(3) Beach, -1.L., Guldner, W.G., ANAL. CHEXI. 31, 1722 (1959). ( 4 ) Booth, E.. Bryant, F. S., Parker, A , , d n a l y s t 82, 50 (1957). ( 5 ) British Iron and Steel Research Association, “1)etermination of Titrogen in Steel,” Special liept. Yo. 62, Iron and Steel Iristitiite, I,ondon, 1962. (6) Chipman, J., J . Iron Steel Inst. 180, 97 (195.5j. ( 5 ) Cost, J. It., FTert, C., ,letu X e t . 2 , 231 (1963). (Si Ilealy, J. lI., Pehlke, R . D., Trans. .1/JfE 227, 1030 (1963). (9) I)iishman, S., “Scientific Foundations of High \‘acuum Technique,” Wiley, Sew York, 1962. ( I O ) Ehrlich, G.] Friedrich, K., r e u e Hiietta 8 . 27 f 1963). (11) El Tnyeb,‘S. i l . , Parlee, X., Trans. AlZfE 227, 929 (1963). (1%)Evens, F. l l . , Fassel, V. A., AKAL. C H E ~ 36, I . 2115 (1964). (13) Everett 1I.Ii.,=Innlyst83,321 (1958). (14) Everett, 11. R., Thompson, G . E., Ibid., 87, 515 (1962). (15) Fagel, J . E., General Electric C o . , private commiinication, Cleveland, Ohio, 1963. (16) Fagel, J. E., Smit,h, H . A . , Witbeck, R. F., ANAL.CHEW31, I l l 5 (1959). f\ -l .i ,) Fast. J. - I).. Verrijp, AI. F., J . Iron Steel Inst. 176,’24 (1954). (18) Hansen, AI., “Constitution of Binary Alloys,” McGraw-Hill, New York, 1958.
(19) Holt, B. D., Goodspeed, H. T., ANAL. CHEM.35, 1510 (1963). (20) Ihida, 31.. Bunseki Kaaaku 8. 786 (1959). (21) Karp, H. S., Lewis, L. L., lIelnick, L. U., J . Iron Steel Inst. 200, 1032 ( l w-~ l2~. \ -
( 2 2 ) Kabhyap, V. C., Parlee, K.,Trans. A l d f E 212, 1 (1958). (23) Klyachko, Yu. A., Chistyakova, E. >I:, Zavod. Lab. 25, 1519 (1960).
124) Kubaschewbki, O., Catterall, J. A., “Thernioc*hemical Data of Allovs.” ” > Pergamon Preys, London, 1956. ( 2 5 ) Kubaschewski, O., Hopkins, B. E., “Oxidation of 11etals and Allovs,” Academic Pre.., Sew York, 1952 (26) Langenberg, F C , Trans AI41E 206, 1099 (1956) ( 2 7 ) AIallett, 11 IT.) Talanta 9, 133 (1962) ( 2 8 ) Xallett, lI W , Griffith, C B , Trans ASJT 46, 375 (1954) (29) 11asioii, C It Pearce, 11 L , Trans dI1fE 224. 1134 (19621 (30) lIcDonald, I- “conditioned” ( 4 4 , 45, 63, 277) before they can be used routinely to obtain good qualitative and quant,it,ative results. 13an-y and Hundley ( 1 7 ) have edited a icide ,\nalytical llanual” conig a c~~mliilation of methods and othw inforiiiat,ioii useful to a residue analyst, Although developed as a guide for chemists in the laboratories of the Food and Drug Administration, it has been distributed to many others as well. I t presents information on sampling, estraction, cleanup, and determinative procedures; techniques for preparing and conditioning columns; and lists of relative retention times and detectable quantities of many pesticides for different chromatographic procedures and detectors. Although gas chromatography with various detectors is now the niost popular technique in residue analysis, other procedures have not been neglected. Thin layer chromatography, with its greater speed of development and increased resolution and sensitivity, has to some extent’ replaced paper chromatography. Colorimetric, ult’raviolet, and infrared procedures allso are used. , 7
G A S CHROMATOGRAPHY-GENERAL
The greatest advances during this period have been made in gas chroniatograiihy. I3ecause of the estreme sensitivity of the electron capture detector. it has been used by many workers. Diiiiick and Hartniann (64) have ~)riblisheda general dewrilltion of electron caiiture gas chromatography as used in 1)esticidr anal! thf 1)rincil)le of electron capture and thc>geomrtry and operating parameters oi thc tietcvtor. Although only one si)wific instrument is described and sonic of the steps in the outlined proccdui.t. have since been improved, this r(,port does provide a good int’roduction
to those unfamiliar with the subject. K i t h the search for ever-increasing sensitivity and speed of ana come the realization that these goals encourage the production of methods in which unreciognized side effects, minor interferences, slight amounts of contaminants, and any lack of care in the use of cquiimeiit or interpretation of responst’s can protluce greatly misleading and inaccurate results. Lovelock (133), in a general discussion of electron absorption detcctors, points out that with complex mistures (.iuch as are usually present in residue analysis) thwe detectors may give ” . . . not only inaccurate hut even totally false results.” Causes of 1-arious false responses, both positive and negat,ive. are discussed and a pulse-sampling technique which minimize3 the errors is described. I3arney, Stanley, and Cook ( 2 6 ) , working with S>-stos, hai-c. shon-n that in a poorly designrd detector, electron capture and ionization may take place a t the same time and that pulse mode of detector operation will not, eliminate interferences froin ionization. Burke and Giuffi,ida (44)point out the need for adeqiiat,e cleanup before a sample extract is injected into the electron capture gas chromatograph. They show that injection of poorly cleaned extracts may contaminate a column and result in weak or spurious responses. Since solvents used niiist be “pure,” redistillation is frequently required. The use of plastic containers for solvents is discouraged, since estractables in the plastiw may cause response of the electron capture detector. In some cases these spurious effects are so strong that responses due to pesticide residues may be completely masked. The need for proper preconditioning of the gas chroinatogralihy column ip also thoroughly discussed. I-nless properly conditioned, the column may cause degradation of some pesticides. S o t only niay the degradation result in loss of the pesticide hut also the degradation products may cause responses a t t,he retention times of some other coliinion pesticides for which they may be mistaken. Equipment and operating parameters described permit detection of chlorinated pesticides, such as heptachlor eposidc, at levels of 0.01 to 0.001 p.p.ni. Relative retention times are listed for 65 pesticides. nelli, Hartmann, and Dimick (,35) ibe two colunins used Ivith electron capture gas chromatogralihy. I’cstirides which manot he rmolveti on one column may be resolved on the other. Operating paraiiieters, sensitivity data, arid retention tiiiies for a number of pesticides, including chlorinated, organophosphorus, and organosulfurs, are given.
Burke and Hol.matfe ( 4 5 ) present similar data for iiiic,roconloiiir,tiic gas chromatography. Iieterition tiiiies rclative to aldrin are 1istc.d for 8; chlorinabed compounds, and t,hosc relative to sulfone for 26 thio cornl)ounds. .Itahle which the amount of each pesticide required to give a hdf-full scale rcwirder found very useful. deflection may Recovery (lata indicaatc. that, rehl)on-;es are linear whcn the pesticide i. present above a d!:finittl iiiininiurn quantity. The w e d for p r o l ) ~cleanup of saml)le extract before injcrtion i.; emphasized, and conditions and I)rwautions for most effcctiw we are dc~-cribrtl. The general levcl of wisitivity ol)tainaIde is givcn as 0.01 l ) . p . i i i l . Shuiiian antl Collie ( 1 77) dewrihe the preparation of a gas chroiiiatogi.al)liy column j they also miphasize the need for 1, ro 1) r r conditioning , T h r y r c(1o 111mend a 6-foot, 6-111m. i d . coluiiin packed with lOT0 Don- C‘orning 200 (12,500 centistokes) qilicoii fluid on -1nakrom A I B . Other ~ v o r k ~ r(44, s 45) have also found thiq type of column superior for pesticide lvork. Dc Faubw-LIaunder. Egnn, and Rohurn (63) give detail< for pre;iaring a column. Good columns. columns which decompose Ix-ticidcs, construction, linearity of reslmnses and rleaniiig of det,ectors, effects of rate of injcction, peak iiieawrenients. antl chroniatograni interprrtation are discussed. The i i c e of a glass injwtion liner is reconiniended. Beckman and I3evenue (8.5) studied the effect of coluiiin tubing composition on recor-ery of chlorinated hydrocarlion?. WT‘orkiiig with 6-foot by ‘4-inchcolumns and a i.liicrocoulometric detector, they checked column. iiiade 1 x 3
It, has been apparent for soiiie time that complete reliance on the retention time for identification of a compound niay yield erroneous reiults. Robinson and Richardson ( 2 70) emphasized the need for caution in interprcting the results of gas chromatography of plant and animal extracts, both a. to idmtity and quantity. when only one coluinn is used. They described four different columns and tabulated the resolution of pairs of pesticides on these various columns. Goulden, Goodwin, and Davie? (88, 89)were also concerned about improving the certaint?. of identification. They found that a column packed nith a 2.57, silicon oil and 0.25% Epikote 1001 on Celite gave good resolution. With column teinperature of 163” and gas flow of 100 nil. nitrogen per minute. they obtained romplet,e .separation of at least 11 pesticides in 30 minutes;. They also proposed the use of simultaneous chromatography wing five parallel VOL. 37, NO. 5 , APRIL 1965
131 R
columns leading to one electron capture detector. The stationary phases of the columns differ so that 3 t,o 5 peaks may be obtained for each pesticide. They called thih a “sl)ect,rochroniatograni” and stated that the pattern is characteristic of the specific I)est,icide. They also described the use of a halogensensitive cell of the type used in detecting refrigerator leaks. The response to individual chlorinated pesticides of this cell differs from that of thc electron rapture cell. Hy connecting this detector in series with the electron capture detector and recording responses from both dPtectors. identification of individual compounds is made more reliable. Prograiiiined temperature gas chromatography is also coming into use as a means of imliroving rwolution and separation, spAeding up runs. and chromatographing mistures containing both veq- fast and very h v l y eluting compounds. Burke 143) used programmed temperature with a microcoulometric detector: he tabulated relative retention times for 22 compounds. Other workers have also used this technique (20, 36, 118). The technique of preparation of a derivative of a Iiesticide before injection into the gab chromatograph has been continued. For some time, it has been the Iiractice to convert 2,4-D and other chlorinated 1)henoxy acid herbicides to their methyl esters because t,he free acids will not ])ass through the common gas chromatographic columns. Derivatives are now being used for othei, reasons. Klein and K a t t s (220) found that Pert,hane, o,p’-DDT: and p,p‘-DDD have similar retention tinies and are difficult to resolve on many gas chromatogral)hic columns; however, olrfins of these three coinpounds prepared by reflusing cleanetiu p sample estract:: with 2% KaOH in ethanol were separated on a 3-foot gas chromatogra1)hic column of Celite 545 with a 2.57, coating of SF-96 and 2.2 - diethyl - 1,3 - propandiolisophthalate polyester (1 : I ) , Klein and M-atts obtained recoveries ranging from 84 to 105% from samples of leafy vegetables containing residues added at levels of 1 to 10 p.1i.m. One striking benefit, from the use of the olefins is that the Perthane olefin gives an electron capture response about ten times greater than Perthane. Beckman and Rerkenhotter (20) used derivatives to increase the reliabilit,y of identification of pesticide residues. They separated the individual conipounds by gas chromatography with a thermal conductivity detector and then dechlorinated the individual fractions with sodium and liquid ammonia. ;\fter that they chroniatographed the dechlorinated portions again and obtained chromatograms. 132 R
ANALYTICAL CHEMISTRY
The results obtained from the two chromatograms can be used to characterize the pesticide. Gutenmann and Lisk (97, .9Y) prepared brominated derivatires which had strong electron capturing ability and chromatographeti these as a nieans of obt,aining increased s(’n>itivity. They worked \vith diphenyl, Guthion, N C P . and MCP13 as pure coml)ounds, and also used this technique to determine residues of CII’C, monuron, diuron, and liriuron in fruit> and vegetables. Recoveries from crops of 76 to 1167, were obtained at levels of 0.05 to 1.2 p.p.ni. ivhen only one l)e>ticitle was present at’ a tinie. word of caution may lie in order about using thi. technique in any 1)rocedure which does not invludc estrnsive cleanup of residue kiefore gaq chroniatography. Valuahle information may be obtained when only one pesticide i; added and when the untreated crop is available so that chromatogram> of sanilile and control can be coml)ared. However, if thr technique is used on crolis of unknown spray history with no control crop availablr for comparison. chromatograms would probably contain so many unidentified and unidentifiahle peaks that arcurate interpretation would he impossible. Bache. Lisk. and Loos (13) prepared nitro derivativcs of MCP, lICPI3, and X-1-J.in order to increase the response of these herbicides on the electron capture gas chromatograph. They used this technique to determine J I C P and MCPB in timothy and pea? and S.i-4 in apples, and reported finding residues of N C P on snap bran plants treated with A\
MCPB. The above d i m w i o n has been concerned ~)riniarily lvith gas chromatograph detectors for halogenated compounds. One of the most esciting and promising developments of the past year was the appearance of two di+ tems, each of which is reported to be highly specific for phosphorus - containing compounds. Giuffrida (5%) modified a conventional flame ionization detector by fusing a sodium salt onto the electrode. The result was a dctector 600 times as responsive to a compound containing 10 carbons and 1 phosphorus atoni as was the conventional flame detector. Response to compounds containing sis chlorine atoms was twenty h i e s as great. while the response to compounds -aim containing neither C1 nor I’ was the : as that of the conventional flame ionization detector. K h e n the estraction procedure of AIills, Onley, and Gaither (246)was used. dinzinon, ronnel. parathion. ethion, and ‘Trithion. when added to broccoli a t levels of’ 0.05 and 0.1 p.p.ni.. were easily detected. There was no interference from crop materials even when the equivalent of 5
grams of original sample was injected. Construction and operating conditions of the det’ector are described, and refor 23 organophosphate compounds are given. 13urchfield, Rhoades, and Wheeler (42) report the develop coulometric detection qiecific for phosphoru from the usual gas chromatographic column is passed through a quartz tube heated to 950” C., with hydrogen as the carrier gas. Organic compound> are reduced to hydrocarbons, water, PH3, HrS, and HC1. ‘The latter three compounds precipitate silver ion and so register on a inicrocouloniet’ric titration cell. Insertion of a short silica gel column removes HC1; substitution of .\la03 for silica gel removes both HCI and H2S and permits measurement of PH3with ahsolute specificity. Response of the cell to PH3.H2S, and HC1 is in the ratio of 2:2:1. Khen a model C-100 microcoulometer at maximum 3enbitivity is used. 0.1 p g . of 1’ gives a peak area of 5 square inches. Cleaned-up estracts from c r o p esaminrd do not interfere wit’h the reduct’ion or det’ection steps. CHLORINATED PESTICIDESGENERAL PROCEDURES
More attention has been given to the development of multiple detection procedures for the chlorinated pesticides than for any other class of pe>tirides. This is only natural, since these coinpounds are widely used and many arr so persistent that trace:: of Rome rainpounds, such a,< DDT. are being found almost everywhere. Moreover, thpse compounds h a r r been found to be inore amenable to this type of analytiral method. AIills. Onley. and Gaither (146)have combined anti modified previouhly reported methods to provide a rapid. simple procedure for estracting and cleaning UI? residues from nonfatty foodi. r‘sed with gas chromatogral)hys thin layer chromatography, or paper chromatography, the 1)roccdurr will determine 21 ehlorinated ~ ~ r h t i c d e s . Good recoverie.; were obtained of 5 pesticides added to 1 I ~)rodu(*t~s at levels from 0.02 to 0.2 I).I).ni. Taylor, Kea, and Kirhy ( I S ? ) estracted chlorinated I)e>tirictc r&lurs from animal tissue by blending tht. t with acctonr and anhydiouh S a The ilesticide!: w r e transfeiwrl t o hesane anti injrcted into a gab c~hromatogralih, Recoveries for lindanc, cmirin, dieldrin. p,p’-I)DE, and 1iel)tarhlor eposide ranged from 7 5 to 99yc at 2.5 to 10 p.p.in. levels. S(,veral prorediire.; have heen 1,q)orted for extracting c.hlorinated iic.
