of U-shaped Pyrex glass tubes packed with different stationary phases; the apparatus is placed in a cylindrical electrical furnace (Adamel type T5HT ) whose ports are closed with asbestos-insulating material in order to thermostate the apparatus. Supplementary heating is provided for the injection port and the end of the column by a heating wire connected to a resistance. The solid stationary phase was Sil-O-Cel brick (30-50 mesh), washed with 2.5 N HNO 3, rinsed with water and dried at IiO °. Some of the liquid stationary phases were: n-butanol, n-decane, glycol, KelF-oil, nitrobenzene, glycerol, A1Br3, Silicone oil-55o, BiC13 and Woods Metal. Samples were labeled with radioactive isotopes, which were distilled in and kept under an atmosphere of dry nitrogen, then introduced into the apparatus as a solution in an organic solvent or as the compound itself. Effluent activity was determined with a liquid beta counter (2oth Century Electronic, Model M6) connected to an Atomic Scaler, Model lO91. Fig. I i illustrates the GC separation of HgC12 and FeC13 on a 8o-cm column coated with 3o% BiCla on Sil-O-Cel brick (30-50 mesh),
h
! I
II It
....
HQC Ia
I
~
Feel
I
3
i
'I
tO 0
v
,
o
I
I
2
4 Time
.......
6
1,,.,
(rnln)
Fig. I I . Gas c h r o m a t o g r a p h y o f HgC1, a n d FeC1 v C o l u m n (80 cm) : Sil-O-Cel brick (30-50 mesh) c o a t e d w i t h 3 o % w[w BiCI v T e m p e r a t u r e : 290 °. Carrier g a s (nitrogen) flow: IO m l / m m .
It is of interest to note that the volatilization and separation of many acetylacetonates and fluorocarbons/~-diketonates (excluding those of mercury) have been accomplished by both conventional n4,n5 and hypelpressure GC techniques. Apparently the analogous mercury derivatives are not volatile enough nor possess sufficient thermal and solvolytic stability to permit GC analysis. 7.
ION-EXCHANGE AND COLUMN CHROMATOGRAPHY
Mercury, zinc and cadmium were quantitatively resolved as their anionic-chloro complexes by an ion-exchange chromatographic procedure ne. The stability of these complexes increased in the order zinc-cadmium-mercury. The metal chtoro complexes were adsorbed on a o.oi M hydrochloric acid solution by the anion-exchange resin
Chromatogr. Rev.,
15 (1971) I 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
227
D o w e x - i (Ct- f o r m ) . Z i n c a n d c a d m i u m w e r e e l u t e d s e p a r a t e l y in t h a t o r d e r w i t h o . o i M h y d r o c h l o r i c acid. M e r c u r y w a s t h e n r e m o v e d w i t h o . o i M h y d r o c h l o r i c a c i d - o . I M t h i o u r e a s o l u t i o n , a n d a n a l y z e d b y p r e c i p i t a t i n g a n d w e i g h i n g t h e m e t a l a s t h e sulfide. Z i n c a n d c a d m i u m w e r e a n a l y z e d b y t h e t i t r i m e t r i c p r o c e d u r e o f MARTELL AND CALVIN 117 i n v o l v i n g t i t r a t i o n w i t h V e r s e n e a t p H i o w i t h E r i o c h r o m e B l a c k T a s a n indicator. RILEY AND TAYLORlls d e s c r i b e d t h e u t i l i t y o f c h e l a t i n g r e s i n s f o r t h e c o n c e n t r a t i o n o f t r a c e e l e m e n t s f r o m s e a w a t e r as w e l l a s t h e i r u s e i n c o n j u n c t i o n w i t h a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y . Two different resins, b o t h of the iminodiacetic acid t y p e , w e r e u s e d i n t h i s i n v e s t i g a t i o n , viz., (a) C h e l e x - I o o ( B i o - R a d L a b . , R i c h m o n d , Calif, 5 o - I o o m e s h ; t h i s is a p u r i f i e d f o r m o f D o w e x A - I r e s i n ) a n d (b) P e r m u t i t S lOO5 ( P e r m u t i t Co., lO-2O m e s h ) . T h e r e s u l t s o f e x p e r i m e n t s u s i n g C h e l e x - I o o a r e s h o w n in T a b l e 30. I n g e n e r a l , b o t h r e s i n s g a v e s i m i l a r r e s u l t s . T h e r e s i n s c a n o n l y b e u s e d f o r the uptake of trace elements from sea water and non-saline waters over a restricted r a n g e o f p H v a l u e s ( t h e l o w e r l i m i t is p H 5.0, b e l o w w h i c h u p t a k e falls off r a p i d l y ) .
TABLE 30 DATA
ON ADSORPTION AND ELUTION OF TRACE AND 20 In] OF ELUTING AGENT
ELEMENTS
FROM
SEA
WATER
WITH
CHELEX-IOO
(5O-lOO mesh)
Trace element
pH for adsorption
Retention (%)
Eluant
Total recovery (%)
A1 uminum Arsenic (AsO4~-) Barium Bismuth Cadnfium Caesium Cerium (Ce3+) Chromium (Cr 3+) Cobalt Copper Indium Lead Manganese Mercury (Hg ~+) Molybdenum (MOO42-) Nickel Phosphorus (PO43-) Rhenium (l~eO~-) Scandium Selenium (SeOa z-) Silver Thallium (TI +) Thorium Tin (Sn 4+) Tungsten (WO42-) Uranium (UO, ~+) Vanadium (VO ~-) Yttrium Zinc
7.6 7.6 5.o a 9.o a 7.6 7.6 9.o a 5.o a 7.6 7.6 9.o a 7.6 9.o a 7.6 5.o a 7,6 7.6 7.6 7.6 7.6 7.6 7.6 a 7.6 7.6 6.o a 7.6 6.o a 9.o a 7.6
o o 25 IOO IOO o IOO 25 IOO Ioo IOO IOO ioo 85 IOO ioo o 90 IOO o IOO 50 ioo 85 IOO o IOO IOO IOO
2N 2N 2N 2N 2N 2N 2N 2N 2N 2N 2N 4N 2N 4N 2N 2N zN 2N 2N 4N 4N 2N 2N
o o 25 IOO IOO o IOO lOb ioo IOO IOO IOO ioo 4 ob IOO ioo o 9° IOO o 9o b 5° IOO 6o b Ioo o IOO IOO IOO
HNO 3 HC104 HNO 3 HNOz tINO~ HC1 HNO 3 HNO 3 HNO~ HNO 3 HNO~ NH4OH HNO 3 NH4OH HNO 3 HNOz HNO 3 H~SO a HNO s NH4OH NH4OH HNO 3 ttNO 3
a Optimum p H value. b Maximum percentage removable from resin,
Chromatogr. Rev., 15 (1971) 195-238
228
L. FISHBEIN
TABLE 31 ANALYTICAL ME T HODS U S e D FOR T H E D E T E C T I O N OF METAL IONS ON D E A E - I O N - E X C H A N G E COLUMNS
Cation
Method
Al(III)a, Ga(III) a, Ni(II)a, V(IV) a, As(III)" Bi(III), Co(II)~, In(III), La(III), Lu(III), Sc(III), Sm(tlI), Th(tV), TI(I), Y(III), Zn(II) Ca(II) Cd(II), i g ( I I ) , Mn(II), Pb (II), Sr(II)
Titration with EDTA using Cu-PAN c as indicator Colorimetrically with ammonium molybdate Titration with EDTA using Xylenol Orange as indicator Titration with EDTA using b as indicator Titration with EDTA using Eriochrome Black T as indicator Colorimetrically as chromate Titration with EDTA using PAN as indicator CotorimetricaUy with NH4CNS , for traces Colorimetrically with phenyl fluorone Colonmetrically with dithizone Colorimetricalty with KCNS-SnCla Colorimetrically with a-nitroso-fl-naphthol Colorimetrically with KI after decomposition of tartaric acid Colorimetrically with SnCI3 Colorimetrically with dithiol Colorimetrically with hydrogen peroxide Colorimetrically with quercetin
Cr(III)a Cu(II) a Fe(III) a Ge(IV) Hg(II) Mo(VI), Re(VII) Pd(II)a Sb(III)~ Se(IV), Te(IV) Sn(IV) a, ~V(VI) a U(VI) a Zr(IV)
Determined after decomposition of thiocyanate with HNO v b 2_ttydroxy_I_(2_hydroxy_4_sulfo_I_naphthylazo)_3_naphthoic acid, c PAN = I-(2-pyridylazo)-2-naphthol. N i t r i c a c i d (2 N ) w a s s h o w n t o b e t h e m o s t g e n e r a l l y - u s e f u l r e a g e n t for r e m o v i n g c a t i o n i c s p e c i e s f r o m t h e r e s i n s a n d h a s t h e a d d e d a d v a n t a g e t h a t i t is e a s y t o r e m o v e s u b s e q u e n t l y . Silver, m e r c u r y , c h r o m i u m a n d tin were v e r y s t r o n g l y r e t a i n e d b y t h e resin and no reagent was found which would elute them completely. However, c o m p l e t e ( 9 9 - 1 o o % ) r e c o v e r i e s o f Bi, Cd, Co, Cu, I n , Mn, Mo, Ni, P t , R e , Sc, T h , W , V, Y, Z n a n d t h e r a r e - e a r t h e l e m e n t s c o u l d b e o b t a i n e d a t t h e i r n a t u r a l c o n c e n t r a tions of sea water when Chelex-Ioo was used.
TABLE 32 DEAE a AS
D I S T R I B U T I O N C O E F F I C I E N T S ( k a ) FOR M E R C U R Y ( I I ) ON AND pH
Serzes
pH
Ka values oM
A
B
F U N C T I O N S OF T H I O C Y A N A T E CO N CE N T RA T I O N
3.o 2. 3 x lO3 2.0 2.3 × lO3 i.o 2.2 × 103 i . o M HC104b I8 3.0 >1o 4 I.O M HC10, b 19
Total conch, of el- (M) o.oxoM
o,o5oM
o.zoM
o.5oM
LoM
2.8 × Io 3 2.5 x lO3 2.9 ~( 103 23 >1o 4 23
1.8 × lO3 2.1 × lO3 2. 5 X lO3 29
9-7 × 1°2 1.2 × lO3 1.2 × Io 3 18
ca.io
4.6 × 103
i.o i.I i.i 41 1.2 37
43 37 43 24 37
o. Io o.Io o. Io o.Io o.o
20
O.O
a D E A E in a thiocyanate form. b Separate experimental determination.
Chromatogr. Rev., 15 (1971) 195-238
30
a
22
× io z x lO2 × io ~ ×
lO 3
(KC1) (KC1-HC1) (KC1-HC1) (NaCI)
CHROMATOGRAPHIC
229
AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
A systematic study of the behavior of many metal ions on the weakly-basic cellulose exchanger D E A E in dilute thiocyanate media revealed that a few metal ions show any marked adsorption while mercury(II) exhibits pronounced adsorption 52. This preferential adsorption of mercury allows its rapid and highly selective separation from about forty metal ions, e.g., ca. o.I mg mercury(II) from milligram amounts of other metal ions and o.I-IO mg of mercury(II) from iron(III) in proportions of mercury(II) : iron(III) = ioo :I to i :8000 can be quantitatively separated on a column containing only I g DEAE. The analytical methods used to detect the various metalcation column effluents are shown in Table 31. Table32 lists the distribution coefficients (K,) for mercury(II) in thiocyanate media on D E A E (Serva; Gallard-Schlesinger Chem. Mfg. Co.) (thiocyanate form) as functions of the thiocyanate concentrations and of acidity. At each thiocyanate concentration, the Kd values for mercury(II) do not vary to any great extent with pH within the range 1-3 (Series A, Table 33). TABLE 33 EFFECT OF TOTAL CHLORIDE CONCENTRATIO.~'~ ON THE DISTRIBUTION COEFFICIENT (/~d) OF
MERCURY(II)ON DEAE pH Ks values oM
O.OlOM
o.o3oM
o.Io3~
o.3oM
o.5oM
I.oM
With DEAE-SCN--form a 3.O b ~ - 1 0 4 I.O c
>104
With DEAE~tl--forrnd 3.o b 9.1 × lO 2 I.I I.O e
×
~--'-104
lO 3
1.2
×
2 . 8 X 103 2 . 9 × 103
lO 3
3 . 0 X 102 3.3 X IO ~
1.2 × 10 ~ 1.2 X IO ~
34 28
6.8
×
lO 2
2.o
×
lO 3
87
27
6.3
x
lO 3
2.2 ×
IO ~
95
28
a The concentration of NH4CNS was kept at O.OLOM throughout. b KC1 was added to pH 3 acetate buffer solution to give the chloride concentration hsted. To KC1 solution of the listed chloride concentration, HCI solution of the same chloride concentration was added to give the chloride buffer solution of pH I. a No thiocyanate present. However, the concentration of thiocyanate or chloride exhibits a marked effect on Ka for mercury(II). The chloride dependence of Ka values for mercury(II) is also demonstrated in Table 32, where Ka values for mercury(II) on D E A E (thiocyanate form) are listed as a function of chloride concentration. Within the lower chlorideconcentration range ( < o . I o M) the Ka values for mercury(II) are well above 2.1o 3 but decrease sharply as the concentration of chloride increases. The form of D E A E is of particular importance in keeping the Ka values for mercury(II) sufficiently high. For comparison, Ka values for mercury(II) in chloride media on D E A E in the chloride form are also listed in Table 33. A radiochemical method for the determination of mercury, arsenic, bromine, antimony and selenium ions in neutron-irradiated biological material has been described by SAMSAHL119,120. The method is based on distillation of the neutron-irradiated sample (max. 200 mg of a dried, soft animal tissue is sealed in a small quartz tube and irradiated together with standards of As, Br, Hg, Sb and Se ions for I to 2 days with a thermal flux of 2. lO13 n/sq. cm/sec), followed after a 2-3 day decaying Chromatogr. Rev., 15 (1971) 195-238
230
L. FISHBEIN
p e r i o d b y a u t o m a t e d ion-exchange a n d e x t r a c t i o n c h r o m a t o g r a p h i c m e t h o d s for s e p a r a t i n g t h e trace elements into twelve groups suitable for g a m m a s p e c t r o m e t r i c m e a s u r e m e n t s . Figs. 12 a n d 13 i l l u s t r a t e t h e d i s t i l l a t i o n a p p a r a t u s a n d scheme of a n i o n - e x c h a n g e s e p a r a t i o n system, respectively. T h e radionuclides 76As (0.55 meV), K
N
A
D
I
N!
.
ii !
Fig. i2. Distillation apparatus. (A) Distillation flask, i5-ml volume, I75 mm long, B 14 joint; (B) receiver flask, 3o-ml volume, i2o mm long, B 14 joints; (C) reflux condenser, I5o mm long, B Io and B 14 joints; (D) and (E) traps for 2 and 5 ml, respectively, B Io joints; (F) and (H) funnels, io x ioo mm; (G) and (I) stopcocks; (J) borosilicate tube, 45 × 380 mm; (K) arrow points to water suction pump; (L) bunsen burner; (M) compressed air. Fig. I3. Scheme of anion-exchange separation system for separation of Sb, As, Hg and Se. (A) Piston barrel, 28 × 15o mm; (13) and (C) piston barrels, 20 × 15o mm; (D) 4 × 5° mm, Dowex 2 (HSO4-, 2oo-4oomesh); (E) 7 × 5o ram, Dowex 2 (CI-, 2oo-4o0 mesh); (F) 18 × 5° mm, Dowex 2 (Br-, CI-, 2o0-400 mesh) ; (G) mixing coils, 5 turns, 15 mm outer diameter; (H) piston with rubber stopper; (I) perspex plate, 15 mm thick. ~°aHg (o.28 meV), l ~ S b (o.56 meV) a n d 73Se (o.14 meV) were s e p a r a t e d in the different groups as follows: G r o u p I : N a O H adsorption--S~Br G r o u p 2: D o w e x 2 (sulfate)--197Hg, ~°SHg G r o u p 3: D o w e x 2 (chloride)--12~Sb, 1~4Sb G r o u p 4: D o w e x 2 (bromide, chloride)m~eAs, 75Se. T h e s i m u l t a n e o u s d e t e r m i n a t i o n of Cu, Zn, Cd a n d H g ions with high s e n s i t i v i t y utilizing n e u t r o n a c t i v a t i o n analysis was described m . A f t e r i r r a d i a t i o n , t h e samples were d i g e s t e d a n d an initial s e p a r a t i o n of t h e four e l e m e n t a l ions m a d e b y m e a n s of a n ion-exchange resin. T h e e l e m e n t a l ions in t h e s e p a r a t i o n fractions were t h e n t r e a t e d to yield a r a d i o c h e m i c a l p u r i t y , p r e c i p i t a t e d a n d their activities measured. TORIBARA AND SHIELDS1~ described a scheme for t h e analysis of s u b m i c r o g r a m a m o u n t s of m e r c u r y ions in tissues involving (I) t h e s e p a r a t i o n of m e r c u r y ion from tissue b y a cold digestion in hydrochloric a c i d - s o d i u m n i t r a t e , (2) collection on an a n i o n - e x c h a n g e column, (3) elution w i t h t h i o u r e a solution, (4) collection in c a d m i u m sulfide, v o l a t i l i z a t i o n a t 55 o°, (5) d e t e r m i n a t i o n p h o t o m e t r i c a l l y w i t h a General E l e c t l i c germicidal U V - i n t e n s i t y meter. The ion-exchange s e p a r a t i o n was accomplished using A m b e r l i t e I R A - 4 o o (5O-lOO mesh) w i t h elution w i t h o . o i M thiourea. T h e s e p a r a t i o n a n d d e t e c t i o n of p i c o g r a m q u a n t i t i e s of c a t i o n s in cells a n d tis-
Chromatog~'. Rev., 15 (1971) 195-238
CHROMATOGRAPHICAND BIOLOGICALASPECTS OF INORGANICMERCURY
231
sues was described b y KLIMES AND BETUSOVAlz3. The sample is first mineralized with HC1-KCI04 and heated, then the cations separated on a strongly-basic ion-exchanger (Wofatit 1-15o, Veb, Farbenfabrik, Wolfen, G.F.R.) or by electrolysis and identified b y means of classic color reactions in a single grain of silica gel support. The procedure is exemplified by the detection of (a) about IO pg mercury cation in o.I-mg samples of kidney and in I - m g samples of adrenal cortex of a rat previously treated with Agronal and (b) the detection of about 7 Pg iron cation in a single cell of A l l i u m cepa. Fig. 14 illustrates the apparatus for the picogram-detection of cations in cells and tissues. 20
40
100
50
,oo~ Glass
(c)
10 \
(a)
(b)
1o0 • \7--
(d)
J "";-"
/Sealed
I
,,,
w~th
lacquer
Fig. 14. Apparatus for picogram-detection of cations in cells and tissues. (a) Pico test tube; (b) mineralization flask; (e) pico electrode; (d) pico electrode pair. All dimensions are in ram. KOPP AND KEENANT M described an ion-exchange method for the complete separation of submicrogram and larger quantities of mercury ions from o.3--o.5 mg copper, lead, cadmium, thallium, zinc and nickel cations. The mercury ions in a digest of a biological sample, e.g., urine, are first adsorbed on a IO × 30o m m column containing a strongly-basic anionic resin (Dowex I-XS, C1--form, IOO-2OO mesh) and then eluted quantitatively with o.002 M thiourea in o.oi M hydrochloric acid. The eluted mercury was then complexed directly with a standard dithizone reagent and read spectrophotometrically at 49 ° m/z, with a sensitivity of 0.3 ~ug. Experiments with *°3Hg and with the stable isotope of mercury in urine yielded recoveries exceeding 92%. The exact chemical composition of the excretory products of mercury in the urine is unknown. There is evidence that organomercurials which have strong C - H g bonds and do not ionize are excreted in unchanged form 125. KURODA et al. 12~ described the separation of thirty-one pairs of metal ions (including C u ( I I ) - H g ( I I ) and Sn(IV)-Hg(II)) using columns of Amberlite CG4B and in the above cases elution with I M hydrochloric acid and 2 M perchloric acid. The chromatographic behavior of Zn, Cd and Hg ions on columns of natural cellulose and substituted celluloses was studied by using SSZn, 1°9Cd and ~°3Hg radio tracers 12~. Traces of Zn and Cd ions were strongly retained b y the functional groups attached on the substituted celluloses, while mercury- ion was not retained to any extent. Amounts of zinc, cadmium and other metal ions were separated from 3 g of mercury on cellulose phosphate in ether.
Chromatogr.. Rev., 15 (1971) 195-238
232
L, FISHBEIN
8. ANALYSIS OF MAMMALIANDISTRIBUTION, TRANSPORT AND EXCRETION OF INORGANIC MERCURY T h e d i s t r i b u t i o n of m e r c u r y a m o n g blood fractions a n d s e r u m p r o t e i n s in t h e r a t following i.v. a d m i n i s t r a t i o n s of o.12 m g ] k g a n d 1.2 m g ] k g of i s o t o p i c a l l y t a g g e d ~°3HgC12 was s t u d i e d b y CEMBER et al. 1~8. T h e m o s t i n t e r e s t i n g o b s e r v a t i o n in this s t u d y was t h a t m o s t o f t h e s e r u m - p r o t e i n - b o u n d m e r c u r y was i n i t i a l l y associated w i t h t h e a l p h a globulins. A t t h e lower dose level, this initial f r a c t i o n a l d i s t r i b u t i o n r e m a i n e d a b o u t t h e same t h r o u g h o u t t h e o b s e r v a t i o n period (9 6 h). F o r tile higher dose level, t h e r e was a shift w i t h increasing t i m e in t h e r e l a t i v e c o n c e n t r a t i o n o f merc u r y from a l p h a globulins to a l b u m i n . Figs. 15 a n d 16 i l l u s t r a t e densitorneter tracings a n d r a d i o c h r o m a t o g r a m s showing in vivo d i s t r i b u t i o n of m e r c u r y in serum p r o t e i n s 3/4 h after injection of 200 # g
Pro tel ps densitometer
tracmg
I I I I J I J I
tl~r~
i
Merc ur y ra d ~ochromatogram
Fig. 15. Densitometer tracing and radiochromatogram showing in vivo distribution of mercury in serum proteins a], h after injection of 2oo/~g of Hg into a rat.
Protelns densitorneter
tracmg
Y Mercury rad~ochromotograrn
Fig. i6. Densitometer tracing and radiochromatogram showing in vitro distribution of mercury in serum proteins 3/4 h after inoculation of whole blood with mercury, at a concentration of 2/~g/ml. Chromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
233
of mercury into a rat, and in vitro distribution of mercury in serum proteins 3/, h after inoculation of whole blood with mercury, at a concentration of 2/~g/ml, respectively. For the above determinations, the serum proteins were separated for mercury analysis by electrophoresis on a cellulose acetate strip in barbital buffer at pH 8.6 for I h at 300 V. After staining with Ponceau S stain, two strips were cleared for graphical protein determination with a densitometer and for graphical mercury determination with a 4~ windowless radiochromatogram scanner. The excretion of volatile mercury (in part from the lungs and in part from body surface) by rats injected with ionic *°3Hg, and the elaboration of the factors that influence the rate of such excretion, including the dose of mercury, the time after inj ection and the intensity of respiration, were described by CLARKSONANDROTHSTEIN39. Although the chemical form of the volatile mercury was thought to be probably metallic-mercury vapor, it was suggested that it could be either one of the volatile organic compounds of mercury such as dimethylmercury. It is possible that enzymic systems in the animal tissues are able to methylate inorganic mercury in an analogous manner as tellurium is converted to methyl telluride and excreted in the breath TM. A more probable alternative suggested by CLARKSONAND ROTHSTEIN is the reduction of the mercury and thence volatilization from the animal as inorganic vapor. The mercury-blood interaction and mercury uptake by the brain after vapor (*°3Hg) exposure has been studied by MAGOS3~. On the wet basis the mercury uptake by the brains of mice exposed to o.oo3-9.9oo #g mercury/1 air for 4 h was little more than two-thirds of the total uptake (inhalation plus adsorption by fur). This high quotient suggests that the maior part of mercury is equilibrated in a highly diffusible form between the blood and the tissues including the brain. The conversion of elemental mercury to mercuric ion by blood exposed in vitro to mercury is a slow process compared with the circulation time from the lung to the brain. It was assumed by MAGOS that the elemental mercury is the highly diffusible form which equilibrates between the blood and the tissues. During the 8-day post-exposure period the relative distribution of mercury was modified b y the differences in organ clearance (8 days after exposure the brain contained nearly as much mercury as the kidneys). Fig. 17 illustrates the diagram of the experimental design for labeling mercury vapor with ~°3Hg based on the Hg-e°3Hg exchange reaction. Labeled mercury in samples of blood, plasma, or saline as well as that adsorbed b y the Hopcalite adsorbers in the generating and exposure apparatus (Fig. 17) was measured in a scintillation detector (Ekco Electronics, Ngg4B ) and scaler (N6IoA) having a counting efficiency for 2°~Hg of 40%. The role of biotransformation in rats was studied by NORSETH AND CLARKSONTM via an examination of the intestinal transport of Z°~Hg-labeled methylmercuric chloride. Inorganic mercury in the presence of methytmercuric salts in tissue samples, fluids and extracts was analyzed by the isotope-exchange method 43. The chemical form of mercury in bile was determined by TLC on Eastman Chromagram 6o61 Silica Gel and electrophoresis on Beckman paper 319328 or 320046. The developing solvents for TLC were n-propanol-water (7° :3o) and ethanol-34% ammonium hydroxide (70:30). Diethyl barbiturate-sodium barbiturate buffer, pH 8.6, was used for etectrophoresis (14 h at 5 mA constant current). Chromatograms were excised for counting or scanned by a gas-flow counter and bile was analyzed by column chromatography of Sephadex G-Ioo to separate diffusible and protein-bound mercury. A Sephadex 15 × 30 column Chromatogr. Rev., 15 (1971) 195-238
234
L. FISHBEIN Water"
pump
NVI
HA2
Me rcLiry-~- f r e e
ale
Fig. 17. Diagram of the experimental design for labeling mercury vapor by exchange reaction (not to scale). The direction of the flow is from left to right. Connection A is used for inhalation experiments and connection B for exposing blood to mercury vapor. The other symbols are as follows: NV1 and NV2, needle valves; Pal, R2 and R3, rotameters; MVGS, mercury-vapor generating system; HA1 and HA2, Hopcalite adsorbers, type 1 and type 2; ER, exchange reagent; ICh, inhalation chamber; BI, impinger containing the blood; T, thermometer; CJ, clamp; PA, permanganate adsorber. was used with sodium-potassium phosphate buffer o.I M at p H 8.0 containing I.O M sodium chloride and 0.o2% sodium azide as eluting and supporting solution. The detection of m e r c u r y in biological material (urine, internal organs) by means of TLC was described b y W y s o c k a 13~. The biological material was first mineralized and the m e r c u r y ion then converted into a dithizonate. Chromatographic analysis was performed on Silica Gel G with n-propanol as the developing agent. After 3 h the separation of the excess dithizone (RF 0.66) from the H g dithizonate (RF 0.82) was completed. A m o u n t s as small as 0.5 # g Hg in large amounts of biological material can be detected b y this procedure.
9. MISCELLANEOUSANALYTICALMETHODOLOGY Methods of analysis for total mercury in biological materials are extensive and have included: neutron activation 133-136, atomic and flameless atomic adsorption 13~14~, isotope exchange45,14s, 144, mercury vapor meter adsorption 145-15°, polarographic 138,151, amperometric15~,lS3, microelectrolysis15,, colorimetric155-1~s, spark-source mass spectrometry 15s, UV p h o t o m e t r y ls°-16~, and thermometric titration TM. The selective determination of inorganic mercury in the presence of organomercurial compounds in biological materials is of noteworthy importance and has recently been achieved b y CLARKSONAND GREENWOOD165 and GAGE AND WARREN150. The principle of the former m e t h o d is the selective reduction of inorganic m e r c u r y
Chromatogr. Rev,, 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURV
235
b y SnCI2 (in t h e p r e s e n c e o f o r g a n o m e r c u r i a l c o m p o u n d s l a b e l e d w i t h t h e *°3Hg isotope) to e l e m e n t a l v a p o r w h i c h is t h e n s w e p t f r o m t h e s a m p l e b y a n air s t r e a m coll e c t e d o n a specific a d s o r b e n t , H o p c a l i t e , w h e r e t h e r a d i o a c t i v i t y is d e t e r m i n e d b y y-scintillation counting. T h e m e t h o d o f MAGOS AND CERNIK 162 in w h i c h m e r c u r y in u r i n e is d e t e r m i n e d by aspirating the vapor through an UV absorptiometer after reduction with stannous c h l o r i d e w a s m o d i f i e d b y GAGE AND WARREN T M b y m a k i n g use o f t h e v a r y i n g l a b i l i t y of o r g a n i c m a t e r i a l in t h e p r e s e n c e o f a c i d c y s t e i n e p e r m i t t i n g t h e d e t e r m i n a t i o n of m e r c u r y a n d o r g a n o m e r c u r i a l s in b i o l o g i c a l s a m p l e s s u c h as urine, feces, b l o o d a n d k i d n e y tissue.
I0. SUMMARY T h e m a j o r a r e a s o f u t i l i t y , a n d t h e ecological a n d b i o l o g i c a l significance o f ino r g a n i c m e r c u r y h a v e b e e n r e v i e w e d a l o n g w i t h s a l i e n t p a p e r , t h i n - l a y e r , gas, c o l u m n a n d i o n - e x c h a n g e c h r o m a t o g r a p h i c t e c h n i q u e s for t h e s e p a r a t i o n a n d i d e n t i f i c a t i o n o f i n o r g a n i c m e r c u r y f r o m e n v i r o n m e n t a l a n d f o r e n s i c sources.
REFERENCES I L. FISHBEIN, Chromatogr. Rev., 13 (197 o) 83. 2 N. NELSON, T. C. BYERLY, A. C. KOLBYE, JR., L. T. KURLAND, R. E. SHAPIRO, W. I-I. STICKEL,
3 4 5 6 7 8 9 lO
ii 12 13 14 15 16 17 18 19 20 2I 22
23 24 25
26 27 28
J. E. THOMPSON, L. A. VAN DEN BERG AND A. WEISSLER, Environ. Res., 4 (1971) I. L. J. GOLDWATER, Sci. Amer., 224 (1971) I5. H. U. MALLING, J. S. WASSOM AND S. S. EPSTEIN, Environ. Murat. Soc. Newsl., 3 (197 o) 7. N. FIMREITE, Environ. Potlut., I (197 o) 119. A. G. JOm~ELS AND T. WESTERMARK,in M. W. MILLER AND G. G. BERG (Editors), Chemical Fallout, Thomas, Springfield, Ilk, 1969, p. 221. C. V. KING, Ann. N . Y . Acad. Sci., 65 (1957) 36o. J. L. WEBB, Enzyme and i~ietabolic Inh~b*tors, Vol. II, Academic Press, New York, 1966, p. 73 o. H. A. SHOEMAKER,Ann. N . Y . Acad. Sci., 65 (1957) 5o4 . H. PASSOW, A. ROTHSTEIN AND T. W'. CLARKS0N, Pharmacol. Rev., 13 (1961) 185. P. L. BIDSTRUP, Toxicity of Mercury and Its Compounds, Elsevier, Amsterdam, 1964, p. 28, Int. Comm. Rep. on maximum allowable concentrations of mercury compounds, Arch. Environ. Health, 19 (1969) 891 . A. SWENSSON, OIKOS (Suppl.), 9 (1967) 27. M. C. BATTIGELLI, J. Occup. Med., 2 (196o) 394. S. C. HARVEY, in L. S. GOODMANAND A. GILMAN (Editors), Pharmacological Basis of Therapeut,cs, 3rd Ed., MacMillan, New York, 1967, p. 98. T. SDFUKI, in M. W. MILLER AND G. G. BERG (Editors), Chemical Fallout, Thomas, Springfield, IlL, 1969, p. 245. N. GRANT, Environment. I i (1969) 18. J. MOUTSCHEN-DAHMENAND N. DEGRAEVE, Experientia, 21 (1965) 200. S. LEVAN, Nature, 156 (1945) 751. M. UMEDA, K. SAITO, K. HIROSE AND M. SATO, Jap. J. Med., 39 (1969) 47. R. H. JE~VSEN AND N. DAVIDSON, Bzopolymers, 4 (1966) 17. H. DAUNE, C. A. DEKKER AND H. K. SEHACHMAN, Biopolymers, 4 (1966) 51. S. KATZ, Biochim. Biophys. Acta, 68 (1968) 24o. D. W. GRUENWEDEL AND N. DAVIDSON, J . Mol. Biol., 21 (1966) 129. R. K. ZAHN, E. TIESLER, H. G. OcHs, B. HEKKE, W. HANSKE AND W. FORSTER, Biochem. Z., 344 (1966) 26. S. KATZ, Nature, 194 (1962) 569. S. KATZ AND V. SANTILL1, Biochim. Biophys. Aeta, 55 (I962) 621. H. DRUCKREY, H. HAMPERL AND D. SCHMAHL,Z. Krebsforsch., 61 (1957) 511. Chromatogr. Rev., 15 (1971) 195-238
236
L. FISHBEIN
29 3° 31 32 33
M. MUROZUMI, Electrochem. Technol., 5 (1967) 236. S. JENSEN AND A. JERNEL6V, N6rdforsk. Biocidinformation, No. IO (1967) 4. S. JENSEN AND A. JERNEL6V, N~rdforsk. B,ocidinformation, No. 14 (1968) 5. J. M. Wool), F. S. KENNEDY AND C. G. ROSEN, Nature, 220 (1968) 173. A. JERNEL6V, in M. W'. MILLER AND O. G. BERG (Editors), Chemical Fallout, T h o m a s , Springfield, Ill., 1969, p. 68. D. K. H. LEE, P e r s o n a l c o m m u n i c a t i o n . J- C. GAGE, Br. J. Ind. Med., 18 (1961) 287. L. MAGUS, Environ. Res., I (1967) 323 . M. 13ERLIN, L+ G. JERKSELL AND H. YON UBISCH, Arch. Env,ron. Health, 12 (1966) 33M. H. 13ERLIN, G. F. NORDBERG AND V. SERENIUS, Arch. Environ. Health, 18 (1969) 42. T. W . CLARKSON AND A. ROTHSTEIN, Health Phys., io (1964) 1115. M. 13ERLIN AND S. ULLBERG, Arch. Environ. Health, 6 (1963) 589. O. I~'ITZHUGH, A. NELSON, E. P. LANG AND F. KUNZE, J. Ind. Hyg. Occup. Med., 2 (195 o) 443. T. NORSETH, Studies on the Biotransformat~on of Methylmercuvy Salts *n the Rat, P h . D . Thesis, Univ. o f Rochester, Rochester, N.Y., 1969. T. NORSETH AND T. W . CLARKSON, Biochem. Pharmacol, 19 (197 o) 2775. T. •ORSETH AND T. W. CLARKSON, Arch. Environ. Health, 21 (197 o) 717 . T. W. CLARKSON, in M. W . MILLER AND G. G. 13ERG (Editors), Chemical Fallout, T h o m a s , Springfield, II1., 1969, p. 274. M. LEDERER AND C. MAJANI, Chromatogr. Rev., 12 (197 o) 239. H. ROMANOWSKI, K. IZDEESKA AND Z. POZECZlEK, Farm. Pol., IO (1961) 452; C.A., 57 (1962) IOI45L P. 13. JANARDHAN AND A. PAUL, Indzan J. Chem., 7 (1969) 66. E. 13. SANDELL, Colorimetric Determination of Traces of Metals, 3rd Ed., I n t e r s c i e n c e N e w York, 1965, p. 621. Y. KURAYUKI AND K. KUSAMOTO, Bunseki Kagaku, 16 (1967) 815. W . ]3. LINK, K. S. KEINE, JR., J. H. JONES AND P. WATTLINGTON, J. ASS. Off'C+Agr. Chem., 47 (1964) 391. R. KURODA, T. KIRIYAMA AND K. ISHtDA, Anal. Chim. Acta, 4 ° (1968) 305. C. L. KAO, M. SIN AND Y. C. SING, Hua Hsueh Tung Pao, i i (1963) 46; C.A., 6o (1964) II369. D. N. TRIPATHI AND S. N. TE~*ARI, J. Prakt. Chem., 9 (1959) I. H. NAGAI, N*ppon Kagaku Zasshi, 80 (1959) 617; C.A., 54 (I96o) 19297. R. INDOVINA AND B. M. RICOTTA, Ann. Chim. (Roma), 45 (1955) 241. F. H. BURSTALL, G. R. DAVIES, R. P. LINSTEAD AND R. A. WELLS, J. Chem. Soc., (I95 o) 516. R. INDOVlNA, E. DELEo AND ]3. M. RICOTTA, Ann. Ch*m. (Roma), 45 (1955) 244. A. R. V. MURTHY AND V. A. NARAYAN, Naturwissensehaften, 42 (1955) 439M. 13. CELAP AND Z. RADIKOVJEVIC, Bull. Soc. Chim. (Beograd), 23-24 (1958-I959) i ; Anal. Abstr, (196o) 3115 . V. K . M. RAO, J. Sci. Ind. Res. (India), I g B (196o) 171. V. K. M. RAo, J. Sc,. Ind. Res. (India), l i b (1952) 277. S. kNL TEWARI, Z. Anal. Chem., 141 (1954) 4Ol. F. H. POLLARD, J. F. "vV. McOMBIE AND G. NICI~LESS, J. Chromatogr., 2 (1959) 284. H. S. R. BARRETO, R. C. R. BARRETO AND ][. P. PINTO, J. Chromatogr., 5 (1961) 5A. R. V. MURTHY, V. A. NARAYAN AND M. R. A. RAO, Curr, Sci., 24 (1955) 158. J. F. W . MCOMBIE, F. H. POLLARD AND I. I. M. ELBEIH, DiscusS. Faraday Soc., 7 (1949) 183. E. ]3LASIUS AND W. G6TTLING, Z. Anal. Chem., 162 (1958) 423 . i~. H. POLLARD, J. F. \ ¥ . McOMBIE AND I. I. M. ELBEIH, Nature, 163 (1949) 292. J. HALMEROSKI AND F. SUNDHOLM, Suom. Kemistil., 1336 (I963) 63. R. P. 13HATNAGER, K. D. SHARMA AND R. P. VARSHNEY, Ind,an J. Chem., 4 (1966) 47H. NAGAI AND T. DEGUCHI, Nippon Kagahu Zasshi, 86 (1965) 516. K. FUKUDA AND A. MIZUIKE, Bunsehi Kagahu, 17 (1968) 65. T. AKIYAMA AND M. SHIOKAWA, Kyoto Yahka Daigahu Guhuho, 12 (1964) 51. M. QURESHI AND M. A. KHAN, Z. Anal. Chem., 232 (1957) 194. H. NAGAI, Kumamoto J. Sci., Ser. A, 6 (1964) 138. N. SUGAWARA, I~agah~ Keisatsu Kenkyusho Hokohu, 2o (1967) 176. G. ALBERTI, Chromatogr. Rev., 8 (I966) 246. M. L. THAKUR, Sep. Sci., 5 (197 o) 645. H. TANAKA, Y. SUGIURA AND A. YOKOVAMA, Bunsek* Kagahu, 17 (1968) 1424. Y. KURAYUKI AND K. KUSAMOTO, fap. Anal., 16 (1967) 815. T. WESTERMARK, D. HAGMAN, A. VESTERMARIC AND K. LJUNGGREN, Nord. Hyg. Tidskr., 5 ° (1969) 79. T. FUKUDA, K. MIYAKAWA AND Z. UEKI, Eisei Kagaku, 13 (1967) 342. G. CATONI, Attar. Acad. Sci. (Torino), 91 (I956-I957) 23. H. SEILER AND M. SEILER, Helv. Chim. Acta, 43 (196o) 1939.
34 35 36 37 38 39 4° 41 42 43 44 45 46 47 48 49 5° 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 7° 71 72 73 74 75 76 77 78 79 80 81 82 83
84 85
Chromatogr. Rev., 15 ( I 9 7 i ) ~ I 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
237
H. SEILER AND M. SEILER, Helv. Chim. Aeta, 44 (1961) 1753. E. PFEIL, A. FRIEDRICH AND T. WACHSMANN, Z. Anal. Chem., 158 (1957) 249M. LESIGANG-BUCHTELA, Mihroch~m. Acta, (1966) 4o8. K . H. KOENIG AND K. DEMEL, J. Chromatogr., 39 (1969) IOI. I4~. M. OL'SHANOVA AND L. A. KUNITSKAYA, IZV. Vyssh. Uchebn. Zaved. Khim. Teknol., II (1968) 637; C.A., 7 ° (1969) 4367of. 91 A. E. SHERWOOD, Metallurgia, 8o (1969) 2o9. 92 S. TAKITANI, M. SUZUKI, M. YOSHIMURA, S. SATO AND M. SKEIYA, Eisei Kagaku, 14 (1968) 32493 M. HRANISAVLJEVIC, I. PEJKOVIC-TADIC, K. JAKOVJEVIC AND J, MILIJKOVIC-STOJANOVIK, Glas. Hem. Drus. Beogr., 29 (1964) 115; C.A., 64 (1966) 16599. 94 B. UNTERHALT, Deut. Apoth. Z., lO9 (1969) II43. 95 P- KuNzI, J. BA/JMLER AND J. I. OBERSTEG, Z. Gerichtl. Meal., 52 (1962) 605. 96 N. SUGIV,rARA, Yamaguch, Igaku, 15 (1966) 3o6. 97 K. N. JOHRI, H. C. MEHRA AND N. I42. KAUSHIK, Chromatograph*a, 3 (197 °) 347. 98 J. w . FAIRBARN AND S. EL MASRY, J. Pharm. Pharmacol., 19 (1967) 935. 99 J. C. MORRISON AND J. G. CHATTEN, J. Pharm. Pharmacol., 17 (1965) 655. IOO H. WEISZ, Microanalysis by R,ng Oven Technique, P e r g a m o n Press, L o n d o n , I961, p. 123. i o i H. BURNE, Analyst (London), 76 (1951) 22o. lO2 K. N. JOHRI, N. K. KAUSHIK AND K. SINGIt, Mikrochim. Acta, (1969) 737. lO 3 M. SEILER, Helv. Chim. Acta, 53 (197 °) 1893. lO 4 F. W. K o s s AND G. BEISENHERZ, Radiochim. Acta, 3 (1964) 22o. lO 5 Z. MASOOMI AND D. T. HAWORTH, J. Chromatogr., 48 (197 o) 581. lO6 V. D. CANIC AND S. M. PETROVIC, Z. Anal. Chem., 211 (1965) 251. lO 7 V. D. CANIC, S. M. PETROVIC AND A. K. BEN, Z. Anal. Chem., 213 (1965) 251. lO8 T, DEGUCHI, Bunsekz Kagaku, 15 (1966) 352. lO9 M. H . HASHMI, M. A. SHADID, A. A. AYAZ, F. I{. CHUGHTAI, N. ['IASSAN AND A. S. ADIL, Anal. Chem., 38 (1966) 1554 . IiO P. W. WEST AND A. K. MUKHERJI, Anal. Chem., 31 (1959) 947. I I I H. J. SENF, J. Chromatogr., 21 (1966) 363 . 112 I~. TAKESHITA, H . AKAGI, M. FUJITA AND Y. SAKAGAMI, J. Chromatogr., 51 (197 o) 283. 113 T. TADMOR, J. Gas Chromatogr., 2 (1964) 385 . 114 A. A. DUSWALT, Diss. Abstr., 20 (1959) 52. 115 W. J. BIERMAN AND H. GESSLER, Anal. Chem., 32 (196o) 1525. 116 E. W. BERG AND J. T. TRUEMPER, Anal. Chem., 3 ° (1958) 1827. 117 A. E. MARTELL AND M. CALVIN, Chemistry of the Metal Chelate Compounds, P r e n t i c e - H a l l , N e w York, 1963, p . 487 . 118 J. P. RILEY AND D. TAYLOR, Anal. Chim. Acta, 4 ° (1968) 479I 19 K. SAMSAHL, Anal. Chem., 39 (1967) 148o. 12o I4~. SAMSAHL, Ab. Atomenergi Stockholm, AE-82 (1962). 121 M. D. LIVINGSTON, H. SMITH AND ~N-. STOJANOVIC, Talanta, 14 (1967) 5o5 . 122 T. Y TORIBARA AND C. P. SHIELDS, Amer. Ind. Hyg. Ass. J., 29 (1968) 87. 123 I. KLIMES AND M. BETUSOVA, Anal. Biochem., 23 (1968) lO2. 124 J. F. Ko~P AND R. G. KEENAN, Amer. Ind. Hyg. Ass. J., 24 (1963) i. 125 H. A. SHOEMAKER, Ann. N . Y . Acad. Sc,., 65 (1957) 504 • 126 R. KURODA, K. ISHIDA AND T. KIRIYAMA, Anal. Chem., 4 ° (1968) 15o6. 127 tt. A. A. MUZARELL1, Talanta, 13 (1966) 8o9. 128 H CEMBER, P. GALLAGHER AND A. FAULKNER, Amer. Ind. Hyg. Ass. J., 29 (1968) 233. 129 T. CLARKSON AND A. ROTHSTEIN, Health Phys., IO (1964) 1115. 13o F. HOFMEISTER, Arch. Exp. Pathol. Pharmakol., 33 (1964) 198. 131 T. NORSETH AND T. W . CLARKSON, Arch. Environ. Health, 22 (1971) 568. 132 B. WYSOCKA, D~s$. Pharm., 17 (1965) 99. 133 B. UNDERDAL, Nord. Vet. Med., 20 (1968) 9. 134 B. SJ•STRAND, Anal. Chem., 36 (1964) 814. I35 T. WESTERMARK AND B. SJOSTRAND, [nt. J. Appl. Rad. tsot., 9 (196o) i. 136 0 . JOHANSEN AND E. GTEINNES, Int. J. Appl. Rad. Isot., 20 (1969) 751. 137 W . IR. MATCH AND ~¥. L. OTT, Anal. Chem., 4 ° (I968) 2085. 138 E. G. PAPPAS AND L. A. RUSENBERG, J. Ass. O~c. Agr. Chem., 49 (1966) 792. 139 t3. 13. MESMAN, 13. S. SMITH AND J. O. PIERCE, II, Amer. Ind. Hyg. Ass. J . , 3I (197 o) 7Ol. 14o A. D. RATHJE, Amer. Ind. Hyg. Ass. J., 3 ° (1969) 126. 141 M. J. FISHMAN, Anal. Chem., 42 (I97 o) 1462. 142 J. F. UTHE, F. A. J. ARMSTRONG AND M. P. STAINTON, J. Fish. Res. Bd. Can., 27 (197 o) 805. 143 T. W. CLARKSON AND M. R. GREENWOOD, Talanta, 15 (1968) 547. 144 L. MAGOS, Br. J. Ind. Med., 23 (1966) 23o. 86 87 88 89 9°
Chromatogr. Rev., 15 (1971) x 9 5 - 2 3 8
238
L. FISHBEIN
145 M. B. JACOB$, S. YAMAGUCHI, L. J. GOLDWATER AND H. GILBERT, Amer. Ind. Hyg. Ass. J . , 21 (196o) 475. 146 M. M. SCHACHTER,.[. ASs. Q~c. Anal. Chem., 49 (1966) 778. 147 W. W. VAUGHN, U.S. Geol. Sure. Circ., 54 ° (1967) 22. I48 T. Y. TORBARA AND C. P. SHIELDS, Amer. Ind. Hyg. Ass. J., 29 (1968) 87. 149 G. LINDSTEDT, Analyst, 95 (197 o) 264. 15o J. C. GAGE AND J. M. WARREN, Amer. Occup. Hyg., 13 (197 o) 115. 151 I. M. WEINER AND O. H. MULLER, J. Pharmacol. Exp. Ther., 113 (1955) 24. 152 J. T. STOCK (Editor), Amperometric T, tratzons, tnterscIence, N e w York, 1965. 153 B. C. SOUTHWORT•, J. H. HODECKER AND K. D. FLEISCHER, Anal. Chem., 3 ° (1958) 1152. 154 D. PAVLOVlE AND S. ASPERGER, Anal. Chem., 31 (1959) 939. 155 D. M. GOLDBERG AND A. D. CLARKE, J. Clin. Pathol., 23 (197 o) 178. 156 V. L. MILLER AND V. S~¥ANBERG, JR., Anal. Chem., 29 (1957) 391. 157 W*. H. GUTENMANN AND D. J. LISK, J. Agr. Food Chem., 8 (196o) 306. 158 E. P. LAUG AND K. W . NELSON, J. Ass. Off. Agr. Chem., 25 (I942) 3o9. 169 S. S. C. TONG, V~r. H. GUTENMANN AND D. J. LISK, Anal. Chem., 41 (1969) 1872. 16o F. N. KUDSK, Acta Pharmaeol. Tox*eol., 23 (1965) 263. 161 F. N. KODSK, Acta Pharmacol. Toxicol., 27 (1969) 149. 162 L. MAGOS AND A. A. CERNIK, Br. J. Ind. Med., 26 (1969) 144. 163 M. B. JACOBS, Amer. Ind. Hyg. Ass. J., 21 (196o) 475. 164 K. C. BURTON AND H. M. N. H. IRVING, Anal. Chim. Acta, 52 (197 o) 491. 165 T. W. CLARKSON AND M. R. GREENWOOD, Anat. Biochem., 37 (197 o) 236.
Chromatogr. Rev., 15 (1971) 1 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
LAWRENCE FISHBEIN National Institute of Enwronmental Health Sciences, National Institutes of Health, Public Health Service and Department of Health, Educat,on and Welfare, Research Triangle Park, N.C. 277o 9 (U.S.A.) (Received June Ilth, 1971)
CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 2. Occurrence, utility and ecological aspects . . . . . . . . . . . . . . . . . . . . 196 3. Biotransformation, metabolism and toxicity . . . . . . . . . . . . . . . . . . . 199 4. Paper chromatography and electrophoresis . . . . . . . . . . . . . . . . . . . 2oo 5. Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . 213 6. Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 7- Ion-exchange and column chromatography . . . . . . . . . . . . . . . . . . . 226 8. Analysis of mammalian distribution, transport and excretion of inorganic mercury . . 231 9. Miscellaneous analytical methodology . . . . . . . . . . . . . . . . . . . . . . 233 io. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
I. INTRODUCTION A n earlier review I highlighted the salient chromatographic a n d biological aspects of the organomercurials with p r i m a r y emphasis on m e t h y l m e r c u r y because of its u n i q u e u b i q u i t o u s p o l l u t a n t a n d t o x i c a n t n a t u r e . The objectives of this review are to (I) focus on the diverse chromatographic techniques t h a t have been utilized for the analysis of inorganic forms of m e r c u r y (e.g., elemental, mercurous a n d mercuric salts a n d their complexes) in a d m i x t u r e with other metals a n d cations a n d in diverse biological a n d e n v i r o n m e n t a l sources a n d (2) delineate m a j o r aspects of their adsorption, b i o t r a n s f o r m a t i o n a n d t r a n s p o r t , tissue d i s t r i b u t i o n , retention, excretion, mode of action a n d toxicity. The general considerations of inorganic m e r c u r y t h a t are r e l e v a n t to this review i n c l u d e : sources a n d chemistry 2-8, p h a r m a c o l o g y 9,1° a n d toxicology n-l~. The genetic considerations include: the synergistic effect of mercuric chloride with e t h y l m e t h a n e s u l f o n a t e in increasing chromosome aberrations in H o r d e u m sativurn 18 a n d V i c i a f a b a l S , 19, the cytotoxic effects of mercuric chloride on H e L a cells~° a n d i n t e r a c t i o n of inorganic mercurials with nucleic acids21-2L The carcinogenicity of m e r c u r y in tile rat has also been d e m o n s t r a t e d 28.
Chromatogr. Rev., 15 (1971) 195-238
196
L. FISHBEIN
2. OCCURRENCE, UTILITY AND ECOLOGICALASPECTS Mercury is comparatively a rare element, ranking I6th from the bottom of elements in abundance in the earth. Sufficient concentration of mercury for commercial extraction is found as mercuric sulfide (frequently as the red cinnabar and less often as the black metacinnabar). I m p o r t a n t deposits are located in Spain, Italy, U.S.A., Canada, Mexico, Brazil, Peru, China, Japan, Russia, Hungary, Yugoslavia and Germany. A less common ore is the mercurous chloride found in Texas. The ore found in Spain has the highest mercury content with an average of o.5-1.2 % mercury with values occasionally as high as lO% mercury. I t has been estimated that the outer i6-km-thick layer of the earth's crust contains 2. 7 × IO-a% mercury and the oceans contain 3.0 × lO-9 % mercury or approx. 50 million metric tons. In soils, it generally ranges from o.oi to 0.06 p.p.m., in rocks from o.oi to 0.09 p.p.m., in air from 2 to 5 ng/Ina, and in water and beverage from 0.002 to 0.006 p.p.m. Mercury has been known and utilized since antiquity. Today there are over 3000 recognized applications for mercury and its inorganic and organic derivatives. The world production of mercury amounts to about IO,OOO tons per year of which about 3000 tons are used in the United States. The main areas of utility of mercury include: the electrolytic preparation of chlorine and caustic soda, agricultural chemicals, pharmaceuticals (diuretics, cathartics, antibacterial agents), hair dressings and preservatives in cosmetics, pulp and paper making (slimicides and algicides), paints (anti-fouling, pigment), electrical apparatus, catalysts, dental preparations and amalgamations. Production of chlorine and caustic soda is an electrolytic process where large amounts of mercury are used as a flowing cathode (75,ooo-15o,ooo lbs for a plant with a capacity of ioo tons of chlorine a day) 5. It is estimated that the chlorinealkali industry loses to the environment approx. 0.45 lbs. of mercury per ton of chlorine produced ~9. This loss, based on projected tonage figures, m a y be as much as 3300 lbs. per day or 1,2oo,ooo lbs. per year. Most of the lost mercury finds its way into streams and lakes; traces of mercury are also carried into the atmosphere with hydrogen gas (20-30 mg/m 8) while usually less than 5 p.p.m, of mercury is retained b y the caustic soda. Mercury has also been detected in chlorinated hydrocarbons, glycols, acetic acid, carbon dioxide, fertilizers, sulfuric acid, sulfide ores, industrial catalyst wastes (e.g., in production of acetaldehyde and vinyl chloride) and in bituminous shales and crude oils. Fossil fuels in the United States contain mercury in concentrations ranging from a few parts per billion* to several parts per million. The annual consumption of 500 million tons of coal per year (containing an average concentration of at least I p.p.m.) would contribute one million pounds of mercury per year to the environment, about 45 ° metric tons 2. This value does not include the mercury contributed from the refinement and use of products from crude oil which m a y contain higher concentrations of mercury. The key inorganic mercury compounds used in agriculture (as insecticides, seed disinfectants and fungicides) and in home, garden and lawn are mercuric acetate, chloride and oxide (red and yellow) and mercurous chloride and nitrate. Elemental mercury is one of the most toxic elements used in the control of crop pests. Quick* Throughout this article the American p.p.b. = IO-~ is meant.
Chromatogr. Rev., 15 (I97 I) 195-238
197
C H R O M A T O G R A P H I C A N D B I O L O G I C A L A S P E C T S OF I N O R G A N I C M E R C U R Y
silver (metallic mercury) has itself been employed in India to fumigate grain in closed containers. There is a large variety of miscellaneous sources that present possible mercurial contamination to the environment and include the following: (a) disposal of thermometers, aerometers, barometers, relays, rectifiers, switches, fluorescent tubes, mercury lamps and batteries; (b) refuse from hospitals, laboratories and dental clinics; (c) processing or use of raw materials containing mercury such as carbon, coal, chalk, phosphate and pyrite; (d) use of mercurial compounds to prevent mildewing in commercial laundries; (e) manufacture of and residues from paints and impregnating agents which contain mercury to impart mildew resistance; and (f) refining or redistillation of mercury.
(CH3)2Hg
(C6Hs)2Hg C6HsHg+
~ Hg2
CH30 (CH2)2Hg+ Fig.
I.
Conversion o f m e r c u r y H g ° --+ H g t+ in t h e e n v i r o n m e n t a3.
Normal human tissues contain mercury because there is a daily intake in the food. Bread, flour, milk, pork and beef contain 0.02-0.04 p.p.m, of mercury and certain vegetables a good deal more, depending on soil conditions and sprays used. Ingestion of fish and fowl with high levels of mercury (as methylmercury) via environmental fallout from sources described above introduces yet another dimension to the ever-increasing realization that compounds of mercury present a substantial hazard.
IImport, 22% 1 L
IM,ni.g Re.erve. 3,% I I I
IStockpilo.2.%l I
IRecyc'"' °'oF J
ITo'a'oomoo'oo%l
DISSIPATIVE USES
Paints
26°/° ~ 12% Pulp and
Agriculture
Dental~t Catalysts
RECYCLABLE
4%
I
~ Electrtcal
I
IMa°ufa:t°relTJ ~ ~
Soda
&Control Laboratory
ceutlcals 1% I
2°/° Metallurgy I and I Mining
TO Environment 26%
USES
;33% Chlorine-- I ~
5%
Pharma-
cO
1°o I
L
1 I~ ~
Y
41%
27Olo I
rr
11% 3% [
Addition to
Inventory 10%
To Environment 23%
To Inventory and Environment 23°/o
Fig. 2. D i s s i p a t i v e and recyclable uses of m e r c u r y in t h e U n i t e d States in i9683.
Chromatogr. Rev., 15 {1971) 195-238
198
L. FISHBEIN MEDICAMENTS Hg COMPOUNDS METALLICMERCURY Hg METAL- OTHER USES in INDUSTRY m INDUSTRY LURGY METALLICMERCURY COSMETICS I
AGRICULT, AGENTS
•rrtX~ttngJ F~"Spe~t~4
I/
r WOste$-
~i
iI
fl
I /
.
FS~llage-
.
.
t- ~
.
.=V-rSpillage~
rt-Spillage -
Ti I r~,~'~
T! /
Ti
/:!
T! /
I~ i
I: ~Vapor---~ ~:.Vapor--
-~!,I
TI
.
l-~-,-Sp,llageJ
T:..-Vapor ~
.
.
.
Til
J
l~
:~
.
~i ;i
1 ~,_,_,_,_~z,~_,_,_~_,~_,_,_,_,_,,_,~ ",~
\ ".... \ "'"..
"~
~.~S~FARM
AOq0ATIC ~
\
EXCRETA.I--
/
MAN
Fig. 3. Principal pathways of mercury contamination and environmental movement3~. -w~r ] !
t i
/I~
I
Leakage and..- I lage~
I
rnetallic merc ury in container I i
i
lOrev,ce~ I I~
I
c,orh,ng ~.~a, s 11 amalgams Hands
WORKER
SO d
wastes
Eff~ent I
t- Sett hng tank
Outlet Stream ~1----- - --I~ Bed i I~-Littoral mud
Lak~
Me~hylmer~cury Solution m ~ fake water
Farm animals
Fig. 4. Ecological aspects of mercury distribution 34.
Chromatogr. Rev., 15 (1971) 195-238
~'~Micro- orga nisms
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
199
Of major significance has been the recent realization that certain biotransformations ~°-s3 may take place in the environment (Fig. I) and in the body, changing one form of mercury to another, viz., (a) the changing of inorganic mercury to methylmercury when oxygen is limited or absent by microorganisms, (b) the release of inorganic mercury from phenylmercuric compounds with subsequent conversion to methylmercury, and (c) the conversion of phenylmercuric compounds to inorganic mercury in the body. Figs. 2 and3 illustrate the dissipative and recyclable uses of mercury in the United States in 1968 and the principal pathways of mercury contamination and environmental movement, respectively. Fig. 4 depicts the ecological aspects of mercury distribution.
3" BIOTRANSFORMATION, METABOLISM AND TOXICITY
It is important to distinguish between different forms of mercury (e.g., elemental, ionic, alkyl and aryl) in any evaluation of the potential hazards of mercury residues as well as the development of requisite analytical methodology for their analysis. Inhaled mercury vapor (in many cases a mixture of vapor and aerosols) is mainly an industrial hazard and there is evidence that the vapor is adsorbed through the lungs '~5. The process of adsorption involves oxidation of elemental mercury with the subsequent appearance of ionic mercury in the blood, then distribution in the body in analogous manner to subcutaneous-injected mercuric chloride, with the kidney as the primary site of deposition. The oxidation of elemental mercury occurs partly in the blood (mainly in the erythrocytes) and partly in the tissues 36. The higher uptake of mercury in brain following exposure to vapor of animals as compared to subcutaneous injections of mercuric nitrate has also been reported3L3s. Diffusion of elemental mercury into the tissues and across cell membrane is facilitated by its lipid solubility and its lack of charge. In the plasma divalent mercury is bound to plasma proteins and in the erythrocytes to hemoglobin. Although there is some evidence that mercuric ions can be reduced to elemental mercury in the body 39, the extent of this reduction is not considered of practical importance. Mercurous ions are probably oxidized to mercuric ions in the body. Following acute administration of inorganic salts of mercury to man and animals, the highest level of mercury is found in the kidneys, and the next highest in the liver. Elimination of mercury from brain, thyroid and testis is slow4°, permitting accumulation of mercury in these organs. With chronic exposure to mercuric salts, it is uncertain whether mercury levels in brain or testis reach toxic concentrations before the onset of severe renal damage. Although inorganomercury salts are more acutely toxic than organomercury salts, chronic-oral studies 41 have shown for example that phenylmercuric acetate was more toxic to the rat than mercuric acetate. Inorganic mercury salts are poorly adsorbed, e.g., the adsorption has been estimated to be approx. 2% while for methylmercury the available data (man and rat) indicate an intestinal adsorption of more than 90%. Insoluble inorganic mercurous compounds such as Calomel (Hg2C12) can undergo oxidation to more soluble, adsorbable compounds. Inorganic mercurials, in
Chromatogr. Rev., 15 (i97I) I95-238
200
L. FISHBEIN
suitable vehicles, can be adsorbed through the intact skin on passage into the blood; they become firmly bound to plasma proteins and in the erythrocytes to hemoglobin. Mercury readily redistributes to the tissues and within several hours is found in human and animal tissues (in the approximate order of decreasing concentration): kidney, liver, blood, bone marrow, spleen, upper respiratory and buccal mucosa, intestinal wall, skin, salivary glands, heart, skeletal muscle, brain and lung. Mercury is excreted by the kidney, by the liver via bile, by the intestinal mucosa, sweat glands and salivary glands; urinary and fecal routes are the most important for elimination. Biotransformation has been shown 4~-44 to be important for the excretion of mercury after exposure to methylmercuric chloride. The carbon-mercury bond may slowly undergo cleavage in vivo, resulting in the release of inorganic mercury and its preferential excretion via the gastrointestinal tract (as distinct from the kidney, where a relatively higher amount of the intact organomercurial was found in urine). Biotransformation within the intestinal lumen in the cecum was shown to be the major source of inorganic mercury in feces. CLARKSON45 has suggested that the nephrotoxic action of organomercurials may be related to the rate of biotransformation with enzyme systems participating in the breakdown of organomercurials.
4-
PAPER CHROMATOGRAPHY AND ELECTROPHORESIS
Although paper chromatographic data for inorganic substances have recently been tabulated by LEDERER AND MAJANI4s, a variety of selected methods are included in this review with primary emphasis on specific methods for mercury. The utility of paper chromatography and hydrazine sulfate and hydroxylamine hydrochloride as specific reagents for the separation and detection of mercury and silver ion in toxicological analysis was described by ROMANOWSKI et al. 47. Mercury and silver were separated using ascending paper chromatography in two solvent systems: (a) butyl acetate and (b) acetone-conc, hydrochloric acid-isobutanol-glacial acetic acid-water (58 :I :2o:6.4:14.6); the RF values were 0.86 and 0.4 ° for mercury and silver, respectively. The paper was first sprayed with a 5% ethanolic solution of either hydrazine sulfate or hydroxylamine hydrochloride and then heated. Mercury produced a dark brown spot and silver one of yellow-brown. The presence of other metals did not interfere and the elements could be successfully detected in the presence of biological or mineral materials. The limits of detection of both mercury and silver were o.I and 0.3 #g with hydroxylamine hydrochloride and hydrazine sulfate, respectively. The radial-paper chromatographic separation of mercury and antimony valence states has been reported by JANARDHAN AND PAUL48. Trichloroacetic acid (TCA) was used as a complexing agent for the separation of mercurous from mercuric ions. TCA forms a semicovalent compound (I) with mercurous ion by sp hybridization, which alone moves with the solvent (benzene-TCA) leaving mercuric ions at the origin. On electronic considerations alone, formation of a similar chelate with mercuric ion is not possible. The formation of the chelate structure (I) has not been supported by dipole moment measurements and a callinear configuration (II) was postulated by JANARDHAN AND PAUL. This necessitates the assumption of additional charge on the
Chromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
201
oxygen of the hydroxyl of the COOH in TCA due to three chlorine atoms (this charge prefers only the mercurous ion, which is less positive as compared to Hg*+). Hence, TCA effects a resolution of the Hg2~+ and Hg 2+. The solubilizing effect of benzene on the covalent complex of Hg~2+ was suggested to be due to the association of benzene molecules with TCA (III). In the case of polar solvents such as water and aliphatic alcohols containing TCA, occurrence of double spots due to the disproportionation Hg2e+ ~ H@ + + Hg was observed.
Cl
0
O--C CI -C I I OI ~1
Cl~C_Cx// O"Hg-- Hg-- O;C-CC t3
~g
(~1
7 ( Z)
,o
I
o
Hg--Hg-O-C,~C---CI.---H--~
~ > . . . . H--CI
0~ \ I ('nT)
The radial-paper chromatographic separations were carried out on circles of Whatman No. I filter paper (diameter 15 cm). The spraying reagent for mercury consisted of fleshly-prepared 20% sodium hydroxide, which yields a brown color with mercurous ions, and a 1% ethanolic solution of 1% S-diphenyl carbazone, which yields a violet to blue color with mercuric ion. (Both reagents are sensitive to a microgram of mercury.) Fig. 5 illustrates chromatograms for the separation of mercury oxidation states in different solvent systems. Table I depicts the paper chromato1
2
4
3
5
Fig. 5. Chromatograms for the separation of mercury oxidation states. Elution of Hg2*+ and Hg2+ by o.I N HNO~ (I); EtOH + HNO3 (ioo:i) (2); BuOH-HNO3 (Ioo:i) (3); o.I N HCI (4); and benzene-TCA (ioo:Io, v/w) (5).
Chromatogr, Rev., 15 (1971) 195-238
:20:2
L. FISHBEIN
I I
l
I
l
I I
I I
I
1
1
lq
i I 1
1
I
i
I I
oh
bl
tt~ ~-
I I
1
1
t
f
1
t
1
1 o
I
1
I
I
I
t m
II
I
I
I
I1
I
1
I
I1
1
I
I
It
I
I
1
li
i
o o o o
0
0
0
0
o o
o
0
0
~
~
I
I
o
~
~
I
1
1
1
I
I
~
o
g t
I
l
I
I
I
I
l
I
I
I
I
o
o~ o~ o ~ ~
o~
6°d°d°o~oo
5
o
~oh O~
dd
I I
0
d
I
5
I
5
l
~
1 t
dd
I I
d
I
d
o
I
d
d
d
d ~
d
d
d 0
~d
I i
I
I
I
I t
t I
~
1
~"
d
I
0
±
I
I
I
I
1
I
0
o
~
_.~0 ~
o~
.~ o~
Chromatogr. Rev.
15 (1971) 1 9 5 - 2 3 8
>
2o3
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY TABLE
2
ANALYSIS
OF MULTIPLE
Spot: 4oL2/*g
(so#l,
Chromatogram
SPOTS OBTAINED
FROM MERCUROUS
ION
o.2 M H g , ~+ s o l u t i o n ) .
Solvent system
Found (~,g)
R~, v a l u e a
NO.
Hgb
Hg~a+~
Hg'+
ng='+b,'t Hg~ and Hg=+
i -
o.i N HNO a MeOH-HNO 3
o.o o.o
o.91 0.75
0.95 0.86
400 37 °
8 15
2
EtOH-HNO,
o.o
0.74
0.83
390
15
3
BuOH-HNO~
o.o
o.23
0.46
360
3°
-
o.I M TCA MeOH-TCA
o.o o.o
0.95 o.83
0.98 0.92
380 41o
6 q
-
EtOFI-TCA
o.o
o.64
o.75
39o
5
(1oo:I) (ioo:I) (ioo:i)
(Ioo:5, v/w) (Ioo:5, v/w) a Average of at least three chromatograms. b By disproportionatiou reaction. e By oxidation during chromatography. a Estimated polarographically. e E s t i m a t e d c o l o r i m e t r i c a l l y b y t i l e d i t h i z o n e m e t h o d 49 o n a B e c k m a n model B.
spectrophotometer,
graphic resolution of mercury and antimony valency states using 2o solvent systems and Table 2 shows the analysis of multiple spots obtained during the chromatographic determination of mercurous ion. Dithizone (S-diphenyl thiocarbazone) as well as S-diphenyl carbazone, are extremely useful reagents yielding very sensitive color reactions with many ions (amenable to extraction with immiscible organic solvents). In the case of mercury, the reaction with dithizone results in the colored chelate
(iv). H
Hg~
C=S N=N
The adsorption and development of nineteen mercury compounds were investigated on Amberlite SB-2 anion-exchange paper by using 1.6 N nitric acid as the developing solvent 5°. The RF values were o.19-o.33 for alkylmercuric compounds, o.o9-O.ll for inorganic salts, and O.Ol-O.O3 for phenylmercuric compounds, hence pernfitting the differentiation of organic and inorganic mercury compounds. An ion-exchange paper-X-ray emission procedure for the determination of microgram quantities of mercury has been described by LINK et al. 51. The procedure permitted the determination of I #g of mercury in IOO ml of aqueous acid solution with a precision of ± 0.25/~g. The mercury is first adsorbed by anion-resin-loaded Chromatogr. Rev.,
15 ( 1 9 7 1 ) 1 9 5 - 2 3 8
204
L. FISHBEIN
paper and then determined by X-ray emission spectroscopy. The method which was applied to the determination of mercury in inorganic pigments used in foods and drugs gave 75-125% recoveries of I / , g mercury from acid solutions containing I O g of Na,SO 4, NaC1, Fe20 ~, MgCO s and CaCO s and satisfactory recoveries from the HC1 extract of C, BaS04, chromic oxide, bentanite, kaolin, talc, Ti02 and magnesium stearate. KAO et al. 53 separated mercury from other ions on W h a t m a n No. I paper using methyl acetate-methanol-water (85:5 :IO) or methyl acetate-water (95:5)- The band was first indicated b y spraying with a reagent containing 5% K I - 2 o % Na2SO~ (I :I), then with a reagent of 5% CuSO, in I N HC1 to form orange-red Cu2HgI ,. (Gold interferes, but can be removed with hydroquinone.) For quantitative determination, the mercury band is removed, extracted with I N H~SO4, dithizone and chloroform and determined colorimetrically. TRIPATHI AND TEWARIs4 utilized paper chromatography for the determination of metallic poisons in forensic toxicology. Lead, copper, bismuth, zinc and mercury gave characteristic colors following separations with n-butanol saturated with I N HCI at 20 ° and detection with diphenyl carbazide. NAGAI5s separated mercury(II), copper(II), bismuth(III), cadmium(II) and lead(II) on circular paper impregnated with dithizone and developed with o.I N HNO3-acetone (IO:I). The cations were detected as follows from the center to the periphery: mercury, orange; copper, black; bismuth, orange; cadmium, red; unreacted dithizone and lead, pink. INDOVINA AND RICOTTA5s described a xo-min circular-chromatographic separation of copper, cadmium, bismuth and mercury ions using n-butanol-acetic acid12 N hydrochloric acid-water (45 :IO:I :44) and n-butanol-3 N hydrochloric acid as developers. Detection was achieved using dithizone. Table 3 shows the RF values of copper, bismuth, cadmium and mercury cations on paper discs using both developers above. TABLE RF
3
VALUES
OF COPPER,
BISMUTH,
CADMIUM
AND
MERCURY
IONS ON
PAPER
DISCS
Developing solvents: (I) n-butanol-acetic acid-i2 N HCl-water (45:Io:I:44) and (2) n-butanol3 N HC1. Metal cation
Copper Bismuth Cadmium Mercury
R F value Solvent z
Solvent 2
o. 18 0.46 0.26 o.9o
o.4° 0.80 0.83 o.9o
BURSTALL e[ al. 57 described the paper chromatographic separation of a number of metal cations and acid radicals on W h a t m a n No. I or 3 paper. The best separations were achieved in Group I I A (Pb, Cu, Bi, Cd and Hg) and Group I I I B (Co, Ni, Mn, Zn, the platinum metals and gold). More difficult separations were found in Group I I B (As, Sh and Sn) and Group IV (Ca, Sr and Ba). The separation of Group I I A metals as their chlorides was best achieved using n-butanol saturated with 3 N Chromatogr. Rev.,
15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
205
hydrochloric acid as developer (RF values were: Cu, o.2o; Pb, o.27; Bi, o.6o; Cd, 0.77; and Hg, o.81). A dithizone-in-chloroform spray detected the above metal cations as follows: mercury, pink; cadmium, purple; bismuth, purple; copper, purplish-brown. Lead, which gave a weak color with dithizone, was detected with an aqueous solution of rhodizonic acid as a bright-blue spot. The paper chromatographic separation of mercury, lead, copper, bismuth and cadmium cations has been accomplished using W h a t m a n No. 4 paper and (I) nbutanol-acetic acid-I2 N hydrochloric acid-water (45:1o:1:44) and (2) n-butanolhydrochloric acid (I :I) as developers 5s. Detection was accomplished with dithizone or hydrogen sulfide. Table 4 shows the effect of concentration of hydrochloric acid on the RE values of the metal cations, developed with system 2 (RE values increase with increasing acid concentrations). Table 5 depicts the R F values and spot colors of mercury, lead, bismuth, copper and cadmium cations developed with system I. The separation of lead, mercury, bismuth, copper and cadmium ions as their sulfates, chlorides, nitrates or acetates has been achieved by circular-paper chromatography using either n-butanol saturated with 4 N acetic acid or with 3 N hydrochloric acid at 25-27 °59 (Table 6). An effective separation of Ag +, T1+, Pb *+ and Hg 2+ was achieved 8° in approx. 3 h at 20 ± I ° by ascending chromatography on W h a t m a n No. I paper using a solvent mixture of m e t h a n o l - I I N nitric acid (60:40). The RE values were: Ag ¢, 0-29; T1+, 0.39; Pb 2+, o.51; and Hg 2+, 0.75. The spots were detected by dipping the chronlatogram in a solution of ammonium sulfide; the lower limit of detection was I X 10 -5 g .
RAO61 described the qualitative separation and identification of lead, silver and monovalent-mercury (Group I) cation mixtures on W h a t m a n No. I paper and using 0.2 N and 0. 5 N sulfuric acid and ammonium nitrate-ammonia mixtures for development, as well as a modified RUT~ER'S technique ez. Table 7 lists the RE values of cations of Group I using the above solvents. TEWAR163 described the effects of: (a) various n-butanol-pyridine-water mixtures, (b) temperature, (c) cation concentration, and (d) extent of development on the separation of lead, silver and mercury cations. Tables 8 and 9 list the RF values obtained utilizing varying mixtures of n-butanol-pyridine-water at 3 °0 and nbutanol-pyridine-water (20 :i :4) at temperatures ranging from IO to 4 o°, respectively. Table io depicts the RE values for varying amounts of the cations obtained in an I8-h development at 3 °° using n-butanol-pyridine-water (2o:1:4). POLLARD et al. 64 described the separation and determination of some heavy metal cations in admixture with lead ions by descending chromatography on Whatman No. I paper. Table I I shows the RF values of fourteen ions chromatographed with diethyl ether-methanol-water-nitric acid (50:30:20:2) and the spray reagents employed for their detection. The table indicates that it is possible to separate completely the heavy metal ions of major interest, e.g., Zn(II), Cd(II), Cu(II), Fe(II), Ni(II), Bi(III), S g ( I I ) from Pb(II). The chromatography of ten cations on Macherey-Nagel No. 261 paper developed with 96% ethanol-2 N acetic acid (80:20) and acetone-8 N acetic acid (9 o:Io) and detected with chloranilic acid was described by BARRETO et al. ~5. Table 12 lists the RE values and sensitivity of chloranilic acid toward ten cations separated with the solvent mixtures mentioned above. Chromatogr. Rev., 15 (1971) 195-238
:206
L. FISHBEIN
TABLE 4 R F VALUES OF Hg, Pb, Bi, Cu AND Cd CATIONS D e v e l o p i n g s y s t e m : n - b u t a n o l - H C l (I :I) w i t h v a r y i n g acid c o n c e n t r a t i o n s .
Metal cation
Concentration of H C I
Mercury Lead Bismuth Cop per Cadmium
IN
2N
3 N
0.75 o.ii 0.52 0.08 0.63
0.76 o.16 0.54 o.I I 0.67
0.80 0.28 0.58 o.2o 0.76
TABLE 5
R F VALUES AND SPOT COLORS OF Hg, Pb, Bi, Cu AND Cd CATIONS D e v e l o p i n g s y s t e m : n - b u t a n o l - a c e t i c a c i d - I 2 N H C l - w a t e r (45 : i o : i :44).
Metal cation
R ~ value
Mercury Lead Bismuth Copper Cadmium
Spot color
o. 78 o.o 5 o.4o o. 13 o.2o
H~S
D~thzzone
black -brown brown lemon-green
rose rose violet brown rose-purple
TABLE 6 R F VALUES OF Pb, Hg, Bi, Cu AND Cd WITH DIFFERENT ANIONS S o l v e n t s : (I} n - b u t a n o l s a t u r a t e d w i t h 4 N a c e t i c a c i d ; (2) n - b u t a n o l s a t u r a t e d w i t h 3 N h y d r o chloric acid. T e m p e r a t u r e : 25-27 °. Circular c h r o m a t o g r a p h y ,
Amon
Sulfate Chloride Nitrate Acetate
Solvent z
Solvent 2
pb*+
Hg*+
B~3+ Cu*+
Cd*+
pbu+
Hg~+
Bz3+
Cu2+
Cd~+
o.46 o.51
0.57 0.84 0.69 o.82
o.61 o.62 -
o.21 0.42 0.52 0.54
halo halo
0.82 o.83 o.89 o.82
o.72 0.73 -
0.36 o.36 o.38 o.35
0.79 o.79 0.79 o.79
0.27 0.40 o.54 o.57
TABLE 7 R p VALUES OF CATIONS OF GROUP I S o l v e n t s : (i) N H 4 N O s s o l u t i o n d i l u t e d 8o t i m e s - N H 4 O H (2o:8); (2) 0.05 N H2SO,; (3) o.z N
H2SO4. Catwn
Ag + Hg + P b ~+
Strap
Rutter' s method
Solvent r
Solvent 2
Solvent 3
Solvent 3
o.92 0.94 0.02
0.85 0.95 0.04
0.92 0.98 o. 17
0.82 o.91 o.o5
C h r o m a t o g r . R e v . , 15 (1971) 1 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
207
TABLE 8 R~, VALUES OF
P b ~+, Ag +,
AND
Hg 2~ W I T H
V A R Y I N G n - B U T A N O L - - P Y R I D I N E - - W A T E R M I X T U R E S AT
3 °o Ascending development for 6 h. Mixtures: (I) (2o:1:4), (2) (I5:I:9), (3) (lO:1:14) and (4) (5:I:19) • Cat, on
P b ~+ Ag + Hg 2+
Mixtures
o.o 4 0.22 0.59
2
3
4
0.0 5 0.52 o.92
0.0 5 0.45 o.7o
o.o 3 0.20 0.5o
TABLE 9 E F F E C T O F T E M P E R A T U R E ON W A T E R ( 2 0 : I :4)
R F V A L U E S O F P b 2+, Ag+ A N D Hg 2+ U S I N G n - B U T A N O L - - P Y R I D I N E - -
A 3u-rain circular-paper development. Cation
P b 2+ Ag + Hg ~+
Temperature (°C) IO
20
30
40
o.31 0.59 0.92
0.27 0.55 o.81
o.2I 0.53 0.82
o.i 5 o.5I 0.74
T A B L E 10 E F F E C T O F C A T I O N C O N C E N T R A T I O N ON GRAPHY
R F V A L U E S O B T A I N E D AT 3 0 ° B Y A S C E N D I N G C H R O M A T O -
Solvent system : n-butanol-pyridine-water (20 :I :4). Development time : I8 h. Cation
P b ~+ Ag + Hg 2+
Catzon concentration (12g) o.z
0.2
0.3
0.4
0.5
0.05 0.23 0.4 8
o.o 7 0.24 0.50
o.o8 o.25 0.52
0.09 0.26 0.55
o.Io 0.27 0.58
The separation of silver, mercurous and lead ions by circular-paper chromatog r a p h y w a s r e p o r t e d b y MURTHY et al. 8~ u s i n g n - b u t a n o l - p y r i d i n e - w a t e r (IOO :20:20) ~ i n a 2-tl (9-cm) d e v e l o p m e n t w i t h a m m o n i a c a l h y d r o g e n s u l p h i d e u s e d f o r d e t e c t i o n . T h e R E v a l u e s f o u n d w e r e : Ag+, 0 . 8 6 ; H g +, 0 . 6 2 ; a n d P b 2+, 0.34. Chromatogr. Rev., 15 (1971) 195-238
208
L. FISHBEIN
T A B L E 11 R F VALUES AND SPOT COLORS OF SOME HEAVY METAL CATIONS IN ADMIXTURE WITH LEAD IONS O b t a i n e d w i t h d e s c e n d i n g c h r o m a t o g r a p h y on W h a t m a n No, I paper. S o l v e n t m i x t u r e : die t h y l e t h e r - m e t h a n o l - w a t e r - n i t r i c acid (5 °: 3 ° : 2o :2).
Cation
Spray detector
Spot color
R v value b
TI(I)
0.5% 8 - H y d r o x y q u i n o l i n e in abs. alcohol. H o l d over a m m o n i a a n d u n d e r U V light. 5 % w]v T a n n i c acid in w a r m 60% aqueous methanol, W a r m t h e d a m p strip. 3.0% R h o d i z o n i c acid. H o l d over a m m o n i a . 0.5% 8 - H y d r o x y q u i n o l i n e in abs. alcohol. H o l d over a m m o n i a a n d u n d e r U V light. o . 1 % wlv R u b e a n i c acid in abs. alcohol. H o l d o v e r ammonia. 4 % Salicylaldehyde in 50% a q u e o u s ethanol. H o l d over a m m o n i a a n d u n d e r U V light. o.5% w/v Aqueous potassium ferrocyanide. IO% w / v F r e s h l y p r e p a r e d a q u e o u s s o d i u m dlthionite (warm). 0.5% w / v A q u e o u s p o t a s s i u m ferrocyanide. 0.05% w / v D i t h i z o n e in chloroform,
Yellow-green fluorescence
o.25-o.17
Brown stain
0.36-0.27 e
Red
o.41-0.22
Yellow fluorescence Yellow fluorescence
o.66-o.55 o.66--o.55
Green Yellow-brown Blue Dark spot against a yellow-green fluorescent background Blue Blue Brown
0.66-o.56 0.68-0.55 0.68-0.55 0.70-0.56
Brown
0.78-0.72
Pale p i n k
0.95-0.87
Ag(I) Pb(II) Zn(II) Cd(II) Cu(II) Co(II) Ni(II) Mn(II) Fe(II) Fe(III) Bi(III) a U(VI) Hg(II)
0.70-o.58 0.70-0.58 H y d r o l y z e d M1 d o w n t h e p a p e r to 0.8
a B i s m u t h m a y be c h r o m a t o g r a p h e d successfully o n l y if t h e original solution c o n t a i n e d 5 % v / v nitric acid. T h e n no h y d r o l y s i s p r o d u c t s are formed, a n d t h e R e v a l u e of Bi is 0.78-o.67. b H e a d a n d tail o f spot. e D a r k h y d r o l y s i s p r o d u c t s also p r e s e n t a t s t a r t i n g time. T A B L E 12 R e VALUES AND SENSITIVITY OF CHLORANILIC ACID AS DETECTOR S e p a r a t i o n of IO c a t i o n s on M a c h e r e y - N a g e l No. 261 p a p e r u s i n g d e s c e n d i n g d e v e l o p m e n t w i t h (I) 9 6 % e t h a n o l - 2 N acetic acid (8o:2o) a n d (2) a c e t o n e - 8 N acetic acid (9o:1o). D e t e c t o r : o. i % chloranilic acid in ether.
Cation
Fee+ Ni ~+ Co S+ Cu 2+ Zn *+ Ag+ Cd ~+ H g 2+ P b 2+ U 4+
RF value Solvent x
Solvent 2
o.o8a 0.34 0.42 0.27 0.78 0.37 o.71 o.o o. i o 0.83
I,OO o.07 0.07 o.2o o.31 0.08 o.20 I .oo o.o9 o.86
Sensztivity (ttg)
Color of spot
o.oI o.1o 0.20 o.Io 5.00 4.00 2.00 5.oo o. I o I.OO
brownish greenish yellow light green greenish brownish red brownish brownish brownish brownish
a Tailing.
Chromatogr. R e v . 15 (197 I) 1 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY BLASIUS AND G6TTLING ss d e s c r i b e d t h e c i r c u l a r - p a p e r c h r o m a t o g r a p h y
209 of a
v a r i e t y of cations. T h e following RF values were f o u n d following a 3-h d e v e l o p m e n t on S c h l e i c h e r & S c h t i l l N o . 2045 p a p e r w i t h I O % a q u e o u s a m m o n i u m a c e t a t e : P b *+, o.95; Ag+, 0.80; T1+, 0.75; a n d H g ÷, 0.65. D e t e c t i o n w a s a c c o m p l i s h e d w i t h 5~o a m m o n i u m s u l p h i d e (viz., T1+ a n d P b 2+, b r o w n a n d A g + a n d H g +, g r e e n ) . POLLARD et al. ~9 d e s c r i b e d t h e c h r o m a t o g r a p h y o f t w e n t y - f o u r c a t i o n s u s i n g c o m p l e x - f o r m i n g m i x t u r e s . T a b l e 13 l i s t s t h e R F v a l u e s o f c a t i o n s o b t a i n e d u s i n g n - b u t a n o l - a c e t i c a c i d - a c e t o a c e t i c e s t e r - w a t e r (5 ° :IO : 5:35) for d e v e l o p m e n t . TABLE 13 RF
VALUES OF CATIONS WITH A COMPLEX-FORMING MIXTURE
Solvent system : n-butanol-acetic acid-acetoacetic ester-water (5° :i o : 5 : 35). Cation
R F value
Cat,on
RF value
Ag + Hg + Pb z+ Hg 2+ Bi 3+ Cu *+ Cd 2+ Asa+ Sb 3+ Sn ~+ Sn 4+ AP +
o. I 0.09 o.18 0.84 0.34 o.65 0.29 o. 17 o.i6 o.16 o.18 o. 17
Cr a+ Fe 8+ Zn ~+ Mn ~+ Co s+ Ni *+ Ca 2+ Sr ~+ Ba i+ Mg ~+ K+ Na +
o.64 0.73 0.30 0.23 0.22 0.22 0.20 o. 18 o.16 o.2o 0.25 0,23
T h e s e p a r a t i o n o f n i n e c a t i o n s o n W h a t m a n No. I a n d No. 42 p a p e r s u s i n g cyclohexane-hydrochloric acid and methyl ethyl ketone-hydrochloric acid mixtures w a s d e s c r i b e d b y HALMEROSKI AND SUNDHOLM 70. T a b l e 14 l i s t s t h e R F v a l u e s o f some metal ions with methyl ethyl ketone-hydrochloric acid mixtures. TABLE 14 RF VALUES X IO0 OF SOME METAL IONS W'ITH M E T H Y L E T H Y L KETONE--HC1 Paper: W h a t m a n No. I and No. 4 2. Solvents (mixtures of aqueous HC1 and methyl ethyl ketone) : (I) i ml cone. HC1, and (2) 2 ml cone. HCI in IOO ml of the solvent mixture. Development: descending. Time of run: 7 h with methyl ethyl ketone. Cation
t'Igz+ Cu ~+ Cd 2+ BP + As~+ Sn z+ Sb 3+ FeZ+ UO22+
Whatman No. I
Whatman No. 42
Solvent z
Solvent 2
Solvent z
Solvent 2
74 6 22 3° 13 a 21 38 xo
75 13 25 46 29 a 37 41 22
84 4 16 56 5 79 26 78 7
87 6 28 60 IO 84 49 80 io Chromatogr. Rev., 15 (I97I) I95-238
210
L. F I S H B E I N
BHATNAGER et al. ~1 described the paper chromatography of a number of metal dithizonates. The dithizonates of Ag, Pb, Hg(II), Bi, Cu(II) and Cd were studied in the following solvent systems : isopropanol; acetone; ethyl acetate; methyl acetate; chloroform-acetone (I :I, 1:3 and 1:9) ; chloroform-methyl acetate (I :3 and 1:9) ; chloroform-ethyl acetate (I :3) ; chloroform-ethyl acetate (I :9) ; carbon tetrachloride-acetone (I:I, 1:3 and 1:9); carbon tetrachloride-methyI acetate (I:I, I:3 and 1:9)- The solvent system and the binary pairs which were separated were: isopropanol, Hg-Ag; methyl acetate, Cu-Bi; chloroform-methyl acetate (I:3), Ca-Bi and P b - H g ; chloroform-methyl acetate (I:9), Ag-Hg, Cd-Bi, and P b - H g ; carbon tetrachloride-acetone (1:3 and I:9), Cu-Cd; and carbon tetrachloride-methyl acetate (I :9), Pb-Hg. The chromatographic separation and detection of silver, mercury, copper, cadmium, bismuth, cobalt, nickel and zinc ions with a dithizone-impregnated filter paper was described by NAGAI AND DEGUCHI7~. At the center of a dithizone-impregnated filter paper was placed a drop of an ion mixture of Hg-Cu-Cd-Co-Zn, Ag-CuCd-Co-Zn, Hg-Cu-Cd-Ni-Zn, Ag-Cu-Cd-Ni-Zn, Hg-Cu-Cd-Bi-Zn or Ag-Cu-CdBi-Zn and developed with 3 N acetic acid-acetone (5:6). Each metal ion yielded a circle with a characteristic color and the order of formation of colored circles (precipitated dithizone chelates) agreed with the order of stability of metal chelate. The separation and collection of traces of metals on paper impregnated with a few milligrams of a water-insoluble organic reagent was described ~. The reagent paper was prepared by dipping filter paper, 7 cm in diameter, in 1% dithizone-inchloroform or in o.05% p-dimethylaminobenzylidene rhodamine-in-acetone followed by o.I N nitric acid. Approx. 98% of Au, Ag and Hg in amounts of less than I/~g to 5/~g could be recovered after washing from lO-25o ml of O.l-O. 5 N mineral-acid solution by a single or by repeated filtrations at a rate of 6 ml/min. The method was found applicable for the rapid separation of several p.p.m, of Au, Ag and Hg in Cu and Pb where the concentration factor of Ag with respect to Cu was approx. 200o. The reagent paper could be stored for more than 80 days without any deterioration. AKIYAMA AND SHIOKAWA74 reported the separation of nfixtures of AI(III)Hg(II)-Cu(II), Hg(I])-Co(II)-Zn(II), Fe(III)-Bi(III)-Sb(III), Ug(II)-Ni(II)-Zn(II), Ag(I)-Cd(II)-Pd(II) and Hg(II)-Cu(II)-Cd(II)-Pb(II) by circular chromatography using Toyo filter paper No. 2 or 5B (diameter 9 cm) pretreated with 0.5% dithizonein-acetone solution and developed with o.I N nitric acid-acetone (io:I). QURESHI AND KHAN75 described the precipitation chromatography of various cations on strontium chromate-impregnated paper. Strontium nitrate (0.2 M; pH 7-8) was impregnated on Whatman No. I circles of 7-cm diameter, dried in an oven at 70°, then kept in 0.2 M solution of potassium chromate for about 5 min. After the complete precipitation of strontium chromate on the paper, it was dried, washed with water and redried. For quantitative determinations (20 min) 0.005 ml of the cation solution was applied and 0.2 M strontium nitrate (b) and 10% ammonium hydroxide (a) were used as developers. The RF values were as follows: (a) In ammonium hydroxide solution: Hg22+, Hg 2+, Pb 2+, Cd *+, Bi ~+, As 3+, Sb ~+, Sn ~+, La 3+, V 4+, Be 2+, Au 3+, Ti4+, Fe a+, A13+, Cr~, Mn*+, Co2+, Zn*+, Ca*+, Ba 2+, Mg2+, Ce3+, UO2*+, Th 4+, y3+ and Zr 4+, all were 0.00; Ag + and Ni 2+, 1.0; and Cu 2+, 0.41. (b) In strontium nitrate solution: Ag +, Hg, *+, Pb 2+, Bi 3+, As s+, Sb~+, Sn 2+, V 4+, Be 2+, Au s+, Ti 4+, Fe 3+, A13+, Cr8+, Mn 3+, Zn 2+, Ca ~+, Ba *+, Mg*+, U02 ~+, Th 4+ and Zr 4+, all were 0.00; Cd 2+, La 3+, Chromatogv. Bey., 15 (1971) 195-238
C H R O M A T O G R A P H I C A N D B I O L O G I C A L A S P E C T S OF I N O R G A N I C M E R C U R Y
211
Co2+ and Ni *+, I.OO; Cu ~+, o.41; Ce3+ and ys+ tailed. The main application of this technique is to separate cations with RF values of o.oo from those of RF I.OO. These two RF values can be made to differ by the choice of a l'roper complexing agent for elution. However, to achieve a specific separation, it is necessary to use a paper impregnated with a particular precipitating agent. Thus, using strontium chromate-impregnated paper it is possible to separate silver, mercury and lead cations from a number of different metal ions. The utility of paper precipitation chromatography for the separation of metal ions was described by NAGAI~6, Hg2~+, Ag+ and Pb 2+ were separated as their chlorides on filter paper impregnated with lO% sodium chloride with 4 M aq. ammonium acetate and ammonium hydroxide used as developing agents. Pb ~+ moved with the developing front and was identified with sodium rhodizonate. The Ag+ band which moved next to Pb ~+ was identified by a potassium iodide reaction. Hg~*+ was identified by the appearance of a black spot in the center of the chromatogram. Hg 2+ and Bi a+ were separated on potassium iodide-impregnated filter paper using 3 M propionic acid as developer. SUGAWARA~ described the separation of a number of heavy-metal ions of tile Cu group (Hg ~+, Bi 3+, Cd ~+ and Pb 2+) and Sn group (As3+ and Sb 3÷) on ion-exchange paper, Amberlite SA-2 (Na + type). The groups were developed with I M NH4C1 and I N HC1, respectively, and detected with ammonium sulfide-4-(2-pyridylazo)-resorcinol (for the Cu group) and ammonium sulfide-palladium chloride (for the Sn group), respectively. The RF values were: Hg 2+, 0.04; Bi 3+, o . I I ; Cd 2+, 0.52; Cu ~+, 0.27; Pb ~+, 0.47; As z+, 0.84 and Sb 3+, 0.26. The separation of TI(I), Hg(I), Ag(I) and Cs(I) on ammonium molybdophosphate (AMP)-impregnated papers was described by ALBERTI~s. Table 15 lists the RF values of these cations on AMP-impregnated paper using ammonium nitrate at various concentrations in I N nitric acid as developing solvent. TABLE 15 TI(I), Hg(I), Ag(I) AND Cs(t) SEPARATED ON AMP PAPER Developing solvent" NH4NO3, at various concentrations in I N HNO3.
R F VALUES OF
Cation
TI(I) Hg(I) Ag(I) Cs(I)
Molarity of NH4NO 3 o,z
0.5
z
3
5
7.4
r5
o,o o.15 0.46 o,o
o.o 0.40 0.63 o.o
0.02 o.41 0.65 o.o
o.o6 0.58 0.73 o.io
o.13 0.68 0.74 o,io
0.20 o.75 o.76 o.13
0.4o o.81 0.86 0.23
The separation of the oxidation states of sixteen elements with the use of trin-butyl orthophosphate-5 M acetic acid-acetone solvent system was reported by THAKUR79. Table 16 lists the RF values of ions with separable valency states obtained on Whatman No. I paper developed with tri-n-butyl orthophosphate-5 M acetic acidacetone (I :I :3). The basis on which ions of different valency states can be resolved is that metals form ionic or covalent complexes depending upon their oxidation states Chromatogr. R e w , 15 (1971) 195-238
212
L. F I S H B E I N
a n d t h a t the ionic complexes associate with polar solvents a n d covalent complexes with non-polar ones. The solvent system used in T a b l e 16 is n o n - p o l a r a n d as shown the higher v a l e n c y states exhibit increased migration, a t t r i b u t e d to the formation of their covalent complexes. TABLE 16 RF V A L U E S O F I O N S W I T H S E P A R A B L E V A L E N C Y S T A T E S Values obtained on Whatman No. i paper, developed with tri-n-butyl orthophosphate-5 M acetic acid-acetone (I :I :3) (ascending, 15 cm, 2 h at 19-22°). Ion
R v value
Ion
RF value
As3+ AsS+ Cr3+ Cr6+ Fe 2+ Fe 3+ Hg+ Hg~+ Mn~+ Mna+ Mn7+ TI+ T18+
0.42 0.57 o. I o.26 o.o 0.56, o. 7 o.oa o.92 o.I8 o.o o.o o.05 o. 3
Sba+ Sbs+ P,O: 4PO43PO sFC1C103BrBrO8IIO,-
o.o 0. 5 o.o o.37 o.o7 o.o o.I 3 0.32 o.2 o. 13 o.28 o.o
a Streaking observed. TANAKA et al. 8° described a radioisotope dilution m e t h o d for the determin a t i o n of inorganic a n d organic m e r c u r y b y paper chromatography. Trace a m o u n t s of mercuric chloride a n d p h e n y l m e r c u r i c chloride were first d e t e r m i n e d chelatometrically with penicillamine. T h e chelate was satisfactorily separated b y paper chromatog r a p h y from u n r e a c t e d m e r c u r y a n d the r a d i o a c t i v i t v of the chelate spot on the paper was measured with a well t y p e scintillation counter. A k n o w n a m o u n t of Z°3Hglabeled s t a n d a r d solution a n d substoichiometric a m o u n t of penicitlamine was t h e n added to a n u n k n o w n sample solution, the p H a d j u s t e d to 6-1o a n d the m e r c u r y chelate separated b y paper c h r o m a t o g r a p h y on Toyo filter paper No. 5IA, 2 × 4 ° c a , with isopropanol-conc, a m m o n i u m h y d r o x i d e - w a t e r (7:1:2) as developer. The a m o u n t of m e r c u r y in the u n k n o w n is t r e a t e d in a n analogous m a n n e r a n d the a m o u n t of m e r c u r y in the u n k n o w n calculated from the activities measured. Mercury was detected down to I/~g when lO -6 M of penicillamine a n d lO -8 M of Z°3Hg-labeled mercuric chloride or phenylmercuric chloride were used. Ni, Fe(II), Cd, Z n a n d Co ions did n o t interfere with the d e t e r m i n a t i o n . KURAYUKI AND KUSAMATO81 described the chromatographic separation of inorgano- a n d o r g a n o m e r c u r y compounds on anion-exchange paper, Amberlite SB-2 (R-C1), using 1.6 N nitric acid a n d 2 M a m m o n i u m n i t r a t e as developers a n d dithizone-in-chloroform for detection. The RF values (using 1.6 N HNO3) were: inorganom e r c u r y compounds, o.o9-o.11; alkylmercuriats, o.19-o.33; a n d phenylmercurials, O.Ol-O.O3. The limits of detection were 0.2 #g. I n a n a d d i t i o n a l s t u d y on the determ i n a t i o n of inorganic m e r c u r y c o m p o u n d s b y measuring the area of the colored zone on strips of Amberlite SB-2 (R-C1) the a m o u n t of m e r c u r y (1- 4 rag) was f o u n d to be p r o p o r t i o n a l to the colored area at p H 5.0. Chromatogr. Rev., 15 (1971) i95-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
213
WESTERMARK et al. 8~ described a concentration-electrophoretic method for the separation of charged mercury compounds in aqueous solution. In electrophoresis, the microcomponents concentrate in very narrow bands on the thin-layer strip, the postulated diffusion forces are counteracted or even balanced by the electrical-field strength. This phenomenon was applied to Hg 2+ and CH3Hg+ in an apparatus consisting of an aqueous mercury-containing insert, surrounded by concentration zones and buffer vessels, by using histidine and E D T A ions. The latter move from the cathode and form a negative complex with Hg ~+. This complex moves from the inert zone towards the anode, being partly concentrated there. Under suitable conditions CH3Hg + migrates to the cathode and concentrates behind the insert. At present the method of WESTERMARK and co-workers is limited to 2o #1, but this can apparently be increased. An advantage of this technique compared to gas chromatography is that inorganic mercury and alkylmercury can be measured in the same operation. FUKUDA et al. 83 described the qualitative analysis of metals b y paper electrophoresis following the initial separation of a sample containing nine metal ions into three portions and their precipitation into sulfides with NH4HS, thence treatment of the sulfides with acids into three groups of metal ions. Cu, Zn, Ag, Cd and Pb in the first group were separated b y paper electrophoresis with I N citric acid, 5 N acetic acid and 5 N formic acid as the electrolytes. For the second group (Hg and Bi) o.I M lactic acid or 0. 5 N formic acid were used as the eleetrolvte and for the third group (As and Sb), o.I M lactic acid was used. The paper electrophoresis of Hg(II) and twenty other cations was described b y CATON184. The electrolytes used were : (A) 25 ml 4 N lactic acid and 25 ml 2 M Na2CO~ made up to I 1 with water, (B) 40 ml 0.2 N HC1 and 250 ml 0.2 M potassium hydrogen sulfate made up to I 1 with water; (C) 468 ml o.I M sodium citrate and 532 ml o.I N HC1. 5. THIN-LAYER CHROMATOGRAPHY
SEILER AND SEILER85,86 separated the copper family (Cu 2+, Cd ~+, BP +, Pb ~+ and Hg 2+) on MN-Silica Gel G - H R using n-butanol-I. 5 N HC1-2,5-hexanedione (lOO:2O :0.5) as developer and 2% potassium iodide, ammonia and hydrogen sulfide, reagents for visualization. The addition of Pthe weak comptexing agent hexanedione reduces tailing. The RF values increase in the order of Cu~+
Hg 2+
Cd 2+ - -
[ ] B t3 + ] P b 2+
o,
1
2
3
4
5
6
Fig. 6. S e p a r a t i o n of t h e copper g r o u p on i N - S i l i c a Gel G - H R d e v e l o p e d w i t h • - b u t a n o l - I , 5 N H C I - 2 , 5 - h e x a n e d i o n e (IOO'2O'O.5).
Chromatogr. Rev., 15 (1971) 195-238
214
L. FISHBEIN
3-4-h development. The RF values of the cations increase in the same order as in paper chromatography, e.g., Ag+
Solvent front
@
0
@ 0
0
©
0 0
© StQrt
Bl(~r[) Cd(ZI)Cu(]Z) Pb(]I) Hg(]Z) Mixture
Fig. 7- TLC of metal ions on cellulose MN-3oo developed with tert.-butanol-acetone-water-6 N HNOs-acetylacetone ( 4 : 4 : i . i :o,45:o.45 ). Detection with 2% ethanolic oxine, ammonia vapors and UV inspection.
Chromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY" TABLE TLC
215
17 O F Hg(ll)
AND
METAL
IONS ON
ZIRCONIUM
HYPOPIIOSPHATE
CHROMATOPLATES
Development in (i) o.i N HC1 and (2) 0. 5 N HC1. Cation
R F value Solvent I
Solvent 2
S n z+ P b ~+
o o
o o
F e 3+
0
0
La 8+ Oa a+ Y~+ In s+ Cs+ Cu ~+ Co2+ Cd ~+ Fe 2+ Ca ~+ Zn 2+ Ni 2+ Hg 2+ Ba ~+ Na + Sr 2+
o o o o 0.05 0.05 0.0 7 o.Io o.oi o. io o.15 o.25 o.so o.3 ° 0.35 0.35
o.io o.o o.15 o.2o o.Io 0.55 0.55 0.53 0.56 o.42 0.52 0.50 o.73 o.63 o.55 0.60
Co 2+, Ni 2+, Cu 2+, Z n 2+, C d 2+, H g 2+, S n 2+ a n d P b 2+ w e r e d e t e c t e d w i t h 1 % dit h i z o n e i n c a r b o n t e t r a c h l o r i d e ; F e z+ a n d F e 3+ w i t h i M K3[Fe(CN)6 ] o r i M K a [ F e (CN)~] a n d L a z+ a n d Y ~ w i t h 0 . 1 % I - ( 2 - p y r i d y l a z o ) - 2 - n a p h t h o l i n e t h a n o l . N a +, Cs +, Ca ~+, S r ~+, B a ~+, L a 3+ a n d y a + w e r e i d e n t i f i e d b y u s i n g r a d i o a c t i v e t r a c e r s . T L C w i t h a 0 . 4 - m m l a y e r o f Alz03 a n d silica gel s o r b e n t s w a s a p p l i e d t o t h e d e t e c t i o n o f H g *+ a n d f i f t e e n i o n s o f m e t a l s o f t h e 3 r d a n d 4 t h a n a l y t i c a l g r o u p s 9°. The ions d e t e c t e d , t h e r e a g e n t s used a n d t h e m i n i m u m d e t e c t a b l e a m o u n t s i n / t g are s h o w n in T a b l e 18. TABLE TLC
18 O F Hg(II)
AND
METAL
CATIONS
O F TIIE 3rd A N D
4th A N A L Y T I C A L
GROUPS
Metal catwn
Detect,ng reagent
M i n i m u m detectable amount (,ug)
Cu 2+ Ag + P b ~+ Hg ~+ Bi 3+ Hg, *+ Cd~+ Fe a+ Fe ~+ Co~+ Ni *+ Zn *+ Mn *+ Cr 3+ Ap+
rubeanic acid HCt, SnCI,, NH4OH Na-rhodizonate KI, biphenyl (NH4)~SCN (NH4),SCN Na2S K4Fe(CN), or KSCN NH 3 and dimethylglyoxime a-nitroso-fl-naphthol rubeanic acid (NH4) ~ [Hg(SCN)4] KIO,-HNO 3 2 M Na,HPO 4 NH3_ahzari n
0,24 1.6 o.9 7.5 1.3 6.02 3.4 o.34 0.04 0.038 0.o87 0.30 o. 18 o.18 o.o3
Chromatogr. Rev., 15 (1971) 195-238
216
L. FISHBEIN
SHERWOOD91 described the TLC behavior of thirty cations using Eastman Chromagram No. 6o61 sheets with the following solvent systems: (I) 5% hydrochloric acid-methyl isobutyl ketone-amyl acetate (i :2 :i). Table 19 shows the RF values of the cations as their chlorides arranged in rough order of movement, viz. very fast, fairly fast, slow and very slow--non-movers, following a 2-h development. Visualization of the cations was achieved using nine sprays (the most universal of these being TABLE 19 RF VALUESOF METALtONS ON EASTMAN CHROMAGRAM No. 6061 SHEETS
Developing system : 5% HCl-methyl isobutyl ketone-amyl acetate (I :2 :I). Order of movement
Very fast
Fa,rly fast
Slow
Very slow and non-movers
Cation
RF value
Cat,on
RF value
Cat,on
R~, value
Cation
RF value
Mo Fe
o.91 0.89
Hg As
0.6 o.51
Mn Bi
0.06 0.05
Ni Ti Ce Ca
]
Au
o.89
Cd
o.31
Pb
o.o2
Sr
Sb Te Sn
o.85 o.82 0.78
Zn Cu Pt Co
o.27 o.i8 o.19 o.13
V
o.o2
A1 Zr Li K Na Mg Cr
Ba
All o.o
o.o5% dithizone in carbon tetrachloride) with the following appropriate color reactions: (I)as sprayed: yellow, Fe, Te, As; yellow-orange, Sb; orange, Au, Hg, Cd, Bi, and Pb; red-orange, Mo; cherry red, Sn, Zn, V; yellow-green, Cu; purple, Co; (2) after exposure to ammonia vapors and heating: yellow, Au, Te and As; yellow-orange, Sb and Bi; orange, Fe, Hg, Cd and Pb; red-orange, Mo; cherry red, Sn, Zn and V; pale green, Pt; olive-green, Cu; purple, Co. The detecting reagents used were: (a) 5% ammonium thiocyanate in water-acetone (5:95) ; (b) 5% potassium iodide in wateracetone (lO:9O); (c) 0.5% oxine in ethanol; (d) 5% silver nitrate in water-acetone (30:70); (e) o.5% diphenyl carbazide in ethanol; (f) 1% dimethyl glyoxime in ethanol; (g) o.1% aq. solution of chromotropic acid; and (h) 5% aq. solution of sodium rhodizonate. The limits of detection of the metal ions developed with solvent system I and visualized with dithizone/ammonia vapors/heating were: Hg, 0.5-2 #g/#l; Cd, As and V, 0.2-0.5/~g//,l; Zn, Pb and Pt, O.l-O.2 #g/#l; Cu, Bi, Co, Mn and Ni, all detected down to 0.05 #g//~l. The TLC of a number of metal dithizonates was described by TAKITANI et al. 9~. Pd, Hg, Cu and Ag ions are extracted as dithizonates with o.1% dithizone in chloroform at pH o - i ; then Bi, Cd, Co, Ni, Pb and Zn ions at pH 7-8; and T1, Fe ~+, Mn, and Sn *+ ions at pH 9-1o. The ist- and 2nd-group metals are separated on an MNSilica Gel G-HR layer with carbon tetrachloride-methylene chloride-benzene (i :7:4) and identified by color. The 3rd-group metals are re-extracted with 20% nitric acid and separated with acetone-hydrochloric acid (99 :I) and detected by spraying with appropriate reagents. Identification limits in p.p.m, are: o.2 for Cu, Ni, Co; 0.3 for Hg; 0. 4 for Pd, Ag, Cd; 0.8 for Zn; 3 for Pb; and 5 for Bi. The TLC of metal dithizonates of Hg, Cu, Bi, Zn, Pb, Cd, Co and Ni was deChromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
2I 7
scribed by HRANISAVLJEVICet al. 98. The metals were extracted from aq. solution with o.1% dithizone in chloroform (viz., Hg, Cu and Bi extracted at pH 3; Zn, Pb and Cd at p H 5; Ni and Co at pH 7). The three chloroform solutions obtained were then separated on Silica Gel G. UNTERHALT94 described the TLC of a number of metal dithizonates and cations on Silica Gel G and cellulose MN-3oo F2s4 layers. Fig. 8 illustrates a chromatogram of the dithizonates of Cu(II), Bi(III), Cd(II), Zn(II), Hg(II) and Pb(II) at pH 5 on Silica Gel G developed with benzene-dichloromethane (5 :I) (3° rain, 15 cm). A 0.2%potassium iodide reagent detected Cu(II), Pb(II), Bi(III) and Hg(II) as yellow-brown to red spots, while o.I M sodium sulfite detected Cd(II) and Pb(II).
0 c) o
o
©
Fig. 8. TLC of metal dithizonates at pH 5 on Silica Gel G developed with benzene-dichloromethane (5:I). The utility of TLC in the forensic analysis of metals was described by KUNZI et al. 95. Silica Gel G (Merck) was used with acetone-benzene (75:25) saturated with tartaric acid and 6% nitric acid to effect the separation of the metals in the order of Re: Hg > B i > S b > C d > A s > P b > C u >T1. Both dithizone and ammonium sulfide reagents were used for detection (limit of detection with both reagents was 2 #g). Table 20 illustrates the spot colors of Cu, As, Cd, Sb, Hg, T1, Pb and Bi obtained with the above reagents. SUGIWARA9s reported the separation and identification of metallic poisons (viz., Pb, T1, Cu, Cd, Sb, Bi, As and Hg) on silica gel plates developed with nbutanol-I.5 N hydrochloric acid-acetylacetone (lOO:2O:O.5) and n-butanol-benzene z N nitric acid (70:3:2). The Re values were in the order: H g > T I > B i > S b > C d > A s >Cu>Pb. The determination of Ge(IV), Sn(II), Pb(II) and Zn(II), Cd(II), Hg(II) by ring colorimetry after separation by TLC has been described by JOHRI et al. 9~. Table 2I lists the R~, values and spot colors of the metal ions when developed on Silica Gel G plates with (I) isobutanol-conc, hydrochloric acid-ethyl methyl ketone (Io :8 :I) and (2) isobutanol-acetic acid (3:1) and detected with (a)o.o5% phenylfluorone, (b) o.o5 M aq. potassium thiocarbonate (PTC), and (c) dithizone and ammonia. For the evaluation of different metal ions, the resulting delineated spots as viewed in UV Chromatogr. Rev., 15 (1971) 195-238
218
L. FISHBEIN
TABLE 20 S P O T C O L O R S O F M E T A L S D E T E C T E D W I T H D I T H I Z O N E A N D (NH4),S Developing solvent: acetone-benzene (75:25) saturated with tartaric acid and 6% nitric acid on Silica Gel G.
Metal
Cu As Cd Sb Hg T1 Pb Bi
Dzthizone Acid
A mmoniacal
(NH4) ,S
yellow-green yellow lilac red rose ---purple
yellow-brown -orange bright brown orange-red rose rose orange-red
brown yellow yellow orange black black brown brown
light were r e m o v e d using a micro v a c u u m cleanergS, 99 an d the collected m a t e r i a l t ra n s f err ed to a paper set on a ring oven a n d d e t e r m i n e d b y ring c o l o r i m e t r y utilizing t he t e c h n i q u e of WEISZ 1°°. SEILER103 s e p a r a t e d H g ( I ) a n d H g ( I I ) f r o m a c a t i o n m i x t u r e b y T L C on silica gel using n - b u t a n o l - 4 N nitric acid (80:20) as d e v e l o p i n g solvent. A f t er separation, m e r c u r y was d e t e r m i n e d b y n e u t r o n - a c t i v a t i o n analysis b y y - s p e c t r o m e t r y o f 13o ke V 197Hg(meso). O n e / ~ g o f m e r c u r y was d e t e r m i n e d in t h e presence of I5oktg o f c a t i o n m i x t u r e w i t h a precision of ~ 4 % . TABLE 21 RE × IOO VALUESAND SPOT COLORSOF Ge(IV), Sn(II), Pb(II), Zn([I), Cd(II) AND Hg(II) ON SILICA GEL G Solvent systems: (I) isobutanol-cone, hydrochloric acid-ethyl methyl ketone (lO:8:1); (2) isobutanol-acetic acid (3:I). Detectors: (a) o.o5% phenyl fluoronO°I, (b) o.o 5 M aq. potassium thiocarbonatO °* and (c) dithizone and NH 3. Metal ion
Ge(IV) Sn(II) Pb(II) Zn(II) Cd(II) Hg(II)
RF × ~oo value Solvent i
Solvent 2
io.o 94.4 76.5 ----
---85.7 71.4 50.5
Reagent
Spot color
Limit of identification (ttg)
a b b c b b
pink brown pink-red pink yellow brown-black
6.5 ° I 1.2o 3.9o 3,60 2.20 4. io
K o s s AND BEISENHERZ104 described t h e d e t e r m i n a t i o n o f h e a v y - m e t a l ions as r a d i o a c t i v e sulfides following t h e i r initial separation on Silica Gel G-coated slides, thence t r e a t m e n t w i t h H2ssS. Fig. 9 illustrates the separation of c a d m i u m , copper a n d m e r c u r y salts on a Silica Gel G - c o a t e d microscope slide d e v e l o p e d with n - b u t a n o l 1. 5 N hydrochloric a c i d - a c e t o n y l a c e t o n e (ioo :20:0.5). T h e T L C s e p a r a t i o n of m e t a l - E D T A complexes on microcrystalline cellulose (Avicel) has been described b y MASOOMI AND HAWORTH1°5. T w e n t y - t h r e e m i x t u r e s of Chromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
2I 9
Fig. 9. Separation of cadmium, copper and mercury salts on a coated microscope slide. Layer: Silica Gel G; solvent: n-butanol-I. 5 N HCl-acetonylacetone (lOO:2O:O.5); time of run 2 h; detection with H235S. The chromatogram was evaluated by means of a Geiger-Mfiller counting tube (B) and the autoradiograph evaluated photometrically (A). the metal-EDTA complexes were separated using seven different solvent systems of v a r y i n g c o m p o s i t i o n (Table 22). T a b l e s 23 a n d 24 s h o w t h e R v v a l u e s o f v a r i o u s m i x t u r e s o f m e t a l - E D T A c o m p l e x e s (viz., Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd a n d H g c o m p l e x e s ) a n d t h e d a t a for specific d e t e c t i o n t e c h n i q u e s a n d r e a g e n t s u s e d for e a c h EDTA complex, respectively. T A B L E 22 TLC
S O L V F N T SYSTEMS FOR
Solvent system
SEPARATION OF M E T A L - E D T A
COMPLEXES ON A V I C E L
Compos,tion W'ater-n-butanol-glycol monomethyl ether-conc, ammonia ~ (4.5 : 3.5 : 2.o "o.25) Water-glycol monomethyl ether-methyl ethyl ketone-acetone-n-butanol-conG. ammoniaa (4 .0 : 3-o:1.5 :o.5 :i.o:o.Io) Water-glycol monomethyl ether-methyl ethyl ketone-acetone-conc, ammonia a (4:2:2:2
4 5A 5B 6A 6B 6C 6D 6E 6F 7
:O. I 5 )
Water-n-butanol-acetone-conc. ammonia a (4:4 : 2 :o, Io) \Vater-methyI ethyl ketone-n-butanol-acetone-conc, ammoma a (4.0: 2.5 : x.o: e. 5 :o. i o) Water-methyl ethyl ketone-n-butanol-acetone-cone, ammonia a (4.0 : I.O : I .o :4.° :o. 15) Water-acetone-conc. ammonia • (I.3 : 8.7:0.o5) Water-acetone-conc. ammoma a (2:8:o.o5) Water-acetone--cone. ammoma a (2.5:7.5 :o.05) Water-acetone-conc. ammoma a (3:7:o.o5) Water-acetone-conc. ammoma a (3-5:6.5 :o.o5) Water-acetone-conc. ammoma a (5:5 :o.io) Water-butyronitrile-methanol-HC104a (3 ml : 7 ml : xo drops) a The conG. ammonia and HC104 are on a basis of IOO ml of solvent.
T h e s e p a r a t i o n a n d i d e n t i f i c a t i o n o f P b ~+, A g +, H g 2+ a n d B a 2+, Sr 2+, Ca ~+ a n d Mg 2+ ions has b e e n a c h i e v e d b y T L C o n m a i z e - s t a r c h u s i n g a c e t o n e - 3 N n i t r i c a c i d (I :i) a n d a c e t o n e - 3 N h y d r o c h l o r i c a c i d (2:3) as solventsl°~, 1°~ w i t h t h e d e t e c t i o n o f I × IO-8-5 × IO -9 e q u i v , o f t h e i o n s a c h i e v e d d u r i n g a I 5 - c m 2-h d e v e l o p m e n t . I M a m m o n i u m sulfide s o l u t i o n w a s u s e d for t h e d e t e c t i o n o f t h e g r o u p o f m e t a l s P b ~+, H g 2+ a n d A g + a n d o . 5 % s o l u t i o n o f 8 - h y d r o x y q u i n o t i n e f o l l o w e d b y e x p o s u r e to a m m o n i a v a p o r s a n d d e t e c t i o n u n d e r U V for t h e g r o u p B a ~+, S r ~+, Ca 2+ a n d Mg ~+. T a b l e 25 lists t h e R E v a l u e s a n d s p o t colors for t h e T L C o f e i g h t m e t a l ions on m a i z e s t a r c h c h r o m a t o g r a m s u s i n g a c e t o n e - 3 N n i t r i c a c i d (I :I) as d e v e l o p i n g s o l v e n t a n d I M aq. a m m o n i u m sulfide a n d o . 1 % d i t h i z o n e - i n - c h l o r o f o r m as d e t e c t i n g r e a g e n t s . T h e s e p a r a t i o n o f t h e d i v a l e n t c a t i o n s of m e r c u r y , c o p p e r , c a d m i u m , n i c k e l a n d zinc b y c i r c u l a r - c h r o m a t o g r a p h y w i t h d i t h i z o n e - i m p r e g n a t e d silica gel t h i n l a y e r s w a s d e s c r i b e d b y DEGUCHI 1°8. T h e p l a t e s w e r e p r e p a r e d b y a d d i n g 3o m l o . 3 % di-
Chvomatogr. Rev., 15 (1971) 195-238
220
L. FISHBEIN
T A B L E 23 R F V A L U E S OF VARIOUS M I X T U R E S OF METAL-EDTA COMPLEXES V a l u e s o b t a i n e d w i t h T L C s e p a r a t i o n o n Avicel. S o l v e n t s y s t e m s f r o m T a b l e 22.
Solvent system No.
Metal ion (complexed w,th E D T A ) Cr
Mn
Fe
Co
Ni
Cu
I 2
3 4 4 5A 5B 5B 5B 5B 5B 6A 6B 6B 6B 6C 6C 6D 6E 6F 7 7 TABLE REAGENTS
0.46 0.46
o.54 o.58
0.60 0.67
o.74
o.42 o.42 o.17 o.39 o.65 o.29
0.07
Zn
Cd
Hg
0.70 0.68 0.74
0.65 0.58 0.64
0.62 0.55 0.60
0,59
0.45
0.38
0,II
0.09
0.07
o.12 o.46 o.52
0.09 0.26 o.44
0.54 0.65 0.66 0.78
0.75
o.49
o.57 o.61
o.13
0.07 0.27
0.29 0.54
0.49 0.39
o.13 o.17 0.59 0.60
o.86
0.75 o.i 5 0.22
0.82 0.06 0.09
0.45 0.42
o.58
24 AND
DETECTION
TECHNIQUES
FOR TLC
O F M E T A L - - E D T A COMPLEXES
S e p a r a t i o n o n Avicel. Solvent s y s t e m s f r o m T a b l e 22.
Metal ion (complexed wzth EDTA )
Complex color Reagent and detect,on techmque
Hg Cr
Colorless Violet-blue
Mn
Colorless
Fe Co
Yellow-brown Violet-red
Ni Cu
L i g h t blue Blue
Zn
Colorless
Cd
Colorless
A f t e r s p r a y i n g w i t h 5 % NazS yields a Mack spot. U n d e r U V light c o m p l e x a p p e a r s black. W i t h 0.05% (w/v) dit h i z o n e in CC14 yields a d a r k green color. W i t h a s a t u r a t e d solution o f 8 - h y d r o x y q u i n o l i n e in a m m o n i a yields a yellow color. W i t h 1% K C N S (w/v) yields a r e d color. U n d e r U V light t h e c o m p l e x a p p e a r s black. W i t h dithizone yields a grey-yellow color. A f t e r s p r a y i n g w i t h d i t h i z o n e yields a yellow color. A f t e r s p r a y i n g w i t h 8 - h y d r o x y q u i n o l i n e yields a l i g h t g r e e n color. A f t e r s p r a y i n g w i t h a dilute solution o f a m m o n i a a n d dithizone yields a p i n k color. After s p r a y i n g w i t h 5 % (w/v) solution o f N a , S b yields a yellow color.
a For m u l t i p l e s p r a y i n g , zones colored o n t h e T L C plate were first m a r k e d for t h o s e r e a g e n t s w h i c h were colored ; t h e n followed b y s p r a y i n g w i t h t h e d e t e c t i o n r e a g e n t s in t h e order : K C N S , NayS, d i t h i z o n e a n d 8 - h y d r o x y q u i n o l i n e ; e.g., in t h e d e t e c t i o n o f Hg, Cd a n d Zn s e p a r a t i o n , t h e p l a t e w a s s p r a y e d w i t h NayS to d e t e c t H g a n d Cd, zones were m a r k e d a n d t h e p l a t e s p r a y e d w i t h d i t h i z o n e a n d a m m o n i a to d e t e c t t h e Zn complex. (The H g a n d Cd s p o t s d i s a p p e a r e d in t h e p r e s e n c e o f base.) b Occasionally n e c e s s a r y to r e - s p r a y w i t h a dilute-acid s p r a y to develop t h e color.
Chromatogr. Rev., 15 (1971) 1 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
221
TABLE 25 RF VALUES AND SPOT COLORS FOR SOME METAL IONS ON MAIZE-STARCHCHROMATOPLATES Developing system: acetone-3 N hydrochloric acid (I:I); detecting reagents: (I) I M aq. ammonium sulfide and (2) o.i % dithizone-in-chloroform. Ion
RF value
Spot color Detecting reagent r
Pb 2+ Cu 2+ Bi 3+ Cd 2+ Hg 2+ As 3+ Sb 3+ Sn ~+
0.27 o.4o 0.75 0.80 0.84 0.36 0.74 0.84
brown yellow-brown black-brown yellow black ----
Detecting reagent 2
--yellow-red rose rose
t h i z o n e - i n - c h l o r o f o r m s o l u t i o n to I 0 g silica gel. T h e c a t i o n s w e r e t h u s s e p a r a t e d as m e t a l c h e l a t e s y i e l d i n g c o l o r e d c h r o m a t o g r a m s o f c o n c e n t r i c circles. T h e b e s t s e p a r a t i o n s o f Hg(II), Cu(II), Cd(II), Ni(II) a n d Zn(II) w e r e o b t a i n e d b y u s i n g e i t h e r 2 N a c e t i c a c i d - a c e t o n e (I :I) o r 2 - 4 N a c e t i c a c i d - a c i d (I :0.66) as d e v e l o p e r s . T h e c a t i o n s w e r e d e v e l o p e d in t h e a b o v e order, Z n b e i n g in t h e o u t e r m o s t circle. C i r c u l a r - T L C m e t h o d s for t h e i d e n t i f i c a t i o n of f o r t y c a t i o n s a n d n i n e t e e n a n i o n s f r o m 0.5 m l of u n k n o w n s o l u t i o n s w e r e d e s c r i b e d b y HASHMI et al. 1°9. T h e m e t a l i o n s w e r e i n i t i a l l y s e p a r a t e d i n t o five d e f i n i t e g r o u p s I - V b y s o l v e n t e x t r a c t i o n a c c o r d i n g to t h e p r o c e d u r e of WEST AND MUKHERJ111° (the s u b s e q u e n t d e v e l o p m e n t o f a chromatoplate was complete within two minutes). For cations, aluminum oxide (D-5, c o n t a i n i n g 5 % c a l c i u m s u l f a t e as b i n d e r , C a m a g ) a n d silica gel (D-0, w i t h o u t b i n d e r , C a m a g ) w e r e u s e d w i t h o u t f u r t h e r t r e a t m e n t , for t h e p r e p a r a t i o n o f t h e t h i n layers. F o r anions, A l u m i n u m O x i d e S a n d Silica Gel G ( H o p k i n a n d W i l l i a m s ) w e r e used. C i r c u l a r - t h i n - l a y e r c h r o m a t o g r a p h i c a p p a r a t u s l°s w a s u s e d for t h e d e v e l o p m e n t o f c h r o m a t o p l a t e s . T a b l e 26 lists t h e s o l v e n t s y s t e m s for t h e c h r o m a t o g r a p h i c s e p a r a t i o n o f c a t i o n s o f g r o u p s I - V a n d o f a n i o n s , T a b l e 27 lists t h e s p r a y r e a g e n t s for locaTABLE 26 SOLVENT
SYSTEMS
FOR CIRCULAR-TLC
SEPARATION
OF CATIONS
OF GROUPS
I-V
AND OF ANIONS
Separation on aluminium oxide (3-5 for cations; S for anions) and silica gel (D-0 for cations; S for anions). Solvent system
Composition
Group a
A B C D E Fb
Acetone- 4 N HCl-acetylacetone (45:3 : 2) Acetone-4 N HC1 (47:3) Acetone- 4 N HCl-acetylacetone (48 :i.5 :o.5) Acetone- 4 N HC1 (46:4) Acetoue-conc. HC1 (47:3) n-Butanol-pyridine-water-ammonia (8 :4 : 8 : I)
I, II III IV V V Anions
a Metal ions separated by solvent extraction into five groups: I = chloride group; II = thiocyanate group; I I I = acetylacetone group; IV = diethyl dithiocarbamate group; V = aqueous group. b After shaking, the upper layer is used. Chromatogr. Rev., 15 (1971) 195-238
222
L. FISHBEIN
TABLE 27 SPRAY RI~AGENTS FOR LOCATION ON CIRCULAR-TLC
PLATES OF CATIONS AND OF ANIONS OF GROUPS
I-V
Spray reagent
Solution
Tannic acid (TAw) Tannic acid (TAg) Stannous chloride-potassium iodide (SC-KI) Phenyl fluorone (Pf) Dithizone (DZc) 1Rubeanic acid (IRA) Diphenyl carbazide (DC) Potassium ferrocyanide (PF) Alizarin (AZ) Peroxide-benzidine (PB)
lO% in water lO% in glycerine-water (I :i) Dissolve 5 g SnC12in IO ml conc. HC1 and dilute to ioo ml with addition of 0. 5 g KI. 0.05% in 96% ethanol-conc. HC1 (3:1) 2 % in chloroform o.5% in 96% ethanol i% in 96% ethanol 5% in water saturated solution in 96% ethanol 5% Na,O, in water followed by 1% benzidine in glacial acetic acid Quinalizarin (QAZ) o.o5% in 70% ethanol Sodium sulfide (SS) 2% in water Dlmethylglyoxime (DO) 1% in 96% ethanol 1Rhodizonic acid. sodium salt (SR) 1% in water, freshly prepared Hydrogen peroxide-ammonia (HA) H,O, (3o% wtv)-conc, ammonia (I :I) Sodium cobaltinitrite (SCo) cobaltinitritea-methanol (3: i) AgNO~ 1% in water; after short drying followed by o.1% Silver nitrate-fluorescein (SF) fluorescein in 96% ethanol Silver nitrate (S) saturated solution in water Potassium iodide-HC1 (PH) lO% solution containing lO ml HCl (2 M) Ferric chloride (FC) lO% solution containing io ml HCI (2 M) Potassium dichromate (PD) saturated solution in water, containing io ml H,SO4 (2 M) Sodium nitroprusside (SN) 20% in water Ferrous sulfate (FS) lO% in water, containing 20 ml HzSO4 (2 M)
a 11.4 g cobalt acetate (CHsCOO)2Co.4HzO, 16.2 g lead acetate (CH3COO)zPb.3H~O) , 20 g sodium nitrite and z ml glacial acetic acid are mixed, dissolved and diluted to 15o ml, centrifuged, and filtered. tion of the cations a n d anions, a n d Table 28 the R F values, s e n s i t i v i t y a n d m a x i m u m a m o u n t of cations of groups I-V. The s e n s i t i v i t y in this table indicates the m i n i m u m a m o u n t of the m e t a l ion which m u s t be present before e x t r a c t i o n ; the m a x i m u m a m o u n t indicates the u p p e r limit of each m e t a l ion which does n o t interfere with the succeeding rings when all m e t a l ions of a p a r t i c u l a r group are present in their m a x i m u m a m o u n t . F o r circular-TLC analysis, it was f o u n d essential to apply the m e t a l ions in a m o u n t s which lie between the sensitivity a n d the m a x i m u m a m o u n t shown in T a b l e 28. The s e p a r a t i o n of a n u m b e r of m e t a l diethyl d i t h i o c a r b a m a t e s on Silica Gel G plates was described b y SEI~Fi n using a solvent system c o n t a i n i n g n - h e x a n e - c h l o r o f o r m - d i e t h y l a m i n e (20:2 :I). After the plate was heated, in order to remove the die t h y l a m i n e , the spots were developed b y spraying with 5% copper sulfate a n d o.I # g of each m e t a l was detectable. Interfering metals such as Co, Ni, Fe a n d Mn could be r e m o v e d b y controlling the p H range with c i t r a t e - p h o s p h a t e buffer. T h e R v values of the m e t a l diethyl d i t h i o c a r b a m a t e s were: Hg, o.56; Pb, o.oo; Cu, 0.44; Bi, 0.27; a n d Cd, 0.34. TAKESHITA et al. nz described a reversed-phase TLC m e t h o d for the separation a n d detection of the dithizonates of both inorganic m e r c u r y a n d a series of alkyl-
Chromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHICAND BIOLOGICALASPECTS OF INORGANICMERCURY
223
mercury compounds and found the method applicable to the separation and identification of mercury compounds in foods and sewages. The adsorbents used were cornstarch containing liquid paraffin and Avicel SF (FMC, American Viscose Div.) containing 20% liquid paraffin. The solvent systems consisted of methyl celloso]vewater (75:25) and ethanol and methyl cellosolve treated, respectively, with water in the ratios from o to 30%. The study revealed that the best pattern of separation of all the dithizonates was obtained on Avicet SF-liquid paraffin layers with methyl cellosolve-water (7:3), although the development time was longer than with impregnated cornstarch plates (1-1. 5 h). No interference of other metals and the residual TABLE 29 DETECTION
LIMITS OF MERCURIC
AND ALKYLMERCURIC
DITtlIZONATES
AND CHLORIDES
Layer: Avlcel SF-liquid paraffin. Developer: methyl cellosolve-water (75:25). Compound
Detection limit (fig)
Mercury Methylmercury Ethylmercury n-Propylmercury n-Butylmercury n-Amylmercury n-Octylmercury Stearylmercury
Dithizonate
Chloride
o.oi
o.oo4 o.o53 o.o55 o,o56 0,057 0.029 0.006 o.oo7
o.I o. i o.i o.i 0.05 o,oi o.oi
dithizone was observed. Table 29 lists the detection limits of mercury and alkylmercury compounds (as dithizonates and chlorides) on Avicel SF-liquid paraffin layers with methyl cellosolve-water (75:25) as developer. 6. GAS CHROMATOGRAPHY TADMORna described the gas chromatographic (GC) separation of a number of metal halides, e.g. (a) SnC14, SnBr~, SnI,, (b) AsC13 and GeC14, and (c) FeC13 and HgC12, (2)
(1l
V///////////// ,4,-..
,
*\
.......
0
',;L-'HI (8)
Fig. Io. Home-made gas chromatograph. (i) Electrical furnace; (2) gas preheater; (3) chromatographic column; (4) carrier gas; (5) injection port; (6) heating wire; (7) effluent solvent stream; (8) to radioactivity detector and recorder. Chromatogr. Rev., 15 (1971) I95-238
224
L. FISHBEIN
~
o ~o ~
o
= ~o o
~
~
. ~.~
~: ~b~ ~
~
~.~ ~.
0
o~
~
o
~o~
o
~00°°
~.
~
~
oo
d d d d 5
~
&~d d d
d d
d
d
d d
o Z
%
~,~o
~o
0 ~
"~
~
o
"~
'~
~
o
~o
~oo%%oo~
d
d6~6dd6d6
o
Z 0
~
~
g~
~
~:.~
~ ~
"~ ~ ~
"~"0
o
.,:
.~ ~ "n~ ~ " 0
~
~o®
~
o
m
o
0 0 0
N
++
+
m
Chrom~mg~. Rev., 15 (I97 I) 195-238
~ + +
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS O F INORGANIC MERCURY
225
3g o.
o~
o~
~
~
~ ~ .~ ~ o
,.m
~,
t~
o .o o~
~
0
~
0
~+~ "
~
o ~0 O
~00
~ 0 ~ ~o
oqt~O
~oo
G*
6
. . . .
666666d
O~ O0
6
oooo
6666
~
666dMG o
fi~ o o
o
~,
o ~
~ o o~
o~
~
~
"~
~o
.,~
++++~+~
~+
+ + +
+
"~+ + + Ho
Chromatogr. Rev.,
15 (1971) 1 9 5 - 2 3 8
~ ,1:::~
226
L. FISHBEIN
utilizing a laboratory-constructed apparatus. The apparatus (Fig. IO) was comprised of a chromatographic column preceded by a preheater section for the carrier gas. These consist of U-shaped Pyrex glass tubes packed with different stationary phases; the apparatus is placed in a cylindrical electrical furnace (Adamel type T5HT ) whose ports are closed with asbestos-insulating material in order to thermostate the apparatus. Supplementary heating is provided for the injection port and the end of the column by a heating wire connected to a resistance. The solid stationary phase was Sil-O-Cel brick (30-50 mesh), washed with 2.5 N HNO 3, rinsed with water and dried at IiO °. Some of the liquid stationary phases were: n-butanol, n-decane, glycol, KelF-oil, nitrobenzene, glycerol, A1Br3, Silicone oil-55o, BiC13 and Woods Metal. Samples were labeled with radioactive isotopes, which were distilled in and kept under an atmosphere of dry nitrogen, then introduced into the apparatus as a solution in an organic solvent or as the compound itself. Effluent activity was determined with a liquid beta counter (2oth Century Electronic, Model M6) connected to an Atomic Scaler, Model lO91. Fig. I i illustrates the GC separation of HgC12 and FeC13 on a 8o-cm column coated with 3o% BiCla on Sil-O-Cel brick (30-50 mesh),
h
! I
II It
....
HQC Ia
I
~
Feel
I
3
i
'I
tO 0
v
,
o
I
I
2
4 Time
.......
6
1,,.,
(rnln)
Fig. I I . Gas c h r o m a t o g r a p h y o f HgC1, a n d FeC1 v C o l u m n (80 cm) : Sil-O-Cel brick (30-50 mesh) c o a t e d w i t h 3 o % w[w BiCI v T e m p e r a t u r e : 290 °. Carrier g a s (nitrogen) flow: IO m l / m m .
It is of interest to note that the volatilization and separation of many acetylacetonates and fluorocarbons/~-diketonates (excluding those of mercury) have been accomplished by both conventional n4,n5 and hypelpressure GC techniques. Apparently the analogous mercury derivatives are not volatile enough nor possess sufficient thermal and solvolytic stability to permit GC analysis. 7.
ION-EXCHANGE AND COLUMN CHROMATOGRAPHY
Mercury, zinc and cadmium were quantitatively resolved as their anionic-chloro complexes by an ion-exchange chromatographic procedure ne. The stability of these complexes increased in the order zinc-cadmium-mercury. The metal chtoro complexes were adsorbed on a o.oi M hydrochloric acid solution by the anion-exchange resin
Chromatogr. Rev.,
15 (1971) I 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
227
D o w e x - i (Ct- f o r m ) . Z i n c a n d c a d m i u m w e r e e l u t e d s e p a r a t e l y in t h a t o r d e r w i t h o . o i M h y d r o c h l o r i c acid. M e r c u r y w a s t h e n r e m o v e d w i t h o . o i M h y d r o c h l o r i c a c i d - o . I M t h i o u r e a s o l u t i o n , a n d a n a l y z e d b y p r e c i p i t a t i n g a n d w e i g h i n g t h e m e t a l a s t h e sulfide. Z i n c a n d c a d m i u m w e r e a n a l y z e d b y t h e t i t r i m e t r i c p r o c e d u r e o f MARTELL AND CALVIN 117 i n v o l v i n g t i t r a t i o n w i t h V e r s e n e a t p H i o w i t h E r i o c h r o m e B l a c k T a s a n indicator. RILEY AND TAYLORlls d e s c r i b e d t h e u t i l i t y o f c h e l a t i n g r e s i n s f o r t h e c o n c e n t r a t i o n o f t r a c e e l e m e n t s f r o m s e a w a t e r as w e l l a s t h e i r u s e i n c o n j u n c t i o n w i t h a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y . Two different resins, b o t h of the iminodiacetic acid t y p e , w e r e u s e d i n t h i s i n v e s t i g a t i o n , viz., (a) C h e l e x - I o o ( B i o - R a d L a b . , R i c h m o n d , Calif, 5 o - I o o m e s h ; t h i s is a p u r i f i e d f o r m o f D o w e x A - I r e s i n ) a n d (b) P e r m u t i t S lOO5 ( P e r m u t i t Co., lO-2O m e s h ) . T h e r e s u l t s o f e x p e r i m e n t s u s i n g C h e l e x - I o o a r e s h o w n in T a b l e 30. I n g e n e r a l , b o t h r e s i n s g a v e s i m i l a r r e s u l t s . T h e r e s i n s c a n o n l y b e u s e d f o r the uptake of trace elements from sea water and non-saline waters over a restricted r a n g e o f p H v a l u e s ( t h e l o w e r l i m i t is p H 5.0, b e l o w w h i c h u p t a k e falls off r a p i d l y ) .
TABLE 30 DATA
ON ADSORPTION AND ELUTION OF TRACE AND 20 In] OF ELUTING AGENT
ELEMENTS
FROM
SEA
WATER
WITH
CHELEX-IOO
(5O-lOO mesh)
Trace element
pH for adsorption
Retention (%)
Eluant
Total recovery (%)
A1 uminum Arsenic (AsO4~-) Barium Bismuth Cadnfium Caesium Cerium (Ce3+) Chromium (Cr 3+) Cobalt Copper Indium Lead Manganese Mercury (Hg ~+) Molybdenum (MOO42-) Nickel Phosphorus (PO43-) Rhenium (l~eO~-) Scandium Selenium (SeOa z-) Silver Thallium (TI +) Thorium Tin (Sn 4+) Tungsten (WO42-) Uranium (UO, ~+) Vanadium (VO ~-) Yttrium Zinc
7.6 7.6 5.o a 9.o a 7.6 7.6 9.o a 5.o a 7.6 7.6 9.o a 7.6 9.o a 7.6 5.o a 7,6 7.6 7.6 7.6 7.6 7.6 7.6 a 7.6 7.6 6.o a 7.6 6.o a 9.o a 7.6
o o 25 IOO IOO o IOO 25 IOO Ioo IOO IOO ioo 85 IOO ioo o 90 IOO o IOO 50 ioo 85 IOO o IOO IOO IOO
2N 2N 2N 2N 2N 2N 2N 2N 2N 2N 2N 4N 2N 4N 2N 2N zN 2N 2N 4N 4N 2N 2N
o o 25 IOO IOO o IOO lOb ioo IOO IOO IOO ioo 4 ob IOO ioo o 9° IOO o 9o b 5° IOO 6o b Ioo o IOO IOO IOO
HNO 3 HC104 HNO 3 HNOz tINO~ HC1 HNO 3 HNO 3 HNO~ HNO 3 HNO~ NH4OH HNO 3 NH4OH HNO 3 HNOz HNO 3 H~SO a HNO s NH4OH NH4OH HNO 3 ttNO 3
a Optimum p H value. b Maximum percentage removable from resin,
Chromatogr. Rev., 15 (1971) 195-238
228
L. FISHBEIN
TABLE 31 ANALYTICAL ME T HODS U S e D FOR T H E D E T E C T I O N OF METAL IONS ON D E A E - I O N - E X C H A N G E COLUMNS
Cation
Method
Al(III)a, Ga(III) a, Ni(II)a, V(IV) a, As(III)" Bi(III), Co(II)~, In(III), La(III), Lu(III), Sc(III), Sm(tlI), Th(tV), TI(I), Y(III), Zn(II) Ca(II) Cd(II), i g ( I I ) , Mn(II), Pb (II), Sr(II)
Titration with EDTA using Cu-PAN c as indicator Colorimetrically with ammonium molybdate Titration with EDTA using Xylenol Orange as indicator Titration with EDTA using b as indicator Titration with EDTA using Eriochrome Black T as indicator Colorimetrically as chromate Titration with EDTA using PAN as indicator CotorimetricaUy with NH4CNS , for traces Colorimetrically with phenyl fluorone Colonmetrically with dithizone Colorimetricalty with KCNS-SnCla Colorimetrically with a-nitroso-fl-naphthol Colorimetrically with KI after decomposition of tartaric acid Colorimetrically with SnCI3 Colorimetrically with dithiol Colorimetrically with hydrogen peroxide Colorimetrically with quercetin
Cr(III)a Cu(II) a Fe(III) a Ge(IV) Hg(II) Mo(VI), Re(VII) Pd(II)a Sb(III)~ Se(IV), Te(IV) Sn(IV) a, ~V(VI) a U(VI) a Zr(IV)
Determined after decomposition of thiocyanate with HNO v b 2_ttydroxy_I_(2_hydroxy_4_sulfo_I_naphthylazo)_3_naphthoic acid, c PAN = I-(2-pyridylazo)-2-naphthol. N i t r i c a c i d (2 N ) w a s s h o w n t o b e t h e m o s t g e n e r a l l y - u s e f u l r e a g e n t for r e m o v i n g c a t i o n i c s p e c i e s f r o m t h e r e s i n s a n d h a s t h e a d d e d a d v a n t a g e t h a t i t is e a s y t o r e m o v e s u b s e q u e n t l y . Silver, m e r c u r y , c h r o m i u m a n d tin were v e r y s t r o n g l y r e t a i n e d b y t h e resin and no reagent was found which would elute them completely. However, c o m p l e t e ( 9 9 - 1 o o % ) r e c o v e r i e s o f Bi, Cd, Co, Cu, I n , Mn, Mo, Ni, P t , R e , Sc, T h , W , V, Y, Z n a n d t h e r a r e - e a r t h e l e m e n t s c o u l d b e o b t a i n e d a t t h e i r n a t u r a l c o n c e n t r a tions of sea water when Chelex-Ioo was used.
TABLE 32 DEAE a AS
D I S T R I B U T I O N C O E F F I C I E N T S ( k a ) FOR M E R C U R Y ( I I ) ON AND pH
Serzes
pH
Ka values oM
A
B
F U N C T I O N S OF T H I O C Y A N A T E CO N CE N T RA T I O N
3.o 2. 3 x lO3 2.0 2.3 × lO3 i.o 2.2 × 103 i . o M HC104b I8 3.0 >1o 4 I.O M HC10, b 19
Total conch, of el- (M) o.oxoM
o,o5oM
o.zoM
o.5oM
LoM
2.8 × Io 3 2.5 x lO3 2.9 ~( 103 23 >1o 4 23
1.8 × lO3 2.1 × lO3 2. 5 X lO3 29
9-7 × 1°2 1.2 × lO3 1.2 × Io 3 18
ca.io
4.6 × 103
i.o i.I i.i 41 1.2 37
43 37 43 24 37
o. Io o.Io o. Io o.Io o.o
20
O.O
a D E A E in a thiocyanate form. b Separate experimental determination.
Chromatogr. Rev., 15 (1971) 195-238
30
a
22
× io z x lO2 × io ~ ×
lO 3
(KC1) (KC1-HC1) (KC1-HC1) (NaCI)
CHROMATOGRAPHIC
229
AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
A systematic study of the behavior of many metal ions on the weakly-basic cellulose exchanger D E A E in dilute thiocyanate media revealed that a few metal ions show any marked adsorption while mercury(II) exhibits pronounced adsorption 52. This preferential adsorption of mercury allows its rapid and highly selective separation from about forty metal ions, e.g., ca. o.I mg mercury(II) from milligram amounts of other metal ions and o.I-IO mg of mercury(II) from iron(III) in proportions of mercury(II) : iron(III) = ioo :I to i :8000 can be quantitatively separated on a column containing only I g DEAE. The analytical methods used to detect the various metalcation column effluents are shown in Table 31. Table32 lists the distribution coefficients (K,) for mercury(II) in thiocyanate media on D E A E (Serva; Gallard-Schlesinger Chem. Mfg. Co.) (thiocyanate form) as functions of the thiocyanate concentrations and of acidity. At each thiocyanate concentration, the Kd values for mercury(II) do not vary to any great extent with pH within the range 1-3 (Series A, Table 33). TABLE 33 EFFECT OF TOTAL CHLORIDE CONCENTRATIO.~'~ ON THE DISTRIBUTION COEFFICIENT (/~d) OF
MERCURY(II)ON DEAE pH Ks values oM
O.OlOM
o.o3oM
o.Io3~
o.3oM
o.5oM
I.oM
With DEAE-SCN--form a 3.O b ~ - 1 0 4 I.O c
>104
With DEAE~tl--forrnd 3.o b 9.1 × lO 2 I.I I.O e
×
~--'-104
lO 3
1.2
×
2 . 8 X 103 2 . 9 × 103
lO 3
3 . 0 X 102 3.3 X IO ~
1.2 × 10 ~ 1.2 X IO ~
34 28
6.8
×
lO 2
2.o
×
lO 3
87
27
6.3
x
lO 3
2.2 ×
IO ~
95
28
a The concentration of NH4CNS was kept at O.OLOM throughout. b KC1 was added to pH 3 acetate buffer solution to give the chloride concentration hsted. To KC1 solution of the listed chloride concentration, HCI solution of the same chloride concentration was added to give the chloride buffer solution of pH I. a No thiocyanate present. However, the concentration of thiocyanate or chloride exhibits a marked effect on Ka for mercury(II). The chloride dependence of Ka values for mercury(II) is also demonstrated in Table 32, where Ka values for mercury(II) on D E A E (thiocyanate form) are listed as a function of chloride concentration. Within the lower chlorideconcentration range ( < o . I o M) the Ka values for mercury(II) are well above 2.1o 3 but decrease sharply as the concentration of chloride increases. The form of D E A E is of particular importance in keeping the Ka values for mercury(II) sufficiently high. For comparison, Ka values for mercury(II) in chloride media on D E A E in the chloride form are also listed in Table 33. A radiochemical method for the determination of mercury, arsenic, bromine, antimony and selenium ions in neutron-irradiated biological material has been described by SAMSAHL119,120. The method is based on distillation of the neutron-irradiated sample (max. 200 mg of a dried, soft animal tissue is sealed in a small quartz tube and irradiated together with standards of As, Br, Hg, Sb and Se ions for I to 2 days with a thermal flux of 2. lO13 n/sq. cm/sec), followed after a 2-3 day decaying Chromatogr. Rev., 15 (1971) 195-238
230
L. FISHBEIN
p e r i o d b y a u t o m a t e d ion-exchange a n d e x t r a c t i o n c h r o m a t o g r a p h i c m e t h o d s for s e p a r a t i n g t h e trace elements into twelve groups suitable for g a m m a s p e c t r o m e t r i c m e a s u r e m e n t s . Figs. 12 a n d 13 i l l u s t r a t e t h e d i s t i l l a t i o n a p p a r a t u s a n d scheme of a n i o n - e x c h a n g e s e p a r a t i o n system, respectively. T h e radionuclides 76As (0.55 meV), K
N
A
D
I
N!
.
ii !
Fig. i2. Distillation apparatus. (A) Distillation flask, i5-ml volume, I75 mm long, B 14 joint; (B) receiver flask, 3o-ml volume, i2o mm long, B 14 joints; (C) reflux condenser, I5o mm long, B Io and B 14 joints; (D) and (E) traps for 2 and 5 ml, respectively, B Io joints; (F) and (H) funnels, io x ioo mm; (G) and (I) stopcocks; (J) borosilicate tube, 45 × 380 mm; (K) arrow points to water suction pump; (L) bunsen burner; (M) compressed air. Fig. I3. Scheme of anion-exchange separation system for separation of Sb, As, Hg and Se. (A) Piston barrel, 28 × 15o mm; (13) and (C) piston barrels, 20 × 15o mm; (D) 4 × 5° mm, Dowex 2 (HSO4-, 2oo-4oomesh); (E) 7 × 5o ram, Dowex 2 (CI-, 2oo-4o0 mesh); (F) 18 × 5° mm, Dowex 2 (Br-, CI-, 2o0-400 mesh) ; (G) mixing coils, 5 turns, 15 mm outer diameter; (H) piston with rubber stopper; (I) perspex plate, 15 mm thick. ~°aHg (o.28 meV), l ~ S b (o.56 meV) a n d 73Se (o.14 meV) were s e p a r a t e d in the different groups as follows: G r o u p I : N a O H adsorption--S~Br G r o u p 2: D o w e x 2 (sulfate)--197Hg, ~°SHg G r o u p 3: D o w e x 2 (chloride)--12~Sb, 1~4Sb G r o u p 4: D o w e x 2 (bromide, chloride)m~eAs, 75Se. T h e s i m u l t a n e o u s d e t e r m i n a t i o n of Cu, Zn, Cd a n d H g ions with high s e n s i t i v i t y utilizing n e u t r o n a c t i v a t i o n analysis was described m . A f t e r i r r a d i a t i o n , t h e samples were d i g e s t e d a n d an initial s e p a r a t i o n of t h e four e l e m e n t a l ions m a d e b y m e a n s of a n ion-exchange resin. T h e e l e m e n t a l ions in t h e s e p a r a t i o n fractions were t h e n t r e a t e d to yield a r a d i o c h e m i c a l p u r i t y , p r e c i p i t a t e d a n d their activities measured. TORIBARA AND SHIELDS1~ described a scheme for t h e analysis of s u b m i c r o g r a m a m o u n t s of m e r c u r y ions in tissues involving (I) t h e s e p a r a t i o n of m e r c u r y ion from tissue b y a cold digestion in hydrochloric a c i d - s o d i u m n i t r a t e , (2) collection on an a n i o n - e x c h a n g e column, (3) elution w i t h t h i o u r e a solution, (4) collection in c a d m i u m sulfide, v o l a t i l i z a t i o n a t 55 o°, (5) d e t e r m i n a t i o n p h o t o m e t r i c a l l y w i t h a General E l e c t l i c germicidal U V - i n t e n s i t y meter. The ion-exchange s e p a r a t i o n was accomplished using A m b e r l i t e I R A - 4 o o (5O-lOO mesh) w i t h elution w i t h o . o i M thiourea. T h e s e p a r a t i o n a n d d e t e c t i o n of p i c o g r a m q u a n t i t i e s of c a t i o n s in cells a n d tis-
Chromatog~'. Rev., 15 (1971) 195-238
CHROMATOGRAPHICAND BIOLOGICALASPECTS OF INORGANICMERCURY
231
sues was described b y KLIMES AND BETUSOVAlz3. The sample is first mineralized with HC1-KCI04 and heated, then the cations separated on a strongly-basic ion-exchanger (Wofatit 1-15o, Veb, Farbenfabrik, Wolfen, G.F.R.) or by electrolysis and identified b y means of classic color reactions in a single grain of silica gel support. The procedure is exemplified by the detection of (a) about IO pg mercury cation in o.I-mg samples of kidney and in I - m g samples of adrenal cortex of a rat previously treated with Agronal and (b) the detection of about 7 Pg iron cation in a single cell of A l l i u m cepa. Fig. 14 illustrates the apparatus for the picogram-detection of cations in cells and tissues. 20
40
100
50
,oo~ Glass
(c)
10 \
(a)
(b)
1o0 • \7--
(d)
J "";-"
/Sealed
I
,,,
w~th
lacquer
Fig. 14. Apparatus for picogram-detection of cations in cells and tissues. (a) Pico test tube; (b) mineralization flask; (e) pico electrode; (d) pico electrode pair. All dimensions are in ram. KOPP AND KEENANT M described an ion-exchange method for the complete separation of submicrogram and larger quantities of mercury ions from o.3--o.5 mg copper, lead, cadmium, thallium, zinc and nickel cations. The mercury ions in a digest of a biological sample, e.g., urine, are first adsorbed on a IO × 30o m m column containing a strongly-basic anionic resin (Dowex I-XS, C1--form, IOO-2OO mesh) and then eluted quantitatively with o.002 M thiourea in o.oi M hydrochloric acid. The eluted mercury was then complexed directly with a standard dithizone reagent and read spectrophotometrically at 49 ° m/z, with a sensitivity of 0.3 ~ug. Experiments with *°3Hg and with the stable isotope of mercury in urine yielded recoveries exceeding 92%. The exact chemical composition of the excretory products of mercury in the urine is unknown. There is evidence that organomercurials which have strong C - H g bonds and do not ionize are excreted in unchanged form 125. KURODA et al. 12~ described the separation of thirty-one pairs of metal ions (including C u ( I I ) - H g ( I I ) and Sn(IV)-Hg(II)) using columns of Amberlite CG4B and in the above cases elution with I M hydrochloric acid and 2 M perchloric acid. The chromatographic behavior of Zn, Cd and Hg ions on columns of natural cellulose and substituted celluloses was studied by using SSZn, 1°9Cd and ~°3Hg radio tracers 12~. Traces of Zn and Cd ions were strongly retained b y the functional groups attached on the substituted celluloses, while mercury- ion was not retained to any extent. Amounts of zinc, cadmium and other metal ions were separated from 3 g of mercury on cellulose phosphate in ether.
Chromatogr.. Rev., 15 (1971) 195-238
232
L, FISHBEIN
8. ANALYSIS OF MAMMALIANDISTRIBUTION, TRANSPORT AND EXCRETION OF INORGANIC MERCURY T h e d i s t r i b u t i o n of m e r c u r y a m o n g blood fractions a n d s e r u m p r o t e i n s in t h e r a t following i.v. a d m i n i s t r a t i o n s of o.12 m g ] k g a n d 1.2 m g ] k g of i s o t o p i c a l l y t a g g e d ~°3HgC12 was s t u d i e d b y CEMBER et al. 1~8. T h e m o s t i n t e r e s t i n g o b s e r v a t i o n in this s t u d y was t h a t m o s t o f t h e s e r u m - p r o t e i n - b o u n d m e r c u r y was i n i t i a l l y associated w i t h t h e a l p h a globulins. A t t h e lower dose level, this initial f r a c t i o n a l d i s t r i b u t i o n r e m a i n e d a b o u t t h e same t h r o u g h o u t t h e o b s e r v a t i o n period (9 6 h). F o r tile higher dose level, t h e r e was a shift w i t h increasing t i m e in t h e r e l a t i v e c o n c e n t r a t i o n o f merc u r y from a l p h a globulins to a l b u m i n . Figs. 15 a n d 16 i l l u s t r a t e densitorneter tracings a n d r a d i o c h r o m a t o g r a m s showing in vivo d i s t r i b u t i o n of m e r c u r y in serum p r o t e i n s 3/4 h after injection of 200 # g
Pro tel ps densitometer
tracmg
I I I I J I J I
tl~r~
i
Merc ur y ra d ~ochromatogram
Fig. 15. Densitometer tracing and radiochromatogram showing in vivo distribution of mercury in serum proteins a], h after injection of 2oo/~g of Hg into a rat.
Protelns densitorneter
tracmg
Y Mercury rad~ochromotograrn
Fig. i6. Densitometer tracing and radiochromatogram showing in vitro distribution of mercury in serum proteins 3/4 h after inoculation of whole blood with mercury, at a concentration of 2/~g/ml. Chromatogr. Rev., 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
233
of mercury into a rat, and in vitro distribution of mercury in serum proteins 3/, h after inoculation of whole blood with mercury, at a concentration of 2/~g/ml, respectively. For the above determinations, the serum proteins were separated for mercury analysis by electrophoresis on a cellulose acetate strip in barbital buffer at pH 8.6 for I h at 300 V. After staining with Ponceau S stain, two strips were cleared for graphical protein determination with a densitometer and for graphical mercury determination with a 4~ windowless radiochromatogram scanner. The excretion of volatile mercury (in part from the lungs and in part from body surface) by rats injected with ionic *°3Hg, and the elaboration of the factors that influence the rate of such excretion, including the dose of mercury, the time after inj ection and the intensity of respiration, were described by CLARKSONANDROTHSTEIN39. Although the chemical form of the volatile mercury was thought to be probably metallic-mercury vapor, it was suggested that it could be either one of the volatile organic compounds of mercury such as dimethylmercury. It is possible that enzymic systems in the animal tissues are able to methylate inorganic mercury in an analogous manner as tellurium is converted to methyl telluride and excreted in the breath TM. A more probable alternative suggested by CLARKSONAND ROTHSTEIN is the reduction of the mercury and thence volatilization from the animal as inorganic vapor. The mercury-blood interaction and mercury uptake by the brain after vapor (*°3Hg) exposure has been studied by MAGOS3~. On the wet basis the mercury uptake by the brains of mice exposed to o.oo3-9.9oo #g mercury/1 air for 4 h was little more than two-thirds of the total uptake (inhalation plus adsorption by fur). This high quotient suggests that the maior part of mercury is equilibrated in a highly diffusible form between the blood and the tissues including the brain. The conversion of elemental mercury to mercuric ion by blood exposed in vitro to mercury is a slow process compared with the circulation time from the lung to the brain. It was assumed by MAGOS that the elemental mercury is the highly diffusible form which equilibrates between the blood and the tissues. During the 8-day post-exposure period the relative distribution of mercury was modified b y the differences in organ clearance (8 days after exposure the brain contained nearly as much mercury as the kidneys). Fig. 17 illustrates the diagram of the experimental design for labeling mercury vapor with ~°3Hg based on the Hg-e°3Hg exchange reaction. Labeled mercury in samples of blood, plasma, or saline as well as that adsorbed b y the Hopcalite adsorbers in the generating and exposure apparatus (Fig. 17) was measured in a scintillation detector (Ekco Electronics, Ngg4B ) and scaler (N6IoA) having a counting efficiency for 2°~Hg of 40%. The role of biotransformation in rats was studied by NORSETH AND CLARKSONTM via an examination of the intestinal transport of Z°~Hg-labeled methylmercuric chloride. Inorganic mercury in the presence of methytmercuric salts in tissue samples, fluids and extracts was analyzed by the isotope-exchange method 43. The chemical form of mercury in bile was determined by TLC on Eastman Chromagram 6o61 Silica Gel and electrophoresis on Beckman paper 319328 or 320046. The developing solvents for TLC were n-propanol-water (7° :3o) and ethanol-34% ammonium hydroxide (70:30). Diethyl barbiturate-sodium barbiturate buffer, pH 8.6, was used for etectrophoresis (14 h at 5 mA constant current). Chromatograms were excised for counting or scanned by a gas-flow counter and bile was analyzed by column chromatography of Sephadex G-Ioo to separate diffusible and protein-bound mercury. A Sephadex 15 × 30 column Chromatogr. Rev., 15 (1971) 195-238
234
L. FISHBEIN Water"
pump
NVI
HA2
Me rcLiry-~- f r e e
ale
Fig. 17. Diagram of the experimental design for labeling mercury vapor by exchange reaction (not to scale). The direction of the flow is from left to right. Connection A is used for inhalation experiments and connection B for exposing blood to mercury vapor. The other symbols are as follows: NV1 and NV2, needle valves; Pal, R2 and R3, rotameters; MVGS, mercury-vapor generating system; HA1 and HA2, Hopcalite adsorbers, type 1 and type 2; ER, exchange reagent; ICh, inhalation chamber; BI, impinger containing the blood; T, thermometer; CJ, clamp; PA, permanganate adsorber. was used with sodium-potassium phosphate buffer o.I M at p H 8.0 containing I.O M sodium chloride and 0.o2% sodium azide as eluting and supporting solution. The detection of m e r c u r y in biological material (urine, internal organs) by means of TLC was described b y W y s o c k a 13~. The biological material was first mineralized and the m e r c u r y ion then converted into a dithizonate. Chromatographic analysis was performed on Silica Gel G with n-propanol as the developing agent. After 3 h the separation of the excess dithizone (RF 0.66) from the H g dithizonate (RF 0.82) was completed. A m o u n t s as small as 0.5 # g Hg in large amounts of biological material can be detected b y this procedure.
9. MISCELLANEOUSANALYTICALMETHODOLOGY Methods of analysis for total mercury in biological materials are extensive and have included: neutron activation 133-136, atomic and flameless atomic adsorption 13~14~, isotope exchange45,14s, 144, mercury vapor meter adsorption 145-15°, polarographic 138,151, amperometric15~,lS3, microelectrolysis15,, colorimetric155-1~s, spark-source mass spectrometry 15s, UV p h o t o m e t r y ls°-16~, and thermometric titration TM. The selective determination of inorganic mercury in the presence of organomercurial compounds in biological materials is of noteworthy importance and has recently been achieved b y CLARKSONAND GREENWOOD165 and GAGE AND WARREN150. The principle of the former m e t h o d is the selective reduction of inorganic m e r c u r y
Chromatogr. Rev,, 15 (1971) 195-238
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURV
235
b y SnCI2 (in t h e p r e s e n c e o f o r g a n o m e r c u r i a l c o m p o u n d s l a b e l e d w i t h t h e *°3Hg isotope) to e l e m e n t a l v a p o r w h i c h is t h e n s w e p t f r o m t h e s a m p l e b y a n air s t r e a m coll e c t e d o n a specific a d s o r b e n t , H o p c a l i t e , w h e r e t h e r a d i o a c t i v i t y is d e t e r m i n e d b y y-scintillation counting. T h e m e t h o d o f MAGOS AND CERNIK 162 in w h i c h m e r c u r y in u r i n e is d e t e r m i n e d by aspirating the vapor through an UV absorptiometer after reduction with stannous c h l o r i d e w a s m o d i f i e d b y GAGE AND WARREN T M b y m a k i n g use o f t h e v a r y i n g l a b i l i t y of o r g a n i c m a t e r i a l in t h e p r e s e n c e o f a c i d c y s t e i n e p e r m i t t i n g t h e d e t e r m i n a t i o n of m e r c u r y a n d o r g a n o m e r c u r i a l s in b i o l o g i c a l s a m p l e s s u c h as urine, feces, b l o o d a n d k i d n e y tissue.
I0. SUMMARY T h e m a j o r a r e a s o f u t i l i t y , a n d t h e ecological a n d b i o l o g i c a l significance o f ino r g a n i c m e r c u r y h a v e b e e n r e v i e w e d a l o n g w i t h s a l i e n t p a p e r , t h i n - l a y e r , gas, c o l u m n a n d i o n - e x c h a n g e c h r o m a t o g r a p h i c t e c h n i q u e s for t h e s e p a r a t i o n a n d i d e n t i f i c a t i o n o f i n o r g a n i c m e r c u r y f r o m e n v i r o n m e n t a l a n d f o r e n s i c sources.
REFERENCES I L. FISHBEIN, Chromatogr. Rev., 13 (197 o) 83. 2 N. NELSON, T. C. BYERLY, A. C. KOLBYE, JR., L. T. KURLAND, R. E. SHAPIRO, W. I-I. STICKEL,
3 4 5 6 7 8 9 lO
ii 12 13 14 15 16 17 18 19 20 2I 22
23 24 25
26 27 28
J. E. THOMPSON, L. A. VAN DEN BERG AND A. WEISSLER, Environ. Res., 4 (1971) I. L. J. GOLDWATER, Sci. Amer., 224 (1971) I5. H. U. MALLING, J. S. WASSOM AND S. S. EPSTEIN, Environ. Murat. Soc. Newsl., 3 (197 o) 7. N. FIMREITE, Environ. Potlut., I (197 o) 119. A. G. JOm~ELS AND T. WESTERMARK,in M. W. MILLER AND G. G. BERG (Editors), Chemical Fallout, Thomas, Springfield, Ilk, 1969, p. 221. C. V. KING, Ann. N . Y . Acad. Sci., 65 (1957) 36o. J. L. WEBB, Enzyme and i~ietabolic Inh~b*tors, Vol. II, Academic Press, New York, 1966, p. 73 o. H. A. SHOEMAKER,Ann. N . Y . Acad. Sci., 65 (1957) 5o4 . H. PASSOW, A. ROTHSTEIN AND T. W'. CLARKS0N, Pharmacol. Rev., 13 (1961) 185. P. L. BIDSTRUP, Toxicity of Mercury and Its Compounds, Elsevier, Amsterdam, 1964, p. 28, Int. Comm. Rep. on maximum allowable concentrations of mercury compounds, Arch. Environ. Health, 19 (1969) 891 . A. SWENSSON, OIKOS (Suppl.), 9 (1967) 27. M. C. BATTIGELLI, J. Occup. Med., 2 (196o) 394. S. C. HARVEY, in L. S. GOODMANAND A. GILMAN (Editors), Pharmacological Basis of Therapeut,cs, 3rd Ed., MacMillan, New York, 1967, p. 98. T. SDFUKI, in M. W. MILLER AND G. G. BERG (Editors), Chemical Fallout, Thomas, Springfield, IlL, 1969, p. 245. N. GRANT, Environment. I i (1969) 18. J. MOUTSCHEN-DAHMENAND N. DEGRAEVE, Experientia, 21 (1965) 200. S. LEVAN, Nature, 156 (1945) 751. M. UMEDA, K. SAITO, K. HIROSE AND M. SATO, Jap. J. Med., 39 (1969) 47. R. H. JE~VSEN AND N. DAVIDSON, Bzopolymers, 4 (1966) 17. H. DAUNE, C. A. DEKKER AND H. K. SEHACHMAN, Biopolymers, 4 (1966) 51. S. KATZ, Biochim. Biophys. Acta, 68 (1968) 24o. D. W. GRUENWEDEL AND N. DAVIDSON, J . Mol. Biol., 21 (1966) 129. R. K. ZAHN, E. TIESLER, H. G. OcHs, B. HEKKE, W. HANSKE AND W. FORSTER, Biochem. Z., 344 (1966) 26. S. KATZ, Nature, 194 (1962) 569. S. KATZ AND V. SANTILL1, Biochim. Biophys. Aeta, 55 (I962) 621. H. DRUCKREY, H. HAMPERL AND D. SCHMAHL,Z. Krebsforsch., 61 (1957) 511. Chromatogr. Rev., 15 (1971) 195-238
236
L. FISHBEIN
29 3° 31 32 33
M. MUROZUMI, Electrochem. Technol., 5 (1967) 236. S. JENSEN AND A. JERNEL6V, N6rdforsk. Biocidinformation, No. IO (1967) 4. S. JENSEN AND A. JERNEL6V, N~rdforsk. B,ocidinformation, No. 14 (1968) 5. J. M. Wool), F. S. KENNEDY AND C. G. ROSEN, Nature, 220 (1968) 173. A. JERNEL6V, in M. W'. MILLER AND O. G. BERG (Editors), Chemical Fallout, T h o m a s , Springfield, Ill., 1969, p. 68. D. K. H. LEE, P e r s o n a l c o m m u n i c a t i o n . J- C. GAGE, Br. J. Ind. Med., 18 (1961) 287. L. MAGUS, Environ. Res., I (1967) 323 . M. 13ERLIN, L+ G. JERKSELL AND H. YON UBISCH, Arch. Env,ron. Health, 12 (1966) 33M. H. 13ERLIN, G. F. NORDBERG AND V. SERENIUS, Arch. Environ. Health, 18 (1969) 42. T. W . CLARKSON AND A. ROTHSTEIN, Health Phys., io (1964) 1115. M. 13ERLIN AND S. ULLBERG, Arch. Environ. Health, 6 (1963) 589. O. I~'ITZHUGH, A. NELSON, E. P. LANG AND F. KUNZE, J. Ind. Hyg. Occup. Med., 2 (195 o) 443. T. NORSETH, Studies on the Biotransformat~on of Methylmercuvy Salts *n the Rat, P h . D . Thesis, Univ. o f Rochester, Rochester, N.Y., 1969. T. NORSETH AND T. W . CLARKSON, Biochem. Pharmacol, 19 (197 o) 2775. T. •ORSETH AND T. W. CLARKSON, Arch. Environ. Health, 21 (197 o) 717 . T. W. CLARKSON, in M. W . MILLER AND G. G. 13ERG (Editors), Chemical Fallout, T h o m a s , Springfield, II1., 1969, p. 274. M. LEDERER AND C. MAJANI, Chromatogr. Rev., 12 (197 o) 239. H. ROMANOWSKI, K. IZDEESKA AND Z. POZECZlEK, Farm. Pol., IO (1961) 452; C.A., 57 (1962) IOI45L P. 13. JANARDHAN AND A. PAUL, Indzan J. Chem., 7 (1969) 66. E. 13. SANDELL, Colorimetric Determination of Traces of Metals, 3rd Ed., I n t e r s c i e n c e N e w York, 1965, p. 621. Y. KURAYUKI AND K. KUSAMOTO, Bunseki Kagaku, 16 (1967) 815. W . ]3. LINK, K. S. KEINE, JR., J. H. JONES AND P. WATTLINGTON, J. ASS. Off'C+Agr. Chem., 47 (1964) 391. R. KURODA, T. KIRIYAMA AND K. ISHtDA, Anal. Chim. Acta, 4 ° (1968) 305. C. L. KAO, M. SIN AND Y. C. SING, Hua Hsueh Tung Pao, i i (1963) 46; C.A., 6o (1964) II369. D. N. TRIPATHI AND S. N. TE~*ARI, J. Prakt. Chem., 9 (1959) I. H. NAGAI, N*ppon Kagaku Zasshi, 80 (1959) 617; C.A., 54 (I96o) 19297. R. INDOVINA AND B. M. RICOTTA, Ann. Chim. (Roma), 45 (1955) 241. F. H. BURSTALL, G. R. DAVIES, R. P. LINSTEAD AND R. A. WELLS, J. Chem. Soc., (I95 o) 516. R. INDOVlNA, E. DELEo AND ]3. M. RICOTTA, Ann. Ch*m. (Roma), 45 (1955) 244. A. R. V. MURTHY AND V. A. NARAYAN, Naturwissensehaften, 42 (1955) 439M. 13. CELAP AND Z. RADIKOVJEVIC, Bull. Soc. Chim. (Beograd), 23-24 (1958-I959) i ; Anal. Abstr, (196o) 3115 . V. K . M. RAO, J. Sci. Ind. Res. (India), I g B (196o) 171. V. K. M. RAo, J. Sc,. Ind. Res. (India), l i b (1952) 277. S. kNL TEWARI, Z. Anal. Chem., 141 (1954) 4Ol. F. H. POLLARD, J. F. "vV. McOMBIE AND G. NICI~LESS, J. Chromatogr., 2 (1959) 284. H. S. R. BARRETO, R. C. R. BARRETO AND ][. P. PINTO, J. Chromatogr., 5 (1961) 5A. R. V. MURTHY, V. A. NARAYAN AND M. R. A. RAO, Curr, Sci., 24 (1955) 158. J. F. W . MCOMBIE, F. H. POLLARD AND I. I. M. ELBEIH, DiscusS. Faraday Soc., 7 (1949) 183. E. ]3LASIUS AND W. G6TTLING, Z. Anal. Chem., 162 (1958) 423 . i~. H. POLLARD, J. F. \ ¥ . McOMBIE AND I. I. M. ELBEIH, Nature, 163 (1949) 292. J. HALMEROSKI AND F. SUNDHOLM, Suom. Kemistil., 1336 (I963) 63. R. P. 13HATNAGER, K. D. SHARMA AND R. P. VARSHNEY, Ind,an J. Chem., 4 (1966) 47H. NAGAI AND T. DEGUCHI, Nippon Kagahu Zasshi, 86 (1965) 516. K. FUKUDA AND A. MIZUIKE, Bunsehi Kagahu, 17 (1968) 65. T. AKIYAMA AND M. SHIOKAWA, Kyoto Yahka Daigahu Guhuho, 12 (1964) 51. M. QURESHI AND M. A. KHAN, Z. Anal. Chem., 232 (1957) 194. H. NAGAI, Kumamoto J. Sci., Ser. A, 6 (1964) 138. N. SUGAWARA, I~agah~ Keisatsu Kenkyusho Hokohu, 2o (1967) 176. G. ALBERTI, Chromatogr. Rev., 8 (I966) 246. M. L. THAKUR, Sep. Sci., 5 (197 o) 645. H. TANAKA, Y. SUGIURA AND A. YOKOVAMA, Bunsek* Kagahu, 17 (1968) 1424. Y. KURAYUKI AND K. KUSAMOTO, fap. Anal., 16 (1967) 815. T. WESTERMARK, D. HAGMAN, A. VESTERMARIC AND K. LJUNGGREN, Nord. Hyg. Tidskr., 5 ° (1969) 79. T. FUKUDA, K. MIYAKAWA AND Z. UEKI, Eisei Kagaku, 13 (1967) 342. G. CATONI, Attar. Acad. Sci. (Torino), 91 (I956-I957) 23. H. SEILER AND M. SEILER, Helv. Chim. Acta, 43 (196o) 1939.
34 35 36 37 38 39 4° 41 42 43 44 45 46 47 48 49 5° 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 7° 71 72 73 74 75 76 77 78 79 80 81 82 83
84 85
Chromatogr. Rev., 15 ( I 9 7 i ) ~ I 9 5 - 2 3 8
CHROMATOGRAPHIC AND BIOLOGICAL ASPECTS OF INORGANIC MERCURY
237
H. SEILER AND M. SEILER, Helv. Chim. Aeta, 44 (1961) 1753. E. PFEIL, A. FRIEDRICH AND T. WACHSMANN, Z. Anal. Chem., 158 (1957) 249M. LESIGANG-BUCHTELA, Mihroch~m. Acta, (1966) 4o8. K . H. KOENIG AND K. DEMEL, J. Chromatogr., 39 (1969) IOI. I4~. M. OL'SHANOVA AND L. A. KUNITSKAYA, IZV. Vyssh. Uchebn. Zaved. Khim. Teknol., II (1968) 637; C.A., 7 ° (1969) 4367of. 91 A. E. SHERWOOD, Metallurgia, 8o (1969) 2o9. 92 S. TAKITANI, M. SUZUKI, M. YOSHIMURA, S. SATO AND M. SKEIYA, Eisei Kagaku, 14 (1968) 32493 M. HRANISAVLJEVIC, I. PEJKOVIC-TADIC, K. JAKOVJEVIC AND J, MILIJKOVIC-STOJANOVIK, Glas. Hem. Drus. Beogr., 29 (1964) 115; C.A., 64 (1966) 16599. 94 B. UNTERHALT, Deut. Apoth. Z., lO9 (1969) II43. 95 P- KuNzI, J. BA/JMLER AND J. I. OBERSTEG, Z. Gerichtl. Meal., 52 (1962) 605. 96 N. SUGIV,rARA, Yamaguch, Igaku, 15 (1966) 3o6. 97 K. N. JOHRI, H. C. MEHRA AND N. I42. KAUSHIK, Chromatograph*a, 3 (197 °) 347. 98 J. w . FAIRBARN AND S. EL MASRY, J. Pharm. Pharmacol., 19 (1967) 935. 99 J. C. MORRISON AND J. G. CHATTEN, J. Pharm. Pharmacol., 17 (1965) 655. IOO H. WEISZ, Microanalysis by R,ng Oven Technique, P e r g a m o n Press, L o n d o n , I961, p. 123. i o i H. BURNE, Analyst (London), 76 (1951) 22o. lO2 K. N. JOHRI, N. K. KAUSHIK AND K. SINGIt, Mikrochim. Acta, (1969) 737. lO 3 M. SEILER, Helv. Chim. Acta, 53 (197 °) 1893. lO 4 F. W. K o s s AND G. BEISENHERZ, Radiochim. Acta, 3 (1964) 22o. lO 5 Z. MASOOMI AND D. T. HAWORTH, J. Chromatogr., 48 (197 o) 581. lO6 V. D. CANIC AND S. M. PETROVIC, Z. Anal. Chem., 211 (1965) 251. lO 7 V. D. CANIC, S. M. PETROVIC AND A. K. BEN, Z. Anal. Chem., 213 (1965) 251. lO8 T, DEGUCHI, Bunsekz Kagaku, 15 (1966) 352. lO9 M. H . HASHMI, M. A. SHADID, A. A. AYAZ, F. I{. CHUGHTAI, N. ['IASSAN AND A. S. ADIL, Anal. Chem., 38 (1966) 1554 . IiO P. W. WEST AND A. K. MUKHERJI, Anal. Chem., 31 (1959) 947. I I I H. J. SENF, J. Chromatogr., 21 (1966) 363 . 112 I~. TAKESHITA, H . AKAGI, M. FUJITA AND Y. SAKAGAMI, J. Chromatogr., 51 (197 o) 283. 113 T. TADMOR, J. Gas Chromatogr., 2 (1964) 385 . 114 A. A. DUSWALT, Diss. Abstr., 20 (1959) 52. 115 W. J. BIERMAN AND H. GESSLER, Anal. Chem., 32 (196o) 1525. 116 E. W. BERG AND J. T. TRUEMPER, Anal. Chem., 3 ° (1958) 1827. 117 A. E. MARTELL AND M. CALVIN, Chemistry of the Metal Chelate Compounds, P r e n t i c e - H a l l , N e w York, 1963, p . 487 . 118 J. P. RILEY AND D. TAYLOR, Anal. Chim. Acta, 4 ° (1968) 479I 19 K. SAMSAHL, Anal. Chem., 39 (1967) 148o. 12o I4~. SAMSAHL, Ab. Atomenergi Stockholm, AE-82 (1962). 121 M. D. LIVINGSTON, H. SMITH AND ~N-. STOJANOVIC, Talanta, 14 (1967) 5o5 . 122 T. Y TORIBARA AND C. P. SHIELDS, Amer. Ind. Hyg. Ass. J., 29 (1968) 87. 123 I. KLIMES AND M. BETUSOVA, Anal. Biochem., 23 (1968) lO2. 124 J. F. Ko~P AND R. G. KEENAN, Amer. Ind. Hyg. Ass. J., 24 (1963) i. 125 H. A. SHOEMAKER, Ann. N . Y . Acad. Sc,., 65 (1957) 504 • 126 R. KURODA, K. ISHIDA AND T. KIRIYAMA, Anal. Chem., 4 ° (1968) 15o6. 127 tt. A. A. MUZARELL1, Talanta, 13 (1966) 8o9. 128 H CEMBER, P. GALLAGHER AND A. FAULKNER, Amer. Ind. Hyg. Ass. J., 29 (1968) 233. 129 T. CLARKSON AND A. ROTHSTEIN, Health Phys., IO (1964) 1115. 13o F. HOFMEISTER, Arch. Exp. Pathol. Pharmakol., 33 (1964) 198. 131 T. NORSETH AND T. W . CLARKSON, Arch. Environ. Health, 22 (1971) 568. 132 B. WYSOCKA, D~s$. Pharm., 17 (1965) 99. 133 B. UNDERDAL, Nord. Vet. Med., 20 (1968) 9. 134 B. SJ•STRAND, Anal. Chem., 36 (1964) 814. I35 T. WESTERMARK AND B. SJOSTRAND, [nt. J. Appl. Rad. tsot., 9 (196o) i. 136 0 . JOHANSEN AND E. GTEINNES, Int. J. Appl. Rad. Isot., 20 (1969) 751. 137 W . IR. MATCH AND ~¥. L. OTT, Anal. Chem., 4 ° (I968) 2085. 138 E. G. PAPPAS AND L. A. RUSENBERG, J. Ass. O~c. Agr. Chem., 49 (1966) 792. 139 t3. 13. MESMAN, 13. S. SMITH AND J. O. PIERCE, II, Amer. Ind. Hyg. Ass. J . , 3I (197 o) 7Ol. 14o A. D. RATHJE, Amer. Ind. Hyg. Ass. J., 3 ° (1969) 126. 141 M. J. FISHMAN, Anal. Chem., 42 (I97 o) 1462. 142 J. F. UTHE, F. A. J. ARMSTRONG AND M. P. STAINTON, J. Fish. Res. Bd. Can., 27 (197 o) 805. 143 T. W. CLARKSON AND M. R. GREENWOOD, Talanta, 15 (1968) 547. 144 L. MAGOS, Br. J. Ind. Med., 23 (1966) 23o. 86 87 88 89 9°
Chromatogr. Rev., 15 (1971) x 9 5 - 2 3 8
238
L. FISHBEIN
145 M. B. JACOB$, S. YAMAGUCHI, L. J. GOLDWATER AND H. GILBERT, Amer. Ind. Hyg. Ass. J . , 21 (196o) 475. 146 M. M. SCHACHTER,.[. ASs. Q~c. Anal. Chem., 49 (1966) 778. 147 W. W. VAUGHN, U.S. Geol. Sure. Circ., 54 ° (1967) 22. I48 T. Y. TORBARA AND C. P. SHIELDS, Amer. Ind. Hyg. Ass. J., 29 (1968) 87. 149 G. LINDSTEDT, Analyst, 95 (197 o) 264. 15o J. C. GAGE AND J. M. WARREN, Amer. Occup. Hyg., 13 (197 o) 115. 151 I. M. WEINER AND O. H. MULLER, J. Pharmacol. Exp. Ther., 113 (1955) 24. 152 J. T. STOCK (Editor), Amperometric T, tratzons, tnterscIence, N e w York, 1965. 153 B. C. SOUTHWORT•, J. H. HODECKER AND K. D. FLEISCHER, Anal. Chem., 3 ° (1958) 1152. 154 D. PAVLOVlE AND S. ASPERGER, Anal. Chem., 31 (1959) 939. 155 D. M. GOLDBERG AND A. D. CLARKE, J. Clin. Pathol., 23 (197 o) 178. 156 V. L. MILLER AND V. S~¥ANBERG, JR., Anal. Chem., 29 (1957) 391. 157 W*. H. GUTENMANN AND D. J. LISK, J. Agr. Food Chem., 8 (196o) 306. 158 E. P. LAUG AND K. W . NELSON, J. Ass. Off. Agr. Chem., 25 (I942) 3o9. 169 S. S. C. TONG, V~r. H. GUTENMANN AND D. J. LISK, Anal. Chem., 41 (1969) 1872. 16o F. N. KUDSK, Acta Pharmaeol. Tox*eol., 23 (1965) 263. 161 F. N. KODSK, Acta Pharmacol. Toxicol., 27 (1969) 149. 162 L. MAGOS AND A. A. CERNIK, Br. J. Ind. Med., 26 (1969) 144. 163 M. B. JACOBS, Amer. Ind. Hyg. Ass. J., 21 (196o) 475. 164 K. C. BURTON AND H. M. N. H. IRVING, Anal. Chim. Acta, 52 (197 o) 491. 165 T. W. CLARKSON AND M. R. GREENWOOD, Anat. Biochem., 37 (197 o) 236.
Chromatogr. Rev., 15 (1971) 1 9 5 - 2 3 8