Studies with a multichannel flame spectrometer

ANALYTICAL

BIOCHEMISTRY

Studies

with

21, 9-21 (1967)

a Multichannel

Flame Spectrometer

III. Simultaneous Analysis of Magnesium, Calcium, Copper, Manganese, and Chromium: A Method Devoid of Cation and Anion Interferences

CHUZO

IIDA’

AND

KEIICHIRO

FUWA’

Biophysics Research Laboratory, Department of Biological Chemistry, Harvard Medical School, and Division of Medical Biology, Department of Medicine. Peter Bent Brigham Hospital, Boston, Massachusetts 02116 Received January

23, 1967

The design and operation of a multichannel flame spectrometer and its application to the determination of magnesiu’m, calcium, copper, manganese, and chromium have been detailed (1, 2). Satisfactory precision and accuracy were achieved when each element was determined independently. However, when all of these elements were present simultaneously a significant decrease in the emissivities of calcium and chromium was observed. Furthermore, low concentrations of phosphate interfere with the determination of magnesium, manganese, and calciu,m, presenting a serious limitation on the analysis of phosphate-containing biological and other samples. The nature and origin of both cation and anion interferences have been examined extensively both in this (3-5) and in other laboratories (6, 7). Most of these studies have been carried out by examining the effects on the measurement of a single element, particularly calcium, but multicomponent systems of the type described here have not been studied. Means to overcome interference include the addition of perchloric acid (8)) lanthanum chloride in 2% perchloric acid (9), 10% glycerol in 0.1 M perchloric acid (lo), and 0.1 M ethylenediaminetetraacetic acid (11). In the course of the present work, t,hese and other procedures for the elimination of cation and anion interferences were tested and evaluated in the multicomponent system. A solvent system, consisting of half-saturated 8-hydroxyquinoline and 0.4M perchloric acid in 1 M hydrochloric acid, effectively eliminates both cation and anion inter‘Present address: Chemical Institute, Faculty of Science, Nagoya Nagoya, Japan. ‘To whom reprint request.9 should be addressed. 9

University,

ference and at the same time produces significant enhancement of the emission of the elements being measured, thus providing a marked increase in sensitivity. The precision and accuracy of the method was established first using N.B.S. certified standards and, finaliy, the precision and accuracy of the instrument and the suitability of the solvent system for biological and commercial samples was ascertained using rat liver mitochondria and chrometanned leather. MATERIALS

AND METHODS

Standard Solutions. Standard solutions of magnesium, calcium, copper, manganese, and chromium were identical to those described (2). Perchloric Acid. This was a double vacuum distilled, 70% HClO,, leadfree solution obtained from G. F. Smith Chemical Co. All chemicals were reagent grade. Glassware was cleaned and water purified as described previously (2). Unless otherwise stated, the instrumental conditions used were those described (1). Half-Saturated 8-Hydroxyquinoline Solution. A mixed solution of 1 ,!W hydrochloric and 0.8.M perchloric acid was saturated with 8-hydroxyquinoline. An equal volume of this solution was added to the aliquot of standard or sample dissolved in 1 M hydrochloric acid. Lanthanum Chloride Solution. 24.55 gm of lanthanum chloride, 1.7 ml of 7070 perchloric acid, and appropriate aliquots of each metal standard were mixed together and diluted with metal-free water to a volume of 100 ml, so that the solution results in a final conccnt’rat,ion of 10 /lg of each element per milliliter in the presence of 1 M lanthanum chloride and 2% perchloric acid. Rat Liver Mitochondria. These were prepared by the sucrose-gradient centrifugation method (12) .3 Tanned leathers of known chromium content served as biological standards for this clemenL4 RESULTS

Effect of Lanthanum Chloride The conditions previously employed to overcome phosphate ference on calcium measurements (9) were duplicated to study the of lanthanum chloride. Its effectiveness in eliminating the effect of phate was measured by comparin,u the emission of each element “Kindly supplied Medical School.

by

Dr.

I’.

J.

Snodgrass,

Department

of

Medicine,

intereffect phosin its

Harvard

,MULTICHANKEL

FLAME

SPECTROMETER

STUDIES.

III

presence and absence over a range of 1O-3 to 10-l M phosphoric resultant intensities plotted as percentages of those observed nesium, calcium, copper, manganese, and chromium alone are Figure 1. The effect of phosphoric acid is most pronounced on

11

acid. The for magshown in the emis-

Mg

z9 50 'F a 100 ii LT

GkL

/ 0

I

I

I

10-3

10-2

10-l

(H3P041, M FIO. 1. Effectiveness of lanthanum ference of Mg, Ca, Cu, Mn, and Cr ponent system: (-) with LaCh; (---) ppm concentration in the presence of HCIO, solution and the concentration

chloride in preventing phosphoric acid interemission by flame excitation in a single comwithout LaCh. Each element was present in 10 1 M HCl and of 1 A4 HCl + 1 M L&l3 in 2% of phosphoric acid indicated.

sion of calciu’m and magnesium. Lanthanum chloride overcomes the interference up to concentrations of 5 x 1O-2M phosphoric acid. At 0.1 16 phosphoric acid, however, calcium emission is diminished below that of calcium and phosphoric acid analyzed in the absence of lanthanum chloride. Phosphoric acid does not interfere significantly with the emissivity of chromium and manganese over t,he entire range of concen-

12

IIDA

AA-D

trations studied; but the addition significant decrease of the intensity phosphoric acid. Perchloric

Acid

I”UWA

of lanthanum chloride induces a of these elements beyond lo-? M

and 8-Hydroxyquinoline

The chelating properties of ethylenediaminetetraacetic acid (EDTA) which have been exploited for the prevention of anion interference (11 j suggested that other chelating agents might be substituted with profit. From among the many possible ones, S-hydroxyquinoline was selected largely since it is known to form a very stable chelate coBmplex with chromium (13, 14) in addition to those with the other four elements. The effect of &hydroxyquinoline alone and in combination with perchloric acid on the emissivity of each element was studied first. The llesults are shown in Table 1 (lines 1, 3, 5, and 7). Perchloric acid alone

Effect of Perchloric X-Hydroxyqninoline

TABLE 1 Acid, S-Hydroxyquinoline, and Perchloric Acid plus on Emissivities of hlg, Ca, Cr, &In, and Cu and on the Interference of Phosphoric Acid Rclntive

1. 2. 3. 4. 5. 6. 7. 8.

None 0.1 M H,POa 0.4 M HClO, 0.4 M HCIO, + 0.1 M HaPOd 8-OHQ 8-OHQ + 0.1 df H,POd 0.4 M HClOa + S-OH& 0.4 M HClOa + &OH& + 0.1 M HzP04

75 so so 65 130 130 130 130

lS0 155 190 100 285 250 285 290

intensity,

60 65 70 50 140 115 150 135

,~~rnp

150 140 150 105 190 180 200 200

80 90 80 80 90 105 100 115

10 ppm of each element in 1M hydrochloric acid is present together with the compounds indicated. The solution of 8-hydroxyquinoline (8-OH&) is half-saturated. The intensity values represent the average of three measurements and the deviation in e?ch instance is within +5 Iramp.

increases the relative intensities of calcium and chromium but not those of magnesium, manganese, and copper (Table 1, lines 1 and 3), whereas 8-hydroxyquinoline markedly increases the emissivities of all five elements (lines 1 and 5). When both perchloric acid and Shydroxyquinoline

are present,

the

emission

of chromium,

manganese,

and

copper

is

enhanced over and beyond that with 8-hydroxyquinoline alone (Table 1, lines 1, 5, and 7). The addition of 0.1 M phosphoric acid alone variously increases gr decreasesthe emissivity of all five elements (Table 1, line 2i

MULTICHANNEL

FLAME

SPECTROMETER

STUDIES.

IfI

13

The effects of perchloric acid and 8-hydroxyquinoline in the presence of phosphoric acid were evaluated next (Table 1, lines 4, 6, and 8). The addition of perchlorie acid alone enhances the interference caused by 0.1 M phosphoric acid (Table 1, lines 1, 2, and 4)) but the addition of 8hydroxyquinoline increases the emissivities of all elements almost to the level observed with this agent alone (compare lines 5 and 6). When both perchloric acid and 8-hydroxyquinoline are added the emissivities of all elements are nearly the same whether phosphoric acid is present or not (Table 1, compare lines 7 and 8). The emissivities of calcium and chromium each taken separately, then together, and finally in the presence of all five elements in a multicomponent system were measured in the presence of concentrations of 8-hydroxyquinoline up to 0.1 M in order

300

250

0

0.01 0:02 0.04 (8-Hydroxyquinoline),

0.1

M

FIG. 2. Effect of S-hydrouyquinoline on emissivities of calcium and chromium separately and in a multicomponent system; ( n , v) single element standard solution; (0, V) combined Ca + Cr standard solution; (0, 0) five element multicomponent standard solution. Each standard solution contained 80 ppm in the presence of 1 M HCl and the indicated concentration of S-hydroxyquinoline.

1‘4

IlD.4

BND

FUWA

to establish the minimum concentration of this agent which yields maximum enhancement (Fig. 2). In the presence of concentrations of Shydroxyquinoline equal to or greater than 0.04111 there is no significant difference in the emissivities of calcium and chromium whether they are measured alone, paired, or in a multicomponent system. The effectiveness of a half-saturated solution of S-hydroxyquinoline with or without 0.4 M perchloric acid in preventing interference at varying phosphoric acid concentrations was then test’ed in a multicomponent system. The results for the five elements are tabulated in Table 2. Either 8-hydroxyquinoPrevention

of Phosphoric and

TABLE 2 Acid Interference on Emissivit#ies Cu in a Multicomponent Solution

of Mg,

(HsPOa). 0 Element

1 x

10-p

Agent

S-OH& S-OH&

+ HClOd

Ca S-OHQ 8-OHQ Cr

+ HClOd --

8-OH& 8-OHQ

+ HClOd

Rln S-OHQ g-OH&

+ HClOh -

Cl1 S-OHQ &OH&

+ HC104

M 1 x 10-z

1 x 10-1

60 120 120 100 255 270 63 125 145 133 175 175 80 90 100

70 117 120 145 235 265 70 110 130 130 163 175 95 95 100

GmP

75

&

1 x 10-z

Ca, Cr, Mn,

120 120 180 270 270 67 130 145 145 170 180 80 85 95

70 120 120 143 275 275 67 130 140 143 173 175 80 53 95

63 120 120 100 270 275 65 130 140 125 175 175 80 55 95

The concentration of each element in the multicomponent solution was 10 pg/ml in 1 &I HCl. 8-Hydroxyquinoline was half-saturated and HClO, was 0.4 &I. Phosphoric acid was added to similar aliquots of the 10 rg/ml multicomponent standard solution in the concentrations shown.

line alone or when mixed with perchloric acid completely eliminates phosphoric acid interference for all elements except calcium, and, in fact, emissivity is enhanced over and beyond 100% of the control. This is particularly apparent for chromium in the presence of from lo-” to lo-* M phosphoric acid where the enhancement is 200%. 8-Hydroxyquinoline employed as the sole adjuvant is, however, unsatisfactory for calcium with lO-2 M phosphoric acid. Therefore, a half-saturated Skhydroxyquinoline solution in 1 M hydrochloric plus 0.4M perchloric acid has been adopted as the standard

MULTICHANNEL

FLAME

SPECTROMETER

STUDIES.

solvent system for the routine analysis of these alkaline transition metal ions in solutions containing all components. Repeatability

and Precision

of the Working

15

III

earths and

Curve

With 8-hydroxyquinoline and perchloric acid in 1 M hydrochloric acid the repeatability and percision of the working curves was determined by measuring all five elements in the multicomponent standard solutions repetitively at concentrations ranging from 1.0 to 25.0 pg ml. Because of the increased sensitivity of detection of calcium and manganese in

500

200 E 100 l3 i 50

20

1

c

0.5 I.0 2.0 5.0 10.0 20.0 (Me), pg per ml FIG. 3. Working curves for Mg, Ca, Cr, Mn, and Cu excited in the a half-saturated solution of &hydroxyquinoline plus 0.4 M perchloric hydrochloric acid. Open symbols : high-gain photomultiplier settings sensitivities (1 and Table 3). Solid symbols: low-gain photomultiplier 0.1

0.2

presence of acid in l&f to increase settings.

this system they were also ,measured over the range from 0.1 to 1.0 pg/ ml by increasing the amplifier gain. Working curves constructed from the data are shown in Figure 3 and the means, standard deviations, and coefficients of variation for the analysis of each are shown in Table 3. As judged by the coefficient of variation the repeatability at 1 pg/ml ranges from 3 to 129~~ but most of the values at higher concentrations were less than 4%. Accuracy of the Proposed Standard Method

The accuracy of the method was tested by analysis of certified samples from the National Bureau of Standards numbers 88, 157, 161, 162A, and

21 57 89 179

Ca

f standard

+ * zk f

(3%) (2%) (1%)

deviation

(6%) (7%) (4%) (3%)

77 f 2.6 177 f 2.6 345 3~ 2.7 500 500

1.2 4.1 3.5 4.9

(2%)

(274

(7%) (4%) (47,)

0.1 0.3 0.5 1.0

2.6 2.4 4.5 4.1 6.8

Ca

k f k 5 !c

Concn., ppm

37 73 125 223 303

Lfg

The values listed are the mean shown in parentheses.

1.0 2.5 5.0 10.0 25.0

Concn., ppm ppm (12%) (4%) (3%) (2%) (2%)

ppm

2.7 2.2 2.6 2.7 4.1

determinations

0.25 0.5 1.0

Mn

with

80 + 3.2 202 f 4.9 395 k 6.3 500 500 Concn.. ppm

Solutions

of six repetitive

0.1-1.0

1.0-25.0 23 f 47 xi 78 f 138 f 277 +

Cr

TABLE 3 of Standard

in pamp

Range

Range

Repeatability

A zk f f 31 1111

14 33 63 117 247

1.6 2.6 2.6 2.6 2.6

the coefficient

(11%) (8%) (4%) (2%) (1%)

of variations-in

44 f 3,s mc’;r 100 + 4 .5 (4 y-c, I 208 f 6.0 (35:)

~~~ -.-__

(12701,) (2%) (2%)

CU

o/o

2 s

5 u kj

E g

MULTICHANNEL

FLAME

Determination Element

SPECTROMETER

STUDIES.

17

III

TABLE 4 of Accuracy Based on N.B.S. Samples

N.B.S. sample NO.

KIIOWKI, PPm

Determined, PPm

Accuracy, %

88 171 88 161 162A 171 157A 162A 161

12.95 47.77 21.78 2.56 3.20 0.90 29.34 15.30 32.58

12.74 47.50 21.85 2.45 3.04 0.83 27.84 14.76 34.17

98 99 100 96 95 92 95 97 105

Mg Ca Mn cu Cr

171. The values obtained are compared to those provided by the National Bureau of Standards (Table 4). The accuracy ranges from 92% for manganese in sample 171 to 105% for chromium in sample 161. Application

of the Method

to Biological

Samples

Rat liver mitochondria were chosen for analysis because of their welldocumented content of magnesium, calcium, manganese, and copper (15). Tanned leathers of known chromium content were analyzed for this element. The present results for the mitochondria are compared to those by other methods previously recorded in the literature (15) in Table 5. The values obtained for each of the metals by the present method are TABLE 5 Analyses of Metals in Bat Liver Mitochondria and Chromium in Tanned Leathers

Mitochondria

Leather A B

Element

Mean

f

S.D.

Mg Ca Mn cu

6.15 2.41 0.166 0.053

+ + f f

0.19 0.06 0.010 0.004

Cr Cr

10.4 13.3

f 0.24 f 0.49

No. of analyses

Cg.’

5 5 6 6

3 2 6 8

11 11

2 4

Literature

(15)

5.3-15.3 1.9-3.5 o-O.41 0.0294).169 10.9” 13.90

a Value found by the donor of the sample employing a chemical method (16). 20 ml aliquots of the mitochondrial suspension and weighed amounts of the tanned leathers were ashed completely in platinum dishes at 450” in an electric muffle furnace. The ashed samples were then dissolved in 1 M HCl. Equal volumes of a 1 M HCI solution of saturated 8-hydroxyquinoline with 0.8 M in perchloric acid were added to the HCl solutions of the ash. The values for the mitochondria are expressed in mg metal/gm nitrogen and those for the tanned leather in ppm.

18

IIDA

AND

FUWA

well within the range reported in the literature. Chromium analyses of the leathers are in excellent agreement with those found by Dr. J. A. Reid, the donor of the samples, who has kindly permitted the citation of his results obtained by means of a chemical procedure (16). The repeatability of all measurements on these biological samples are similar to those obtained on standard solutions. Finally, the over-all performance of the method was assessed by measure’ment of the repeatability and recovery of standards of magnesium, calcium, manganese, and copper when added to ashed mitochondria and of chromium added to the ashed leathers. The mean, standard deviation, and coefficient of variation of five repeated analyses of mitochondria -L added magnesium, calcium, manganese, and copper and eleven replicate

Repeatability

Sample

Mitochondria

Leather A B

TABLE 6 of Metals Added

and Recovery

Element

found

aahl

f

S.D.

C.V.,

%

to Mitochondria

and Leather Recovery

Calculated

Found

& S.D.

%

Ca Mg cu Mn

5.3 10.8 7.4 2.29

+ ok f zk

0.03 1.42 0.45 0.10

1 4 6 4

6.3 11.8 8.4 3.29

6.4 12.0 8.8 3.3

rk f f f

0.11 0.62 0.45 0.07

102 102 105 100

Cr Cr

11.1 13.5

+ 0.70 f 0.54

6 4

21.1 23.5

20.5 21.9

f 0.42 f 0.57

97 93

Aliquots of the standard solutions containing 1 ppm Ca and 1 ppm Mg were added to 1 ml of mitochondrial suspension and 1 ppm Cu and 1 ppm Mn were added to 20 ml of the mitochondrial suspension prior to ashing and dissolution in %hydroxyquinolineperchloric acid as in Table 5. Aliqnots of the standard Cr solution containing 10 ppm were added to a weighed amount of the tanned leathers and evaporated to dryness before ashing along with the leather as described in Table 5. The mitochondria were analyzed five times and the tanned leathers eleven times.

analyses of the tanned leathers + 6. The repeatabilit’y varied from 1 calculated from the ‘mean difference and without added metals ranges sample B to 105% for copper in is close to 100%.

added chromium are shown in Table t,o 67% while the percentage recovery of the analyses of the sample with from 93% for chromium in leather mitochondria. The average recovery

DIHCUSSION

Phosphate interference on the emission of magnesium, calcium, and manganese on the one hand, and an effect of calcium on chromium emission on the other. have been shown to be the essential limitations in the

MULTICHANNEL

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SPECTROMETER

STUDIES.

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19

routine analytical use of a #multichannel flame spectrometer (2). Based on their known causes, means were sought to overcome these limitations (6, 10, 17). On the basis of similar considerations a number of procedures employing a diversity of agents have been used to eliminate phosphoric acid interference in given instances. Dinnin (Q), e.g., employed lanthanum chloride in 2% perchloric acid to overcome interferences of aluminum and phosphate on calcium emission while Rains et al. (10) used 10% v/v glycerol in 0.1 M perchloric acid for the same purpose and to eliminate interference of aluminum, chromium, iron, and titanium on calcium. West and Cooke (11) found that 0.1 M EDTA at pH 9.0 removed phosphate and sulfate interference on the emission of calcium, magnesium, cobalt, copper, chromium, and manganese. The present results demonstrate that lanthanum chloride in perchloric acid does not adequately compensate for the spectral interactions in this multicomponent system. While the interference of phosphoric acid up to 5 X 1O-2M on calcium and magnesium emission was eliminated completely, at 0.1 M phosphoric acid calcium emission was decreased below that of calcium alone. Phosphoric acid does not suppress manganese and chromium but the addition of lanthanum chloride quenches the emission beginning at 1O-2 M phosphoric acid (Fig. 1). Over the concentration range of 10m4 to 10-l M the chelating agent Shydroxyquinoline, like EDTA, proved excellent for the elimination of phosphoric acid interference (Tables 1 and 2). However, two additional benefits accrue which prove crucial to its use in the multicomponent system: (1) The mutual cationic interference between calcium and chromium is prevented (Fig. 2). (2) A marked enhancement of the emissivities of all elements is observed which results in a significant increase in sensitivity. Perchloric acid potentiates both of these effects. 8-Hydroxyquinoline or perchloric acid used separately either fail to enhance emission or eliminate anion or cation interference completely (Tables 1 and 2 and Fig. 2). The synergistic action of perchloric acid may be a function of the ionic state of perchlorates in solution. Unlike phosphates and chlorides, perchlorates do not form covalent complexes at high concentrations and therefore cannot compete with 8-hydroxyquinoline to reduce the quinolate concentration significantly. The enhancement brought about by S-hydroxyquinoline is apparently not due merely to its chelating properties but may be due to the structure of 8-hydroxyquinoline itself. A systematic study of a series of chelating agents might provide an explanation for the effect of 8-hydroxyquinoline and at the same time illuminate the mode of action of other compounds having greater enhancing properties.

20

IIDA

AND

FUWA

Repeatability and precision of the analyses employing the half-saturated %hydroxyquinoline/perchloric acid mixture as the solvent system are excellent (Table 3). Simultaneously, the marked enhancement of emissivities for calcium and magnesium increases sensitivity and lowers the analytical limit of detection to 0.1 pg/ml (Fig. 3). Comparison of the accuracies obtained in the present system with N.B.S. standards and with those observed when 1 N hydrochloric acid as the solvent (Table 4) (2) demonstrate considerable improvement. This is consistent with the excellent results obtained on the biological samples of known metal content, in spite of the known high phosphate content of mitochondria and the added problem of contamination during ashing. The method has many obvious applications in the analysis of biological samples, providing for the first time a routine method using a flame source for the joint analysis of alkaline earths and transition metals. Furthermore, the ‘measurement of chromium in tanned leathers offers the analyst and the leather technologist a simple, rapid, precise, and accurate method which obviates the difficulties of chromate oxidation, inherent in the available chemical techniques (18). The addition and standardization of channels for other elements should greatly expand the usefulness of flame emission analysis in the analytical chemistry of metals. ACKNOWLEDGMENT This work was supported by of Health of the Department Nutrition Foundation, Inc.

grant-in-aid of Health,

HE-07297 Education,

from the National Institutes and Welfare, and by The

REFERENCES B. L., Anal. B&hem. 17, 444 (1966). IID.4, C., AND Fu~A, K., Anal. Biochem. 21, 1 (1967). MARGOSHES, M., AND VALLEE, B. L., Anal. Chem. 28, 180 (1956). MARGOSHES, M., AND VALLEE, B. L., Anal. Chem. 28, 1066 (1956). VALLEE, B. L., “Flame Spectrometry” in ‘(Trace Analysis” (J. H. Yoe and H. J. Koch, Jr., eds.), pp. 229-254. New York, 1957. DEAN, J. A., “Flame Photometry.” McGraw-Hill, New York, 1960. HERR;WBN, R., AND ALREMADE, C. T. J., “Flame Photometry.” Interscience, New York, 1963. GIBSON;, J. H., GROSSMAN, W. E. L., AND COOKE, W. D., Anal. Chem. 35, 266 (1963). DINNIN, J. I., Anal. Chem. 32, 1475 (1960). RAINS, T. C., ZITPEL, H. ,E., AND FERCUSON, M., Talanta 10, 367 (1963). WEST, A. C., AND COOKE, W. D., Anal. Chem. 32, 1471 (1960). SNODGRASS, P. J., AND PIRAS, M. M., Biochemistq 5, 1140 (1966). MUKHEDK~R, A. J., AND DESPHPANDE, N. V., Anal. Chem. 35, 47 (1963). “Stability Constants,” The Chemical Society, London, 1964.

1. FUWA, 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12.

13. 14.

K.,

AND

VALLEE,

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SPECTROMETER

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21

15. THIERS, R. E., AND VALLEE, B. L., J. Biol. Chem.!2!26, 911 (1957). 16. A. C. Laurence Leather Co., Peabody, Mass. 17. DIPPEL, W. A., BRICHER, C. E., AND FURMAN, N. H., Anal. Chew 26, 553 (1954). 18. SANDELL, E. B., “Calorimetric Determination of Metals,” Interscience, New York, 1958.