Fluorimetric derivatization for pesticide residue analysis

Jam-naI of C~~ronraro~~r~~pl~~. 9s t 1974 233-170 Chronratographic Re~ien-s 8 Elsevier Scientitic Publishing Cotnpmy, Amsterdam CHREV_

-

Printed

in The Netherlands

71

FI_UORIMETRIC

DERIVATIZATION

FOR PESTICIDE

RESIDUE

ANALYSIS

CONTENTS 1. Introduction _ _ _ _ _ _ _ _ _ 2. Fiuorescence analysis _ _ _ _ _ A. Fluoresceuce theory _ _ _ _ B. Factors atkting t?uorescence

_ _ _ _ a. Structurdl &ects _ _ _ . _ b_ Solvent etlkts. _ _ _ . _ _ c. Concentmtion eKects_ . _ _ C. Analytical considerations _ . _ 3_ i\nrtl~sis of pesticides _ _ _ _ _ _ A. Hydrolysis _ _ _ . . _ _ _ _ B. Ligsnd-eschangc reactions _ _ C. pHeIkts. _ . _ . . _ _ _ _ D. Polarity _ - . . . _ _ _ _ _ E_ Heat treatment _ _ _ _ _ _ _

_ _ _ _ _ _

_ _ _ _ _ _

_ _ _ _ _ _

_ _ _ _ _ _

_ _ _ _ _ _

_ _ _ _ _ _

_ _ _ _ _ _

_ _ _ _ _ _

_ _ . _

_ _ _ _ _ _ _ _ .

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ______-________._ _ _ _ _ _ _ _ _ _ _ __ -_ _ -________ _ _ ____ _ _ _ _ . _ _ _ _ _ _ _ _. _ _ _ _ _ _ _ _ . ._ _ _ - _ _ __ _ _ _ - __ _ _ ._ _ _ _ _ _ _ _ _ _ --_ _ _ _ _ _ _ _ _ _ _ _ _ _ - - _ _ _, _ - _ _ _ _ - _ F. Condensation reactions _ _ _ _ _ _ _ _ _ _ _ _ _ ___ 1. Reactions ofdausyl chloride with przticidrs . _ _ _ _ _ _ --_-_ -_-. _ A. N-lletll~lllrbamlrtrc insecticides _ _ _ _ _ _ _ _ _ -____ _ _.. _ B_ WPhen_\-lcarbanmte and urea herbicides _ _ _ _ _ -_ -_ C. Orgnnophosphnte insecticides _ _ _ _ _ _ _ _ _ D. +Trirtzine herbicides _ _ _ _ _ _ _ _ _ _ _ _ _ _ E. Hydrosybiphcnyk _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 5. Reactions of NBD chloride \vith pesticidry _ _ _ _ _ _ -6. Reactions of lluorescamine_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 7. Fluorigenic labelling in solution and TLC and HSLC analysis KConclusions _ . _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ References ___________ ______________

_ _ __ _ _ _ _ _ -_

-.-_ _ . - -. _. -._ - _ _ - _ .._ _ _ . .._ _ _ - _-._ _ _ .._._ -_

_ _ _ . . . _ _ _ _ _ _ . . _ . _ _._._

. .

‘57

_ 257 _ _ _. . ‘57 -__ _ _ 3s _ _. _ 25s _ . _. _._ . . . _ _ _ -_ ___ _

. . _ 159 _ _ _ ‘59 _ _ 260 _ . _ - 261) _ __ 261 _ _ _ 262 _ _ _ 261 _ _ _ 263 _ _ 263 __ _ 765 _ _ _ _ _ 265 _ _ _ _ _ 167 .._..16S

I. 1NTRODUCTION

The increasing concern over the use and effects of pesticides and other potential pollutants in the environment has motivated much resetkch into the trke mkysis of these compounds_ Since the introduction of synthetic pesticides* the problem of harm to non-target organisms from persistent residues has grown considerably_ In recent years there has been a significant increase in the use of the less persistent pesticides, carbnmates, ureas and organophosphates being desirable alternatives. However, the

m

J_ F_ LAWRENCE,

R. W. FREI

methods of analysis of many of these compomlds leave much to be desired by current standards of ease of analysis_ sensitivity and reproducibility_ Thus, there constantly appear in the literature newer and better methods to replace or modify older techniques- The use of fluorimetry in residue analysis has been very limited in the past_ Today- with new instrumentation including better electronics and precise sensitive optical systems, fluorescence measurements are as simply and conveniently carried out as spectrophotometric techniques_ The development of thin-lxper (TLC) scanners has also espnnded the use of1luorimetry to a semiquantitative, and even quantitative. tool for evaluating chromatographiczllly separated fluorescent s~o!s’-~. The recent resurgence of column liquid chromatography utilizin=0 high pressures and flow uelocities, as well as cohmm packins that approach gas chromatographic (CC) levels of efficiency, has also broadened the use of fuorimstry for quantitative analysis. The main restriction on the use of fluorimetry at present is its selectivity. Most compounds of current interest to the residue chemist are not fluorescent enough to be determined in trace quantities- Generally, absorptiometric or GC techniques are the main methods for residue analysis. The concept of fluorescence derivatization for non-fluorescent compounds is very new and has much potential as a trace analytical technique_ Besides the selectivity of tluoresceuce itself. the reactions and reagents can offer selectivity in the formation of derivatives. There are four uses of derivatization reactions in residue analysis. One of the main uses is to impart measurable parameters to the compounds to be anal~sed. This type of dcrivatization has been used for many years in pesticide residue analysis. Early work usually involved the formnrion of colowed ~;pc-‘cies\vhich \v\‘ct-c quttntitatively evalurtted photometric,lll~~-l”_ The use of bromination”. chloro~~cet~l;~tio~I” ability to compounds has and triflilor03cetYI;ttionr3-t~ to impart electron-capture received much attention in pesticide residue rrnalysis b_vGC_ Derivatization techniques in association \\-ithTLC are ;1 necessity for visualization of separated spots_ The use ofspray reagents for such purposes has been cstcnsi\-ely investigated for a wide variety of pesticide compounds*5-2Z_ The second use of derivntization is for idcntikation. Chromatogrtlphic :malysis of compounds is made on the basis of retention time or RF value. However, as more than one compound can exhibit the same characteristics in a number ofdilkrent systems_ chcmktl veriticxrion of the results is often required_ Cochrane and Chtr~‘~ reviewed chemical dcrivatiztltion techniques for organochlorine compounds_ The use of chemical derivati\-tx for the identification of or~anophosphatc insecticides_ alcohols, primary and secondary amines. thiols and other compounds by GC has :xlso been reported’J-‘h_ Thirdly_ derivatiztttion is also used to aid or improse chromatography. Polx compowds such as phenols, thiols. cxbosylic acids and amines are not often directly suited to chromtlto~rtlphy_The use ofsilyl derivativcs”-3’. esterification retlctionsJZ-3’ and ether rextions3”-J’ fk such compounds for GC analysis has been reported_ A fourth use of dcrivatization is to alter the chemical nature of ;I compound in order to make it more easily extractable from sample matrrial~“-J*_ This concept is usually asociatrd \sith steam distillation tcchniqucsJ1~~4J_ Fluorescence dcrivatizntion may be used for enhancement of sensitivity for quantitative zlnalysis. contirmntion of residue data obtained by other snalytictll procsdurts and also to aid chromatography -TLC and high-speed liquid chromatography (HSLC)_ The present work discusses recent developments in Ruorescence reactions for trace analysis. \vith emphasis on pesticide residue determinations.

FLUORl~IETRICANALYSlSOFPESTIClDE 2_ FLUORESCENCE

RESIDUES

255

ANALYSIS

There are many detailed tests on the theory and practical applications of fluorescence in both organic and inorganic tields45-‘“. Cniy a brief account of the fluorescence process and Factors which affect this phenomenon is $ven here. Fhiorescence can be described as the emission of a photon from ;I molecule state from the lowest electronically escited sin$et state. upon its return to tht _ ground The excitation of a nwiecuic is usually achie~cd by irradiation Lvith light of :t spccikic w~veiength_ The iiktirnc in the excited singlet state is of the order of IO-“-IO-’ sec. This is a quantum eKcct and the quantum efficiency is the fraction of excited moleCUICS that Iluorsscc: it reaches 100 :A: for compounds such as tluorcscein or Rhcdamine B. The quantum werg_v thttt is emitted is oflmver sncrsy than the absorbed quanturn. as part of the excited-state energy is dissipated by vibrational rei:m~tion processes. A shit-t to longx- wavelengths rciztive to the absorption spectruni is tillis observed. This phenonicnon is known as the Stokes shift. 14uoresccncc only occurs from the io\vest excited singlet state. if a moiccul~ is exited to ;L hi&r Gnglet-state energy irvci. it undergoes vibrational rciasation until it has reached the lwvest exited sinyiet state. The time for this process is short in comparison to the fuorescence decay time. in mtlny moiecuies \vhich are raised to ;ln exited singlet state_ there xc’ sullicient conipt’tin, tx radiationicss processes for the IVturn to the ground state to prevent tluorescencc. This fact limits the use of tluoresccncc as :m analytical technique but at the s;unc time renders the method more selecti\-s_

;iu exited n~oie~uie nla): ha\-e (tr) Strttc~trrrtrl c:&-ts. As mentioned above. stxmai nwthads ot’cncr~_\; dissipation other th:ln by tluorsscence. These processes are strongly structure depmdcnt. The ssistcnce of rigid. planar aromatic structures is usur~ily l~~vourr~hlc to tluot-esccncc. in addition. the presence of clcctron-donating “roups_ such ;ts -OH :tnd -NH?. tends to whancc tluorescence. Stcric strain and the = presence of electron-\~ithdra~iug groups. such as -NO?. -CN or -Cl substitue:ltS_ tend to quench or signiticantly decrcxse tluorwxncc_ Moiecuies \vhich are able to rot:w_ bend or t\vist have ;I tcndencv to lose excited state energy through moiemlar cailisian and other vibrational processes. Ho~vever. it is not possible to rwign ;I cornpietc Set 0frulcs \vhich drterniines \vhether 3 molecule \vili tluorcsce . r1.i there are riinny csccptions. if>) Sdwrtt cIf_fiws_ The solvent in which ~1 species is dissolved (or material upon \vhich it is adsorbed) may ha\-c ;i considrrt~bie influence upon the iutensity and \vavelcngth at- tiuorescence. Polar solvents such as z~lcoilois or t’stcrs dtcn incretlsc iluort’scencc relative to non-polar hydrocarbon saivalts_ The sol\lcnt cn\.ironil:ent \vill often prevent or decrease triplet state excitation in t‘tlvour of singlet-state acitation and tluorcscence. \vhile at other times the opposite cllkt is true_ ~V’:mr Sontctinles has a quenching cfiict on tluoresccncc through hvdrogm bonding_ Compounds that do ll~~orescr in aqueous mrdia often exhibit marked changes in fluorescence \vith chrmses in pH_ All of these clkcts must be esaminrd in ordrr to dcvciop an analytical mrthod.

256

J. F. LAWRENCE.

R_ W. FREI

(c-j Co~w~~zrrcrtio~~&Y-IS_ Because tluorescence is a quantum elfect. the intensity of the emitted radiation is proportional to the concentration of the compound present_ However_ at high concentrations this relationship is not linear due to an “inner filter*’ ellixt lvherc-2some of the emitted radiation is reabsorbed by the tluorescins molecules or thesolvent moIecules_ This is known as concentration quenching_ For analytical purposes solutions which eshibit this behaviour may simply be diluted to bring the concentration within the linear range of fuoresccnce_

is_ only

The selectivity of fluorescence is obtained through structuctl restrictions: that certain types of molecules m-e able to tluoresce. Fluorescent molecules are

characterized by t\vo \vtlveIengths_ ri-__ the excitation wavslength. Thus. while it is easy to detect Iluoresccnt

\~~velength and the emission substances in the presence of

non-ffuorcscent compounds. it is also possible to dctcrminc Iluorcsccnt materials in the presence of one another if their characteristic \vnuclengths dilli-r enough. The use of the two wavelengths gives an added degree of selectivity compared to absorption techniques_ _4 second characteristic of fuorescence is its sensitivity_ Fluorescence is often IO-100 times as scnsirivc as absorpriomctric methods and frcqucntly has a lincw response retativt’ to concsntrtttion over ;t IOOO-fold range. FIuorimeters and spectrotliloriliirtsrs are avnilablc from :I nun&x of sources_ including Carl Zeiss_ Aminco-Bo\~man_ G_ K. Turntx- and Associarcs. Pet-kin-Elmer and Baird Atomic. Attachments have been devised for these instruments to permit i/z sim scanning &TLC pIatss_ Although the method of dircct quanritari\-c scannin, *x for tluoresccncc on thinlayer chromato~rtlms has not been used to any St-eat extent in residue analysis_ it has been used rstcnsively in air pollution work5’-SJ -and in the wnal~sis of pll~~rmaceuticrtl and biological s_vstems5a-5’_ A theoretical treatment of the distribution of ;I spot adsorbed on thin layers has been carried outh”--hh. 111sifrf tluarimetric me;isurements :1re largely independent of spot size and shapeh’_ As has been demonsrrated b_v :L number of authors”‘-‘“_

linear reltttionships

stance and recorded

area

csist over a wide range between

quantity

16 sub-

of the

peak, as Ion,** k _ the concentration -1s of substance is lo\\ snou~h to avoid tluoresccncc bb- self-absorption. Very little work has been carried out on Iluorcscence an:ilysis br HSLC due mainIy to the fact that HSLC is TVnew technique and that there is an :kbsence ofsuitabk t?uorescence detectors_ Laboratory Data Control have rccentl~ marketed ;t tiuorimster designed for use lvith HSLC. Similar dctcctors for HSLC hsve bcsn produced from a converted Turner fluorimetcr^“. an Amitlco-Bo\~man fiLtc)ritiit’tCI”“-” or built up directly from components’3-7A. The theory of HSLC has been covered in great detail. including the causes of band broadening. elliciency of separations interactions bet\veen solute? mobile phase and sttltiomwy pha~e’~-‘“_ sample

In tluorimetry, and cokction

there are three major contigurxtions of the emitted light, rk_

I I) Irradiation

ofthe

tube at an an$e

of Xi

(1) l’k~ 1-6?I-s‘cI_

for initial

sample at a 90. angIc and collection

to the

sample surfke.

irradiation

and of a

by a photomultiplicr

FLUORIM~RICANALYSIS

OF PESTICIDE

RESIDUES

257

(3) Specitically for some i/zsim TLC scanning: irradiation at an an+ of90’ to the TLC plate surface and emission collection through the= glass support by a photomultiplier tube_ This is limited to emissions in the visible region. For residue determinations. in siru scanning ofTLC plares or HSLC olt‘er more adutmta~es in fluorescence analysis. Solution fluorescence measursnwnts must rely on sample blanks for a background fluorescence correction_ which is not required in the chromatographic merhods. 3_ ANALYSIS

OF PESTICIDES

Although fiuorimctric analysis has been in use for :t nun~ber of >wars in biochemistry. biology and related areas. it has had only limited use in pesticide rrsiduc Lvork and has been restricted to those compounds xvhich exhibit an intrinsic fluorescence strong enough to be anrdytict~lly useful. The initial dark on lluorimetry of pesticides \vas carried out in solution_ The 0uorcsccnce behaviour of compounds such as Guthion (azin~l~osmethyl)_ Pots:m_ W’ttrfarin. piperonyl butosidc and others \v;ls investigated for possible uses in the analysis of their residues”_ Mucl~ \vork has been done by Sa\vicki and co-\vorkcrs in connection \vith Ruorescent air pollutants and has racentl_v been revimved”‘. However_ most 0uorimetric analyses of pesticides require pre-treatment of the co~iipaunds to convert them to fluorescent species.

Most older n~thocis of fluorimetric pesticide analysis involved hydrolysis to forni fiuorescent anions_ Co-t-al (couniaphos)7s \v;is hydrol~sed in alkali to the hydroxybenzopyrnn. \vhich \vas subsequentlv determined by fuorescencc_ Hydrolysis of tlnrtlysis has been ;tcGuthion (azinphosmerll~l) 10 anrh&iiic acid for tluorimctric co~ii~~li~ih~ci”-““_A mcthad t-i)r X-!arctin (N-h~dros_vnaph~h~~limidc dirth~l phosphate) in tilt and nxlt by hydrol?:sis in 0.5 :\’ mcthanolic NaOH for fluaresccncc detcnnination of ihe liberntcd naphthalimidc moict~ has been dsveloped““. Carbm-~1 ( I-naphthyi N-mcth_vlc;trbam;ltc) and its mctabolitss hasc been determined by a number of \vorkers_ \vho used basic hvdrol_vsis and tluorescence of the free nr~phtholats anion for qumtitation in ;I variety of s;t~npleP~-“~_ Frei LVcr/.S”separated cxbaryl and I-naphthol by TLC and used scanning itrsirrrtluorimetr; for the evaluation of the sepaxted spots after hydrolysis on the plate b; spraying \vith 1 LV NtlOH_ The iluoresccnt mphthok~te anion obtained from etlch compound could be detected in low n;mogr;m quantities. Zcctran (~-dimeth_vlan~i~lo-3_S-svlul has been dc- _ N-niethrIcsrba!n~itc) termined by tluorescence ofirs hydrolysis producrS”_Thc tluorcsccncs bshaviour ofother carbamatc insecticides in neutral and basic media has been report&‘. Gibbsrellin spray used on cherries has been determined f~lorimerric~~ll~~ after treatment \vith strong xid’“. Renomyl :meth_vl N-[ I-(hut~lcarbamo~l)-2-he~~~i~~~id~~~~~l~l]c~~rb~~~~~~~te~~ has been analysed by flulwinxtry after hydrolysis to 7-ai~~inobenzimiciaz~~le~“_

The recent us-t‘ of meta chelates for the tluorinwtric mmlysis of pesticides is an interesting :lppro:lch to the determination of these compounds. Loelllsr and MacDoufall”” analysed DEF after hydrolysis to butylmercaptant \vhich was distilled into a

J_ F_ LAWRENCE.

15s

of a palladium

solution

&elate

of S-hydrosyquinolinesulplzollic

R. W. FREI

acid_ The mcrcaptan

complexed with the metal to free an equivalent amount of the sulphonic acid_ Mtlgnesium chloride was fhen added and the tiuorescent &elate formed with the sulphonic acid was determined_ Recently Bidleman et aL9’ simplitied the technique and applied

it fo the TLC analysis of organothiophosphorus pesticides. The method involved the of a TLC separation of the pesticides followed by sprayin, n the plate Gth :I solution calcein chelste of pttlladium. The thiophosphtlte replaced the calcein li$md of the cheltltc to release free calcein_ which is highly fluorescent in the uncomplesed form. Calcein blue xas also sstunined for use with this technique. As little as 10 ng of some o!gmophosphate \vith merctlptans

insecticides

was determined_

The

method

\sorked

cstremtly

\vcll

should provide ;t useful method for their analysis. Frei, Mallet and co-~\;orkersy’-“J 11;~e done much \\-ark on metal chelnte-pesticide interactions. They studied the etkct of bromination of or~~~notl~iopIiosplIc~r~~s insecticides on TLC plates before sprtlyins with ;L metal &elate sol&on_ Brominvtion oxidized the pesticides to liberate HBr_ The HBr then complssed \vith the metal of the cheltlte to free the l&id,

and

which Uuoresced_

\v;ts obscrvcd_

Thus

a tluorcscent

spot on a non-fiuoresctnt

background

A number

-1wnts were examined. The of metals and organic chelating L 2 method was applied to pesticides in \yater and blucberricsv3_ This s;nne process has been used to determine trace metalP_ The method involved estraction of the metals from sample material as their dierhyldithiocarb~liiiatc chelates. The chclatcs wxe then separarrd by TLC and the developed plates sprayed \vith a dilute solution of ;I non-tluorescenr ctdcein chelatr. The reaction imvlvcd a ligand-cschangc process in which the dieth~ldithic~cztrb3matc ligand replaced the cakein. \vhich then ttuoreswd in the uncomplesed form A number of ditli-rent chelates were csamincd for the dctection of the trace metals. Detection limits varied from 0.1-1.0 ng per spot for the metal

ions studied_

The use of pH-sensitil--c fluorescent indicators as spray reagents for the determination of sulphur-containing pesticides and amino acids separated by TLC has of ;L Iigand-eschange recently been eS;mGneduh-9’. The procedure was an adtlptation method of Frei and iUallety’_ The separared pesticides \vcre subjected fo bromination dircxtly on the TLC plate. This treatment osidizcd the pesticides to liberate HBr oT DDQ (I_?as a side product. Upon sprayin g the plate with ;1 neutral solution diehloro--L5dicy:mobenzoquinone)_ which tluoresces strongI>in acid solution_ ;L fluorescent spot \vas observed in the area of the sulphur-containing compound. due to the HBr. The method has general application to stron$v acidic compounds separated by TLC_ Detection knits were less than 100 ng per spot for some of the compounds_ D. P0larit-r

The rspIoi!ation of the elkct of polarity on rhe tluorcsccncc bchaviour of various orsmic compounds has led to an interesting tluorirnetric method for pesticide rinalysis. A number of Shydrosgflavones unsubstituted in the 5 position were found to be practically non-tluorescent in non-polar media. while cshibiting intense tluorescence in polar environmentsyS_ The use of such compounds for pesticide residue tmtll-

FLUORIMETRICANALYSISOFPESTICIDERESIDUES

159

ysis has been evaluated for polar compounds separated by TLCq9_ The developed plates were sprayed with fisetin (I), resultin g in a fluorescent spot where the pesticide was located on a weakly fluorescent or non-fluorescent background.

0 The system was not selective and NXS applicable to ;i wide variety of polar compou~~ds”‘n-*‘“. Detection limits \vcrc often less than 100 ng per spot of compound_ Linear calibration plots in the re$n of 0.1-2.0 jig per spot \VCI-IZobtained.

The analysis of :t number of organophosphorus compounds by heat trct~tmcnt of the developed chromatoplt~tes has recently been es~m~ined’~‘2_ The fluorescence obtained upon heating at sprcitic temperatures and times was quantitatively measured by iir sirlrfuorcscence spsctroscop_v. The xtual mechanism of fiuorescencc production is not vet understood_ although a number of tluorescent compounds \vcrc obtained. The heat tre:ltment of other pesticides \vhich exhibited natural tluorescencc has also been es;iiiiined”‘3. Heat treatment caused sigGtictmt shifts in fuorescencc escitation and emission maxima to longer w~uelengtk The fluorescence intensity of some COIIIpounds also increased greatly upon hctlting. \vhilc others decreased_ The detection limit of Fuberidazole [2-(2-furyl)-henzimid:lzole]_ for cstm~ple. decreased from I !IS per spat to 0_002 ;I% per spot after hcrtt tre;wnent of the developed plates;.

Reactions \vhich alter the chemical structure of compounds in such :1 III~IIIL’Ias to induct the ability to fluoresce have been receiving increasing attention in recent years. ivluch \vork has been carried out on the analysis of sugars by fluorigenic derivtltization. Trioses’“.’ have been detcrmincd by tluorescence after acid treatment and condensation \vith chromotropic acid_ He~oses’“~ havr similarly been tmalysrd after coupling with 5-I~ydros~tetraloIlt to form fluorescent derivatives. Amrline and [;naphthalrtmine \vere reacted \vith ketoses. psntoses and disaccharides in the presence of phthalic acid to produce fluorescence. which \v;ts subsequently mr:lsured>md equated to sugar content’“h_ Rogers er NI.“’ condensed hesoses and pentoses \vith resorcinol to form tluoresccnt furfurals. Detection limits \vere rrporred to be in the lox\ nanogram rang-c under alkalinr conditions. While these reactions may he of some use in the pesticides analysis of certain compounds_ no \vot-k has Frt been reported cm such investigitionsThe use of fiuorigenic lt~brllit~g reagents for the fuorimetric analysis of pesticides and other polhwnts has been recently examined. Lobelling nlay Lx detined as the attachment of a fluorescent “t:ls_” to the compound to be detected by replacing only a proton or single atom ofthe compound_ This dill-e1-s slightly from other conden-

_I. F. LAWRENCE,

160

R_ W_ FREI

sation reactions, which crettte fluorescent structures via the reaction_ Much work has been done on the tlnalysis of ctlrbonyl-containin? compounds. such zs aldehydes or ketones_ by tluorigenic k~belling. The retlctions could be of some use in fluorescence analysis of pesticides. ;IS mttny of these contain carbonyl functional groups. The use of l-diphenylacetyl-1_3-indancdione-I-bvdrazone for the fluarigenic labellinp of cat-bony1 compounds has been used in their analysis’“s_ Other hydrnzino nxgents such ~1s2-hydroxy-Gnethosybenzene hydrtlzide tmd 5-methos~-l-nitmsaIic?lI hydrazide have been investislted as possible tluoresccnt rengents for Gtrbonyl compoundslu“_ The use of o-phenylenediamine for the detection of glycolaldehyde by fhmrimetry and TLC b:ls recently been investigate-dK1”_ Sawicki ~‘1 c~i*“-‘*~ used the fluorescent derivatives of zltdehydrs obtained with o-phrnyIcnedinn~ine_ o-phthairtldcb~de tmd dimedone for the TLC z~naIysis of :I number of air pollutants_ Methyl ketones and c:-keto xids have been determined tufter fxrnin~ fluorescent derivatives using rl-hydrtlzino-2-stilbrtzolc”” The fluorimetric tmt:lvsis of thiols 11~1srecently been and o-nitrobenznldeh_vde”5. atten~ptcd’lh_ A number of lttbelling retlgents have keen eva!uated_ including a ff uorescent labelled malrimide_ This method could be used in pesticide residue tmttl\;sis_ as many sulphur-containins Festicides can yield tbiols upon reduction or hydrolysis_ Extensive studies on fluorimetric analysis have been made in amino acid tmd peptide chemistr_v_ FIuorisenic reagents for the laEelIin~ of biogenic amines. amino acids. .- peptides and proteins include 3,2-naphtI~t~lenedi~~ldel~~d~*‘~ _ sulphoindonyl chloride”S. 7.7.S_8-tetrrtcyanoquinodimrthnne’*~~nt~pl~tl~~l isocyxxltetL? o-phti~t~lt~ldel~~de’~‘~formttldshyde”‘_ dansyIchloride( I-dimeth_vlttn~inon~~pl~tl~~~Ie~~e-~-s~~lp~~~~~~~l chlorideZ”-*-“‘_ N BD chioride (4-chloro-7-nitrobenz-Z_ 1,3-oS;ldi3zole)‘J’-‘3’ and_ most recently. fluoresxmine ~Ltphenylspiro[t~ur~~~~-~(3H)_I-phtht~lan]-3.3-dio~~~~~*3s~*~~_ The latter three compounds show g-tilt promise for the determination of pesticides by fIuorimctry associated with TLC or HSLC_ Dansyl chloride reacts \vith primary :md secondary amines. phenols. some thiols and, to ;I lesser extent, :tliphatic alcohols. The dcrivaiives xc\ estrwiely fluorescent_ whiIe dansyl chloride itself is non-ffuoresccnt_ The reactions are usu:llly simple :md rapid_ N BD chloride is ;I newer_ less used reagenr lixamine analysb_ The interesting ai;iIity of this compound to I?.x-m intcnsety tluorescent derivatives \vitb only primary and srcondzlry rlIiphntic tmiines @-es it a great deal of selectivity for use as an analytical technique_ A further russet of this reagent is that the derivatives fluoresce at very high excitation and emission \vzlvelen_gths (.470-4SO nm excitation530-550 nm emission) which rue often signifkmtly diflerenr from most tluorescent co-extractives. Fluorescamine was only recently discovered as the result of an investigation of the fluorescent ninhydrin reaction LJJ-‘JJ_ This rengent readily produces fluowscent derivatives tvith only primary amines_ \vhile the compound itself and its hydrolysis products are non-ffuorescent_ A detailed discussion ofrach ofthese three labelling reagents as applied to pesticide residue zulalysis is presented in the following sections_ 1. REACTIONS

OF DXNSYL

CHLORIDE

WlTH

PESTICIDES

The dansyl IabelIin~ of these compounds 11~s recently been sccon~plisl~ed”5. The procedure involved the hydrolysis of the carbamates with 0.1 AZ NalCOj to form

FLUORIMETRIC

ANALYSlS

OF PESTICIDE

RESIDUES

261

The t\vo hydrolysis products \vcre then labelled with phenol and ~~~eth~:f;t~~~i~~r“.‘“. dttnsyl chloride and subsequently detected and qttantittttivel_v determined by TLC on The reaction conditions \vrre silica gel layers by if2 situ scrtnnin g spectrolluorimctr~_ An esamincd and optimttm conditions for hydrolysis and Ittbellin~ established’J7. overall reaction schctne is shmvn in Fiz_ l_ The separation of the phenol derivatives of a number of N-meth;lcttrbttiii~ttes has been accomplished by one- and t\vo-dimcnsional TLPS. The tlttoresccnce behaviottr and stability of the derivatives have been e.xan~incd’49_ IMost of the dansyl derivtttiurs fluoresced at shilar uxvslrngths (esciration z 363 mn. emission 2 520 nm)_ Fig_ 2 sho\vs ;t typical dansyl deri\-tttive fuorescence spectt-urn. The method has been applied succcssf~~lly to the antllysis of 10~1 levels of carbanxttes in water and soil samples \vith little or no clean-up reqttired15”_ Amounts as low as 1 ng equivalent of insecticide \vet-e detected instrumentttlly. Visual detection Iinlits uwe reported to appt-oxI1 S-IO ng per spot. it

I

Jo &A_ __-

IL

1

I

350

450

I

600

Nmometres Fig. 2. Excitation Baygon_ -.

and rmission spwtra of the amine Phenol: - - -. methylamine.

:md

phrrlol

hydrolysis

products

ohrained

tiom

J. F. LAWRENCE.

262 B. ~r-PJ~efrr_Jearbarrlats ad

R. W. FREI

w-m Jwrbiciclex

The use of dansyl chioride for the fiuorimetric analysis of N-phenylcarbamate and urea herbicides has been esamined *S _ Tlk herbicides \vere hvdrolysed in Z N N:tOH to release the anilincs. These products were then extracted from the hydrolysis misrure with hesane and spotted on a TLC plate_ An excess of dansyl chloride was added fo each spot and the plate left in the dark at room temperature for 40 min. The dansyl labelling proceeded on the plare. The dansyl derivatives were then separated by developing the plate_ -4 reaction scheme is shown in Fi,.‘1 3. This ittxi~rrreaction proved

to be cleaner

and more quantitative

than labellin s in solution_

Application

of this

l_ -

+

CH3NHOCH3+ CO<

Cl

2.

S02GI

Cl

7”” NH 3 0 Cl

Cl

Fi=. 3. Reaction scheme for the labcllkg of cxbama~c and urea herbicides Hydrotyvsis to anitinez Z =-=1;tbrlIing of aniline with dansyl chloride.

(Esamplc.

Linuron).

I

method to crop sampIe analysis was also esamined’5’. Linuron was detected in potatoes_ beets_ turnips, peas_ strawberries_ tomatoes, corn and oranges at levels of 0. I0.05 ppm. with only a single partition step as ckan-up_ AS litrle as 5-10 11~ofhcrbitide were detected visualiy under a LX lamp at Ion,0 \vavelenpths_ Instrumental dctection limits using a Zeiss chromatogram scanner approached I-1 ng per spot. C. OrgarropJ~ospJrateinsecticides Application of fiuorienic labelling to the determination of some organophosphatc insecricides has been attempted153-*5-L_ Compounds such as Fenthion (O-Odimeth~l-O-[4-~mrtl~ylthio)-~n-tol~l]phosphorot~~i~~~~te)_ Ruelenc (O-4-w-r_-but+?chlorophsnyl-O-methyl methylphosphoramidate)_ GC 64OG [dilllerI’lyl-Jo-(nlcthvlrhin)phenyl phosphate] and several others which yield phenols upon hydrolysis kvt’rc‘ examined- Detection limits for some of these Iabelled derivatives have been reported to be in the low nanogram range. The organophosphare Proban [010-dimethyl-0-( psuLphnmoylphenyI)phosphororhioate] has been directly determined without hydrolysis by dansyIarion of the free amino group on the n~oIecuIez55_ The derivative exhibited

blue fluorescence,

as compared

to yellow for phenol and alkylamine

dansyl derivatives.

D_ s-Tria-_ine herbicides

Identification

of residues of triazine herbicides

by reaction

and dansylation

has

FLUORIMET-RIC

ANALYSIS

OF PESTICIDE

recently been attemptedtjh. and alkylamines according

CH3

263

RESIDUES

The trinzines were to the equation:

found

1 NH

NLN

to hydrolyse

3

-

H

H

acid

N +

l-z=’

” C$N0kNA._‘H2CH3

to cyanuric

CH3CHzNH2

+ (Cil3I2CWlH2

d-N&H

The hydrolysis reaction was carried out in 1 N HCI for 2 h at 150” in :L sealed \Gal’57. The hydrolysis solution wts subsequently made basic and the mixture then dansylttted*56’. Triazines such as att-azine, simazine and propazine yield difi’erent combinations of free amincs on hydrolysis. thus enttblins their characterization using the fluorigenie lttbelling technique.

The analysis of hydrosybiphenyls, which may occur in the environment as mctabolites of biphenyls or po!ychlorinatcd biphenyls. has been investigated using Various dihydrosybiphenyl isomers were direct drtnsylation of the compoundP”_ labelled and separated by TLCtj” and HSLCtS9_ The reaction. detection limits. tluorcsccnce spectra and stability of the derivatives were esamined. 3. RErKTIOSS

OF NBD CHLORIDE

WITH PESTICIDES

The use of NBD chloride for the fuorescence analysis of~tIkyIamine-~encr~tti~~~ pcsticidcs !las been recently investigated Ihn_ A tawphast reaction svstem was employed for the hydt-olzsis and Ittbclling of N-mcthyland N.N-dimethtlcarb3m.I3te pesticides. The residue uxs hydrolyscd in O-1 Al N;12C03 and the liberated amine reacted with NBD chloride in ;LII organic phase ( MIBK) above the aqueous layer. An aliquot of the organic laxer was used for chromatography. Fiz_ 4 illustrates the reactions involved. 1_

2.

-

NH2CH3

+

&,o NO2

OH-

’

OH+ NH2CH3

:&+

+CO3=

HC’ NO2

Fig.4. Reaction scheme for the Isbsllin g of mrth~lcarbattxttr insecticides using NBD chloride. I = Hydrolysis of rhr insecticide)n the aqueous phase: 2 =: labrIling of methylamine in a merhylisobutgl ketone (MIBK) phase.

The method allowed instrumenta detection of in some cases sub-nanogram quantities of pesticides per spot_ Fig_ 5 shows the fluorescence spectra obtained for the NBD fluorimderivatives of methyktminc and dimethylttmine usins an Aminco-Bowman

J. F_ LAWRENCE.

264

Fig. 5. Fluorescence ( - - -_) adsorbed

spectra

of rhc

SBD

dr‘rivxive

of

merh~lamine

(------_)

and

R. \V. FREI

dimeth~lrtmine

on silica geI_

with a TLC attachment_ The analysis of Ztctran and Mataci! by fiuorigenic were the only onss to labelling produced an interestin, cx result_ These fwo carbamntes yield each two fluorescent NBD derivatives by the described ltlbelling procedure’“‘. The second derivative was found to be the NBD derivative of dimeth_vlaminc, resulting from the c1eavag-e of t!lc dimethylamino substituent from the benzene rings of these molecules_ The overall reaction scheme is illustrated in Fig. 6. The authors used NM R_ IR and spot tests to verify the results_ eter

Dansyl or MBD Derivziive Fig. 6. Reacrion scheme for the dam+4 Iabrl!ing of Zectrm_ I = H~drol~sisr 1 -- labelling: 3 ::: conversion of free phenol to the h_vdroquinone: 4 r= Iabrlling of the liberated dimcthglaminer 3 :.= osidstion to rhe corresponding bcnzoquinonc

Determination of triazine herbicides using hydrolysis and NBD-labelling~ has been eS;tmined’4h. Preliminary work indicated that after hydrolysis of the triazines in 1 M HCI for Z h at I50 e the ring amino groups werr clcavcd and thus capable of being of labelled with NBD chloride_ The method appears promisin, cxfor the determination triazine residues_

FLUORlMETRICANALYSlSOF 6. REACTIONS

OF

PESTICIDE

RESIDUES

265

FLUORESCAMINE

Unlike dansyl chloride ry smines to form fluorescent

or NBD chloride tluorescamine rexts derivatives according to the equation:

R--&H2

only with prima-

PH 7-9

The reaction conditions art similar to the drtnsyl It~bcllin~ of amines_ The reaction could be carried out on the amine hydrolysis products of carbamatcs, ureas and tritwines kvith a sin&u- sensitivity to that of dansyl chloride labelling. The analysis of amino acids”” and proteins 13s-13y11-1~ c _ alrc:idv_ shown the utility of tluorcscnmine 17~ the analysis of biological sampIes_ Because of its speciticity_ t!x applic:k~n to primary aminc-~eiierrltili~ pesticide residues appears promising_ 7. FLUORIGENIC

LABELLING

IN SOLUTION

.-\SD TLC

AND

HSLC

XNALYSlS

Most earlier tluorimetric methods of pesticide analysis utilized classical soIution t’fuoresccnce tiie3sltrt’liir’litS”-XJ. This type of qutmtitation is not suitabk fgr tluorcsccncc anal_vsis of pesticide residues because of the rigorous clean-up required in order to keep b!ank tluorescencc ~alurs to a minimum_ TLC associated with lluoresccnce and fluorescence derivatization techniques has solved to a signiticant extent the problem of tluoresccnt co-extractives. Chromato2*vaphv crc‘ates the Illc‘;lllS Of St’p:lrittin, tv t!x desired compound from impurities, thus more ;Iccurilte results. Fluorescent spots ~:L\Y Bern ~cr:tprd from TLC plates I4vini The recent development of scanning fuorescence and then quantified in solution’h’_ spcctrophotoiii~ters, as mentioned earlier. hiIs made spot removal unnecessary for quantitation. as these instruments are capable ofqunntifvin g tluorcscent spots directly on the TLC plattP-““. Holvever. the vnlus of TLC-ilz sitll tluorimetry as a method depends upon the chromatographic prccess itself. The control over the parameters is difficult. Often TLC layers are slightiy diil>r~nt_ the eluting solvent composition varies and the temperature and humidity !luctu;tte, makins plate-to-plate comparisons ditficult. The chr~~mato~raphy itself is often time consuming and irregular_ Extending the use of tluorescence techniques to HSLC has made the field of tluorescencs and tluorigenic labelling an even better alternittive to gas-liquid chromatography. The advantao,s of HSLC over TLC--ill sim scanning are that complete control over solvent composition and flow-rate may be attained. that HSLC has a greater separation e!liciency_ and that it is more amenable to automation and data processing The development of chemically bonded stationary phases for HSLC permits a new degree of selectivity which is m~likely to be developed in TLC. At present. there arc few tluorescence detectors :lv:Gl:lble for HSLC instrurccnts- The use of a converted Turner Model I I1 tluorimctcr for HSLC has keen cvuluatedh’. The detector was found to have a sensitivity range similar to that of the com-

266

I

J_ i=_ LAWRENCE,

I

I

I

I

i

I

R. W. FREI

I

Fig- S- Fluorescence emission spectra and relative intensities for phenol dnnsyl derivatives. Equal amounts of phenols (4 x IO-*’ moles per spot) were reacted. The spectra are given for the dansyl derivatiws of (I) phenol (SOO), (2) 14-dichlorophenol (500). (3) 2.4.5trichlorophenol (473, (4) + chlorophenol (.5101, and t5) pentachlorophenol (470). Figures in parenthestx are wavelengths of emission maxima_

FLUORIMETRIC

ANALYSIS

OF PESTlClDE

RESIDUES

267

mercial HSLC detector marketed by Laboratory Data Control_ The Turner instrument also had a filter system to permit measurements at a variety of fluorescence wrtvelenpths. The analysis of carbamate insecticides by fluorescence analysis and HSLC of their corresponding dsnsyl derivatives has been examined’h3. Fig_ 7 shows a HSLC separation of a number ofdansyl carbamate derivatives_ Fluorescent N BD derivatives of a number of rtlkylamines have been separated by HSLCL”3- Hydrosybipllerlylsl’V have also been determined by fluorigenic labellin~ and some organophosphates”’ and HSLC. The fluorescence characteristics of chlorophenol dtmsyl derivatives are shown in Fig_ S_ With regard to phenol and lower su’ictituted chlorophenol dttnsyl derivatives a hypsochromic shift in the emission mtlsima of the higher chlorinated derivatives is

observed

(Fig_ S)_

The tluoresccnce intensities of the diftkrent phenol dansyl derivatives were compared_ Equal concentrations of the phenols were used for the dansylation reaction. The large ditll-rrnce betlveen the fluorescence intensities of phenol and chlorinated phenol dtmsyl derivatives may be partially due to the lower reactivity of the latter compounds to\vards dansyltltion. Besides that. the phenolic chlorines esert a considerable quenching (heavy atom) et&t on tluorescencelnvesti&on of the chemical nature of the derivatives was carried out by mass spcctrometry. Mass sprctromrtric identification of the dansyl derivatives of biogenic aminrs has ken reported by several workers. Recently_ mass spectrometry of dansyl amino acids \v:ls investi~lted by Seiler or CI/.“~-‘~“.The mass spectra of dansyl derivatives (Fig_ 9) show characteristic major fragments at IIZ!L~170 and 171_ The occurrence of these major fragments indicates ionization of d:msyl derivatives. These molecular ions occur in all the spectra_ with m;tsst’s corresponding to calculated molecular \vcights.

techniques The recent tldvances in fluorimetric analysis usin g derivntization associated with TLC or HSLC have provided an important and varied basis for future work in this Geld_ The use of tluorimetry and HSLC \vill probably expand over a \vider area in the trace analysis of pesticides. industrial pollutants, drug residues and metabolites of these compounds.

J. F. LAWRENCE,

2’6s

R_ W. FREI

REFERENCES I Z 3 I 5 6 7 S Y 10

46 -c7 IS 4Y 50 SI

E_ F_ Knipling, Fuud uml Drug ddmimh-urim Pqwrs. 3 ( 1969) 16. P. R- Beljaars and F- H. XL Fabry. J. .-h_ OfJc_ _-Irrd Ch~wr.. 55 11971) 772. C. R_ Engel. Fhw~scc~rcc* Xw\-. 3 (196s) I_ H. Jerk. Arch. PImrm_ ( IVeinlrcimf.303 (1970) Bl01_ H. H. Chang. J. .-?_w-_Fuutl Cf~t~r.. I7 (1969) 1174. R- Hcndricksson and W_ R_ Meagher. J. &r_ Fuod Ckwr.. 16 (196s) 90X A. K_ Klein and J. D. Link, J_ A.ss_ Oj]ic_ Atrd CJW~JL, 53 (1970) 524 D. J. Sissoti and G. hi. Telling, J_ Ch-o~~~uro.~~-__37 (lY70) 3X D_ J. Sissons and G. %I_ TelIing. J. Chronrcucy-_, 4S (1970) 46s. D. P. Johnson and H. A. Stansburv_ J. Ass_ Olfic-_ And_ Cl~cwr.. 49 ( lY66) 3YY_ \V_ H. Gutennrann and D. J_ Lisk,J_ _-tgr_ FOCJ~~Chat.. 13 (1965) 45. R_ J_ X~auer. _-tmrl_ C~WK, 40 t 196s) I??_ E. R. Holden. W. >I_ Jones and Xl. Bcroza, J. Agr. F&d C~WJJ_, 17 (1969) 56_ C_ S. Lau and R. L. Xlnrsmill. J_ _-fgr_ Firotl C~JCVJJ.. IS (1970) -I13_ D. C. Abbott and P. J. Wtgsralfe. J. Chronmru.~r.. 43 ( lY6Y) 361_ M_ Look and L. R. Whiw. J. Clrronrctrc~gr.. 50 (1970) l-55. Rl_ _A_ El Dib. J_ _-tss_ OJ&_ =Irnrl_ ChcvJ~.. 53 11970) 756. D_ C_ -Abbott, J_ A. Eunting and J. Thomson, .-Irtu&.u f LLJIJ~~JIII. 90 ( 1965) 356. K. C_ Walker and hl_ Bcrozn. J. _-ks_ 0,&c_ _-lnul_ CX~nJ__ 46 ( IY63) 250. D- C- Abbott- K_ \V_ Blake. K_ R. Tarrant and J. Thwmon. J. C~JJ?JJJJCJfOJ.yJ-_. 30 ! 1967) 136. %I. Ramasamy, =Irrul~sr i.!_umkmj_ 91 1lY6Y) lY75_ Xl_ Ziclinksi and L. Fishbcin, 1. &LX CIJF~JJIJU~~J~J-..3 119651 I-P_ W. P. Cochrmr and _A_ S. Y. Chau, Ack_ Clrem. SW_, ( lY7i) 1 I_ D_ _A_ Cronin and J. Gilbert. J. Chmoro~~r_, 71 ( 1971) 151. L. Gasco ad R. Barrma. .-lrral_ Ckir,r_ _-km. 61 (1971) 152. Xl. T. Shatik, D. Brad\\ay and H_ F_ Ems. Bull. Em-inm. Cmrrwn. Tosid., 6 I lY71) 55. L. Eaborn_ D. R. Xl. Wtlton and B. S. Thon~as. C/mm I&_ ILoJJ&JJJ~. ( lY67) s17_ L_ Fishbein and W_ L_ Ziclinski. Jr_. J. Clrrmmrr~~g-., 10 ( 1965) 9. B. S. Thomas. C. Eaborn and D_ R. Xl. Walton. Ciwm. C~JIJJJJJJJJJ..13 ( 1966) 1OS. C. A_ ISache. L. E. St_ John and D. J. Lisk. _-IJJc~. C/J~JJI._ 10 ( IY6S) 1241. G. T. Flint and W. A. Xuc. J. Ch-or~rtrr~~g-_. 32 ( lY70) 175 D. E. Clark. J. _-Igr-_ Ff-‘ood C%rm., 17 ( IYhY) ll6S. P_ E_ Mattssm and \V* J. Kirsten, J. _-1.y. F~JOC~C/WJJ.. 16 ( 196s) YOS. G. Yip. J. .-I.~~_ O&c_ _-IJIuI_ Clr~v~r.. 53 (1971) 3-G. H. A. Moyr J. _-l-lss_O/fir= _-tend Cfw~n., 51 (IY6S) I260_ S. Sumida, 51. Tzlklki and J. Xujanmto_ =Ig,-_ Bid. C/rn~r_. 3 11970) 1576, 1. C_ Cohen and B. B. \Vhtxalsr 1. CImnmrr~~~r.. 43 (ISthY) li3_ XI. Fe Cmruner and J. Freal. L(fi Sci., 9 (1970) 111. J- Askew_ J_ H_ Ruzicka and B. B. \Vhcatr, J. C/JJXJJJrfJfoJ.yJ-_. 41 (1969) ISO. G_ D. P;Iulson and C. E. Portnoy. J. _-lg~-_ ~GJOC/C%L,JJJ._ IS 1 IY70) ISI). H. A. hlclcod and K. A. ~~lc~ully, J. ;t.v.\-_O./&c_ _4Jrtd_ CIWJJJ.. 52 I !Y6Y) 126_ J_ R_ Lme. J_ _-f.q-_ FCCJC~Ckm., IS I IYiO) WY. G_ E. Keppcl. J. Am-_ O_flic_ drrtrl. CIJ~*JJJ., 52 f lY6Y) 161_ A_ W_ \Vt%hke_ F_ A_ Gunthrr and \V_ E. \\'~Adx. J. ;-?g:,-.I-;ux/ C/wm.. 17 t 1969) I l5_ G. G. Guilb~ult [Editor). Fluorawncc: Thcur_v. IJwrJ-nJJrrJrraric,lr ~JJJ~ Pmc-riw. l\ltmxl Dekker. New York. lY67.

FLUORlMETRlCANALYSlS

OF PESTlClDE

RESIDUES

269

W. Frei and H. Ziircher, AfikrocftiJJt. Acts, (1971) 209. Sawicki, T_ W. Stanley nnd H. Johnson. ~~ficrochttt. J.. Y (1961) 257. Niwaguchi and T. lnoue, J. Cltrotttrtr~~gr., 59 (1971) 127_ C. Madsen and H. W_ Latz. J. CfJroJ~tor~g-_. 50 i1970) 2%. W_ Frci, A_ Kunz, G. Pntaki, T. Prims and H. Ziircher_ Ami_ Chint. ilcttt, 19 (1970) 517. 57 M. Doss. B. Ulshiifer. W. K. Phillipp-Dormrton and T. Phil1ipp.J. C~~JJJJUJ~J~J-_, 63 ( 1971) 113. 55 S. SilvLatcr, Pltr~rm. dcru Hdr-., 45 (1970) 3YO. 59 W. Schlcmmrr, J. CJn-untutogr_. 63 11971) 121. 60 N. St&x and M. Wiechmann, Ho~ppcSc_dm--s Z. Ph_vsid. Cltcv~t__337 11964) /?Y. 61 A_ A. Boulton and V. Poll&. J. Chrotmttogr_. 63 11971) 75. 61 N. St&r, J. Cltrmtuftyr-., 63 (1971) 97. 63 E. Sawicki, T. W. Strtnlc_v and W_ C. Elbert, .I_ CItt-u~~trttc~gr.. 10 (1965) 3%. 64 J. Goldnun_ J_ Chrt~tt:ttrc~_~r_. 75 (lY73) 7. 65 Cf. Zfirchcr, G. Pat&i_ J. Borko and R. W_ Frci_ f_ Chor~ttttty-.. 13 ( 196Y) 457. 66 V. PoIlnk and A. A_ Eouhon. J. Cftr~Jt~t~tt~:~~-.. 72 (IY72) 731. 67 ~1. A. Boulton and V. Pollak, J. Citrotmtoqt-_. 15 11969) lSY_ 65 R. Klaus. J. Cf~Jr~/~:tttcJ.~t-__ I6 1196-l) 31 1. 6Y R. Xl. Cnssidy and R. W_ Frci. J. C~~I-~JJJ~~~~J.~J-_. 71 (1972) 2Y3_ 70 E. D. Pcllizzari and C. Xl_ Spnra5no. Amf. CJJLVJL, 45 (1972) 119-l. 71 D. D_ Chilcotc, Chit. C/JLVJ~.,IS 0972) 77s. 72 D. D. Chilcotc, Cht. Ckrtt.. lS (1971) 1376. 73 S. Katz and W. \V’. Pitt. .-~rtctI_Lctt., 5 (1971) 177. 71 L. H. ?‘h~kcr, J. CftJ-~JJJtfttrJ~J-.. 73 ( 1971) 117. 75 L. R. Snyder. Primiph c?f’Aclso~-ptiottCftrotJtut~J.yrufJft~. hlarccl Dckker. New York. 1965. 76 J_ J_ Kirkland 1Editor). .~focfcr-JrPructiw ofliquid Cltrontatcgrttp~~. Wiley. iKc\v York, 19 7 I_ 77 I. Hornstein. J. _-I.w_ fomi Char., 6 ( lY5S) 32. 78 C. A. An&rson_ 1. .-lpr. fia~rl CIJ~JJJ., 7 ( 1959) 256. 79 _I. 31. Adams and D. ~lxDoup;~lI. C/Jorrtr.yJw Cor~J__ Tdt. Rep.. No. 7075. 1961. SO J. X1. Adanis and C_ A. Anderson. J. _-l.w_ I-;Joc/ CitrrJt.. 1-l (1966) 53. Sl R. J. Anderson. C. A. Anderson and hl. L. Yagclowish, J. _4gr_ FotJd Citntt.. 11 (1966) 43. S1 J. B. Knack, X1. S. TAant. W. S. BarrIcy and L. J. Sullivan. .I. .-lgr-_ I-iJocf CfrrrJt.. 13 (1965) 537. S3 R. J. Xrgauer, J. _-ts.v_Ojjk-_ _-lmI. Clr~w., 53 (1970) Il66_ S-l R_ J_ Xrgauer. t-i. Shimanuki and C. C. Xlvarcx. J. .4gr-. I-;mI Cftcv~t., IS 1lY70) MS_ S5 R. \V_ Frri. J_ F. Lnwrcncc and P. E_ Bcllivcau, % .-1trttf.Cftc*ttt..1% ( 1971) 171. S6 1~. 0. Ebcrlc and F_ A_ Gunther. J_ .-lxs. O&. .-l~~ttl. CJtc~~rt..-IS i 1965) 927. S7 51. C. k3owt1s1iand ;\I. Bcrom. RK Rrl-.. 17 11967) 13. SS K_ G. Parka-. L. E. St. John and D. J. Lisk_ J. =Is.s_ O/fc. _-lmtl_Cftrr~t.. 40 (IY63) YS6. SY H. L. Pcasc rend J+ A_ Gardincr. J. A_~J-. F&J~/ Cltrnr.. 17 t iY6Y) 267. 90 13. W. Locttlcr md 1). MacDougall. CIIIVJI~J~~~JGJrp. Tdt_ RcfJ._ No. 51 19, 1960. Y1 T. F. Bidlrman. B. Xowland and R. \V_ Frei_ .-l~td. CltitJt. .-lcrrt. 60 ( lY71) 13. 92 R. \V. Frci and 1’. Xlalkt. Ittt. J. Em-it-on. _-l/tttf. CftoJt.. 1 1lY71) 99. Y3 R_ W_ Frci. V. Xlallet and Xl_ Thii-hault. Itrt_ J. t5twhrt_ .41tct/. CIIPIK. 1 (1971) 141. Y-I B. E. Brllivca~_ V. Xktllct and R. \V_ Frci, J. C/tr~mtut~~g-.. -IS (1970) 47s. YS R- Xl_ Cttssidy. V_ Xlikrtukova and R. W. Frei. =Irtctf. Lctt., 5 (1972) 115. 96 1’. 1% Brllivcau and R. W. Frci. Cltl-~~Jttctro~~t~ttpltitt. 4 ( 1971) ISY. Y7 P. E. Brlliwau. Ph.D. -TIte.~, D:~lhousic University. Halifax. N-S.. lY71_ YS v. Mxllct and R. W. Frci. J. Ch-~~t~tttt~~.~~-_. 5-I ( 107 1) IS I_ YY v. Xktlkt and R. \V;. Frei, J. Chrontcttoqy.. 56 (1971) 6Y. 11111\‘_ XIalIct and R. \V_ Frci. J. Cftt~c~J~trtto~.~t-.. 60 (1971) 113_ 59 (1971) 135. 101 R. \V_ Frei. V. Msllct and C. Pothirr. J. C/trtmurf~~t-.. 101 G. L. Brun and V. Xlallct. ht. J. EminJn. .4Jtttl_ C/J~VJJ..3 (1973) 7j_ 103 v. Mzlllrt, D. Surcttc and G. L. Brun, .I. Clrt-uJ~tutoJgJ-..79 (1973) 217_ 10-l T. Nrtksi. Xl. Koyanm rind H. Drmurq J. Cftro~~tttr~~~~-.. 50 (lY70) 338. 105 Y. Ohkurrt, Y. W’atmahc and T. hlo~nose, Cftr~~t. PJturJ~t. Bull. TCJX-_IW. 19 (1971) lS-l?_ 106 L. C. Lo&-, Uttir. Stttrf_ Tricsrc, FM-. Ecott. C~JJJ., ht. ~~let-c-eel..3 11965). 107 C. J. Rogax. C. W. Chambers and N. A_ Clrtrkr. _-lttrtf_Ch~wt.. 3S 1 lY66) lS51. 10s D. J. Pirtrz~k and E. I’. Ghan, ArtttI. Cftrr~t., 42 11970) 37.

52 53 54 55 56

R. E. T. B. R_

2770 10s) I10 111 IIZ II3 114

.

J. F. LAWRENCE.

R. W. FREI

O- N- Devgan, S_ K_ Gupttl and M. M. Bokadkt. J_ fllrl.k~ Chm_ SM.. 42 (1965) 30.5 T_ Nnkai. H. Demum and M_ Koyama, J_ CJrranrurugr_, 66 (1971) Si’. E. Sawicki and J_ D_ Phti. C/rw~_ Arruf.. 55 11966; 6. E- Saxkki and R_ A_ Carries_ dJikrurJrim_ Acta, (1967) IlS. M. Guyer and E_ !&wick drrul_ Clrim. Actu. 49 11970) 182. S. hlizutani. Y. Wztkuri, N_ Yoshidtl, T. Ntkajimi and 2. T~murzt. Clrmr. Phurm. Bull_ Tokyo, 17 1IY6Y) 2340. I15 D. N_ Kmmer, L. U. Tokntino and E_ B. Hackley, _-fnuf_ C/rmr.. 44 (1977) 2243. II6 T. Sckine. K. Ando. M. Machida and Y. Kanoaka, .4rrul_ Bioclrcnr., 45 (1972) 557_ I Ii’ T. Amino. ~~lktr.ytrkrr Zusslti (J. Plrurtm Sac_ Jcrp_), 86 ( 1966) I_ I IS C. P_ Ivanov and Y. \‘hdo\‘skn-YuIrhno~s~, Biochim. Bioplr_vs_ .4ctu, 19-l (lY6Y) 315. 1 19 Z. DeyI. J. C-hrunrum_yr_. 4s ( 1970) ljl_ 120 I__ Edvinsson. R. Hakanson, A_ L_ Ronnberg and F_ Sundlcr. J. Clrromtrto~~r.. 67 ( 1972) St. Bioclretrr., 46 11972) 473. 121 L_ Edvinsson, R. Haknnson and F. Sundler, Ad_ 14 (IY6S) 302. 122 C. R. Crevling_ K_ Konda and J_ \V_ DaIy_ Clirr. Cknr.. 123 R. Dvir and R. Chn\-den, J. C/rromuro.~r_. 15 (1969) 76. 124 V_ A_ Spikak and V_ V. Shcherbukhin. _&r~d_ Biuchltrwr. 33 (lY7 1) 171125 L_ P_ Penzes and G_ \V- Oertel. J_ Clrrmmzco.~r_. 74 (1972) 35Y_ 126 N. Seiler and M. Wiechmxm. Z_ _-lmrf_ CIrmr., 270 (1966) 109. 127 N. Seiler and A. K. A&w_ I_ Chmnruro~gr.. 63 1197 I) I?[_ IX K_ Seikr. JJ~*:lruci.~ Biuc-iwm Arurl.. IS 11970) 259. I?Y 1\;‘_Seiler and J. Xl. Schroeder_ Bruilz Rex, 21 11970) S I. 130 S. SeiIer and H_ Schneider. Jh Chrcmruro~~r.. 5Y (.I97 i ) 367. 131 P_ B_ Ghosh and XL \V_ Whitchousc, Biuchvm J., 10s I lY6S) 155. 132 J_ Reisch. H. .Aks_ H. J. Kommerr. X:. Santos. El. Mnllnxm and D. Chnsin~. Plrcmmrzi~~_ 15 llV7O) 331_ 133 Anonymous, R&s :~cn- Prducr ;~;-a-A-_Regis Chemicrtl Co__ Chicago. III.. 1971. R. J. Bath and I. Sunshine_ Chr. C%LWI.. IS ( 1971) 13lY. 131 J. hlontfortc. 135 Anon)-mous. ivurcrs .-fss. CIrronru~o~~r. Cur.. IY70. p_ 36. :I36 Xnon~mous, Ifirrcm .-lss_ Clrmuruxo~r_ Cur__ 1970, p_ 37. 137 Anonymous, IKuters _-lss_ Chrormrogr. Cur_. lY70, p_ 3% 51 (1973) $77_ 13s A. Xl_ Felix and Xl. H_ Jimenez. _-i~rctf_Biochx. 13s) P_ Bijhlsn. S. Stein, \V_ D&man and S. Udcnfricnd. _-lrcfr. Bioch~r. Bioplr~~. 155 11973) 213. IN S. Stein. P. Biihlen, J_ Stone. \V_ Drrirman and S. Udenirisnd, _-ircir_ Biochrnr. Biophp-., 155 ( 1973) ‘0’ _ _~naI_ Bicdt~w., -I? ( 1071) 227. 111 K. Sxnejima_ W. Dairman and S. Udenfriend, 1-17 K. Samejima. W_ Dairmtm. J. Stone. S. Udenfriend. .4traf. Bicdrcnr.. 17 11971) X7. IS3 %I_ WeigcIe. S. L. De Bernardo, J. P. Tengi and W. Lcimgruber. J_ _-11m~_ Chtv~r. Sm-.. 9-l llYi1) 5Y27_ 1-l-l S_ Udenfriend- S_ Stein. P. Bijhkn, W. Dairman. W. Iximgruber and XI. Wei~ek. S~iwrw. 17s 11973) S71_ l-?S R. W_ Frei and J_ F_ Lawrence, in A. S. T;lhori (Editor). :\Ir*rlw&x irr Rcsidrrc- =Irur[vsis. Paxkidc Clremtifry. VoI_ 4. Gordon and Breach. London. 1971, p_ lY3. 146 J_ F_ Lawrence and R. \V_ Frei. kr_ J. EtrG-mr. _-l~rui_ Circvn.. I (1971) 317. 147 R_ W. Frei and J. F_ Lawrence. J. C~rrumurtr~r.. 61 ( 1971) 17-I_ 145 J. F_ Lawrence. D. S_ LeGa\- and R_ W. Frsi, J_ Clrromuro_yr__ 66 (1972) 295. 14Y J_ F_ Lawrence and R. W. Frei, J. Chronrurqyr.. 66 (lY72) Y3_ 150 R. W. Fr& and J_ F_ Lawencc. J. Chmmnrrq~r., 67 (1972) $7. I51 R_ W_ Frei, J. F_ Lawrence and D. S. LeGay, _-Irrcr~~.stILurnlmrj. YS 11973) Y I51 1. F_ Lawrence and G. W. Laver. J_ Asx. Offi_ ,411ul. Ckm. in pnss. 153 R. Nr’. Frei and J. F. La\vrence_ J_ Clmmrurogr_. S3 lbY72) 371. 154 C_ Porhisr, M_Sc_ Tksi\_ Dalhousie University. HnIifa.ri, N.S.. 1972. 155 J. F. Lawrence and R_ W_ Frei. unpublished data, lY71. 156 J_ F_ Lawenczt, unpublished data, 1973. . 157 .A_ CLY and J. Gaspxi~. Mi.bmlrir~r. .Actrcr, (iY7~) SZ:_ 15s %I_ Frei-H%tsler. R_ W_ Frei and O_ Hutzinger. J_ Cfrrurnu~q~r., 79 (1073) ZOO’):S-t (lY73) 114. 15Y R. kl_ Cassidy. D_ S. LeGay and R. W_ Frei. J_ Cfmmmrqy.. submitted for publication. 160 J_ F_ Lawcnce and R. W. Frei, .&uf_ C/w~zt., 44 t.1972) 1016. 161 R. W_ Frei ami J_ F_ Lawrence, J. An_ Oflc_ AIIUL Clrrrtr., 35 (1972) IXY162 S. Blackburn, _-lnti,ro Acid Derrrtmkrtion. Marcel Dekker, New York. 196S, p_ 16s. 163 R_ W_ Frei, J_ F_ Lavxcncc, J. Hope and R. M. Cassidy, J. CIrrunruro~r_ Sri.. 12 llY74) -lO