Document qdQgD72ooDGn98b4ybRJZ1BwK

'l <5 mJ) J liittve umy rbidil>Jat epled Mil in rbidiiiclUr There inulti . lid., ri) J. Sym >tt in I0.4C ! \merl- >e*tl* iK* nMI.I.INO A HUtKINII: TLC OF AHOCLOR8, IIALOWA.TO, AND FK8T1CIDKK A INDUSTRIAL CHEMICALS Reverse Phase: Thin Layer Chromatography of Some Aroclors, HaJonuxes, and Pesticides 1UVID ft.STAU.ING and JAMES N. 1IUCKINS f'.th'Ptfiicuie Research Laboratory, Bureau oj Sport Fisheries and Wildlife, Columbia, Mo. 65S01 The components of Aroclors 12.12, 1218, 1254, ami 126Q Halownxe* 1099, 1013, ami 10M( and several chlorinated pesticides aro resolved by reverse phase thin layer chroma tography (KIT1.C), which permit* component separation by partition between a nonpolur stationary phnac ond o polar mobile phase. H/ value* of resulting spots were calculated for 2 of 4 new aolvent systems (mobile phitsr*). lU'TI.C patterns were reproducible and clinr* arterial ic of each material examined. The apola were recovered from the pin lea ami character* lard by gas-liquid chromatography (GLC) and/ or GLC-mas* apeelrometry. In noma cases, single GIAI peak* of Aroclor standards were resolved Into more than one component by MPTLC, whereas some lilTLC spots of Halo* waxes were resolved into as many as 4 GLC peaks. The analysis of environmental residues of chlorinated compounds was facilitated by ihik technique. Polychlorinated biphenyls (PCBs) arc wide spread ami persistent industrial pollutants of tho environment (1). Chlorinated naphthalenes (Hulnwn.\ps*:) arc industrial compounds with a vari ety of uses similar to PCBs (2). GLC of PCBs and chlorinated naphthalenes results in mullicomi>um-nt chromatograms and until recently only GLC or GLC-mass spec trometry (MS) was available for PCH com|>onent resolution. PCD components have been srpuruted by 2-dimensiotmI thin layer chromatography (TLC) (3), although resolving j,p*-l)l)K from 1`CBa was the printary object of these experi ments. Also reverse phase thin layer chromatog raphy (RI*TLC) lias been used to resolve PCB components of I'hcnochlor** 1)P0 or Aroclor** 1200 (!) but, in our exjreriencp, several compo nents of Aroclors 1264,1218, and 1232 were very ii -:ir. the solvent front ond were not clearly resolved. Nevertheless, 11PTLC appeared to offer {potential os mi auxiliary analytical method for "xamimng complex residues of PCBs and other, multicomponent chemical*. Wc modified the ex isting RPTLC method (4) to permit separations of Aroclors 1264, 1248, and 1232; Halowaxcs 1090, 1013, and 1014; and several chlorinated pesticides. In addition, we characterised these components by GLC and/or GLC-MS. Experimental Apparatus (a) Rrrerse phase thin layer chromatographic plates. --Prescored (20 X 20 cm) gin** plates coated with kicsclguhr G (Co$0 binder) to thickness of 250 am (Analtech, Inc., 100 S. Justison Si., Wilmington, Del. 10801). (b) Chromatographic lank.--Closed glass develop ing tank lined with Alter paper. (c) Irradiation tamp.--General Electric G8T.1 germicidal UV lamp. (d) Ga fhromalogroph.--rerkin-Eliner Model 880 with 'Ni-cicclron capture detector. Operational parameters: column---7' X 2 mm id coiled gloss packed with 0.3% (w/w) OV-7 on glass beads; nitro gen carrier gas at 40 ml/min; temperatures (*C)-- injection port 220, detector 350, column 160 or 100. (e) Mass prrtro#irtir.--Perkin-Klmcr Model 270 double-focusing, low-resolution miwg spectrometer coupled through Wnlapii-Hiemann molecular sepa rator to temperature-programmed gas chromutoBrnph. Raagant* and Standard* t (a) Standards.-- Bestieidcs.-- p.p'-DDT, p,p'DDE, aldrin, dicldrin, and technical chlordauc (ob tained from Pesticide Depository Section, Perrine Primate Research Laboratory, Environmental Pro tection Agency, Perrine, Kin. 33157). PCBs.--Tech nical Aroclors 1232, 1248, 1254, mid 1260 (provided by Monsanto Chemical Co., 800 N. Lindbergh Blvd., St. Louis. Mo. 63166). Chlormatrd naphthalenes.-- Halowaxcs 1000, 1013, 1014, 1031, 1051, ami I00Q (secured from Koppcm Co., Inc., Bridgeville, Pa. l.>017). (b) Solvent*.--Pesticide grade, redistilled in glass. (c) ParaJHn oil.'-Liquid petrolatum (Matheson, Coleman & Bell). (4) Silrir nitrate.-- Reagent grade (Fisher .Scien tific Co.). HONE 082760 I ; 3OK ' JOURNAL OP TUB AOAC (Vol. 5G, No. 2, Id/o; . h i.i xr; a It I'rf/xirnh'nn of Hrirrtc I'ltanc 7'liin lAtyrr Cliroinnlun'itplMf f'lnte* Knongh pnrhfliu oil wn* dissolved in n xltnllow troy fill'd with petroleum ether to yield 11 solution of 8% by volume of the oil in the ether. Next, n prreonted ploteyM carefully immersed in the paraUm-petroleum elber solution until the adsorbent hiyer wu* saturated. Then the plnle wn.s removed find allowed to air-dry overnight. Plates thus prepured cim bu stored for avvertvl weeks witlrovtt any loss of separa tion efficiency Solvent System* Four solvent systems were employed to obtain maximum resolution of the various components of the Aroclors, Hnlowaxcs, and chlorinated pesticides (Table I). All developing solvents were saturated with paraffin oil by shaking the solvent and about 5 ml paraffin oil in separatory funnel. Sample Application and Development All reference standards were dissolved in benxene (I nig/ml) and 2-50 pi was spotted at points 2 cm above the bottom edge of the plate. Paralfiii-paturnted developing <>!vont (200 ml of I of the 4 solvent system* listed in Table 1) was added to a filter paperlined chromatographic lank, the tank was covered with n glass plate, and the atmosphere was allowed to become saturated with the solvent. Karh plate was developed to a point 15 cm above the sample origin, removed from the tank, air-dried in u hood, and redeveloped and air-dried twice in a similar manner. All development was done at ambient tem perature (ea 2.5*0. developed mid visualized (Fig. I). This fish rontnined a PCH residue in excess of 200 mR/k whole fish os determined by CI.C (0). Hrsolts and ilUcunlon Visualization sensitivities for the resolved com ponents of Aroclor anti Halowax mixtures and for selected pesticides were determined by using a silver nitrate chromogcnic spruy (the fluorescent indicators tried were too insensitive for the de termination of PCBs und Hnlowaxcs). There appeared to be some correlation of sensitivity with degree of chlorination, particularly for the PCBs and Holowaxcs. Approximately 15 pg Aroclor 1232 or Halowux 1099 was required to achieve visualisation of major components, while only 8 pg Aroclors 1243 and 1254 and Haloivaxes 1013 and 1014 was needed for detection. Sensi tivity for some other compounds or mixtures are as follows: Aroclor 1200,2 pg; p.p'-DDT, <2 pg; p,p'-DlMC, <2 pg; and technical chlordane, 5 gg. Because chlordane is a multicomponent pesticide, a lower detection sensitivity is reasonable. We found that 2 solvent systems (M and IV, Table 1) were adequate for resolving components of the comjwHinds tested (see Figs. 2 and 3). Componrnts (spots) of the resolved Aroclors and Halowaxcs were numbered in ascending order. Standards of Aroclors and Halowaxcs wrro ana lysed by GLC nt 2 diflerent column tenijreralurcs, depending on GLC retention time (Figs. 4-0). Tables. Solvent system II II II ViiuoUttttion After final development and drying, the plnte was moved to a holder for spraying with a chromogcnic spray consisting of 1.7 g AgNOs in 200 ml FtOJI (00%) with ] ml NIIOII added just before applica tion. A uniform spray was applied to the plate; theg the plate was held over a steam bath for s few sec to allow the steam to play over the adsorbent Inyer. Finally, the plate wua placed under a UV lamp for Severn! min. All chlorinated component* appeared M dark spots on a light background. ffmdronmenlol Analytit JUJTLC wo* used to separate PCD residues in a goldfish, C<iras*iu* auratus L., which was collected for the National Pesticide Monitoring Program from the Hudson Ilivet at Poughkeepsie, N.Y. The PCI1 fraction of a petroleum ether extract from this fish (Sample KOI2) wax obtained by using silicic acid column chromatography (5). Approximately 25 Mg PClis from the extract ami standard Aroclor P24X were spotted on an HJ*TLC plate nud the plate was Table 1. Developing solvents lor reverse phese Ihli layer chtomatography of PCBs. chlorinated naphthalenes, and chlorinated pasilcldes Solvent system* Composition (v/v) Compounds* 1 wetcr-ecetonitrfle-MeOH Aroclor 1232* (20 + 40 + 40) II wstc r.acetonitrile. Me(7H Aroclor 1241 (15 + 40 + 45) chlordane dicldi in DOT aidrin in water-acetonitrlle-MeOH Aroclor 1254 (10 4 45 4- 45) IV wator-acetone. acetonitrile MeOM (5 4- 15 4 40 4- 40) Aroclor 1200 Halowax 101) Halowax 1014 Ha lows* (099 Each paraffin-costed kieselguhr G TIC plate * developed 3 times. `p.p- OOe may be resolved by using any of the * developers. * Mixture of Aroclors 122] snd 1242. 0 IV Cond'hons for > ` See r<gs Tend values r>tp* A column temf * I u compontrv < I.C peaks wm* i '>'uiinn tints*. In order to n l" At b >1 m<L uf the *an> i>.: ii'K | fin Upm ' Philo. The p '! nix'd ami a X. MONS 082 761  V.1Qffc) li run : whole 0 com* r< mid . uning M'MTnt ihe d There itivity (<K the Ji MR irrd to >, while imnxrH Seiinirri lire <2rs; . 5 MR. lieidc, nd IV, xHtcnU . Com* ri> mid onler. re nua> attire*, . 4-0J. i# IMn jm IO 17*9 )0U ' a 1014 1099 net* I* l the 4 jif v'. l.tNO * mjCtotNB: TLC OF AUOCLOnS. IMLOWAXK.S. AND I'F.STJCJDHS 309 Table X. Chromatographic cheractorltatlon of polychlarlneUd biphenyl* (Arocior*): correlation of reverse phase Ihln layer chromstoyraphlc (RPTLC) spots a nd GLC peak*" RPTtC GlC' GLC MS Solvent Spot no.* R/` Peak no.* Rt relative to p.p' ODE No. ol chlorines Molecular weight Arocior 1232 If 1 0.35 13 2 0.40 11 1 0.46 12 4 0 55 6 6 0-60 1 6 0.06 10 7 0.72 ( 0.75 9.6 5.70 t 0.76 7A 10 0.44 7,5 11. o.s; 3 12 0.69 . 2 1.46 1.07 1.29 0.64 0.07 0.97 0.76.0.40 0.30.0.52 0.46 0.46.0.30 0.21 0.15 Arocior 1241 6 356 5 374 6 351 S 324 1 189 5 324 5.4 324.290 3.4 256.290 4 290 4.3 290.256 2 222 2 222 il 1 0.37 2 0.42 3 0.47 4 0.51 6 0.57 6 0.63 7 0.69 0.76 9 0.79 It 12.9 6 10 9 6.6 7A4B* 9.2 4 1.06 1.26,0.76 0.63 0.99 0.76 0.30.0.40 0.47.0.51 0.21.0.15 0.24 Arocior 1254 5 6.5 8 5 5 3.4 4 *2 3 924 356,324 324 324 324 256,290 ' 290 222 256 II 1 0.21 2 0.27 3 0.35 4 0.40 S 0.4S 6 0.55 7 0.69 1 0.76 IV 1 O.bO 2 0.60 3 0.66 4 0.76 5 0.67 0.92 7 .0.96 16 13 12.15 14 9 11 1.6 7A 2 36 1.55 1.28.1.95 1.61 0.79 1.06 0.66.0.40 0.46 Arocior 1260 16 5.23 16 3 10 12A, 14.17 1.51.2.12.3 96 120.13,15 1.67.1.69.2.56 9,11 0.82,1.26 10.12A * 1.22,1.51 6.10 0.72,1.12 6 6 6 6 5 6 5.4 4 7 7 6.7 6.7 5.6 6 5.6 356 356 368 358 324 324 324,290 290 39? 392 369.393 358,392 324,358 358 324.358 " Condition* lor RPTlC end GlC are thoae deacribed in text. ` See l ies. 2 and 3 for tool number* and Fig. 5 end 6 for peak numbers. ' H/ velvet ol p.p' tJUfc for solvent tytlem* U end IV were 0.60 end 0.4?. respectively. 'A column lempoiatur* of 1G0*C wa* used, except for GLC of Arocior 1260 which wat resolved at ]90*C. ' Two components under the tame peek. < il.C |n':kv worn numbered in order of increasing time. In "rdcr to relate the retention time of GLC l^ak* obtained from RPTLC separations, 3 stan dard, of thp same material were spotted approxi mately 1 fin apart and 2 cm above the lower edge "I a plale. The plate was developed as previously '*"*::l*r| mid n narrow strip of foil was placed over the center area of the developed plate, cov ering the area of the middle sample. The chrotnogenic agent was carefully applied and the plate was irradiated sufficiently to reveal the 2 outside rows of siMits. The foil was removed and horitoiital tones of the unreaeted middle standard, corresponding to spots of the 2 outside standards, were scraped and eluted with 5% diethyl ether in I 1 HONS 082762 370 JOUtl.NA). or TUB aoac (Vol. 50, No. 2, 1973) Table I. Chremateyrephlc characterlxatlen of chlorinated naphthalan** (Halowax**): correlation of ravers* ph*s* thin layar Chromatofra phlc (RPTLC) apels and GLC peaks'* RPTLC GLC' GLC.MS Solvent system Spot no.* If/ Peak no.* Rt relative to aldrin* Halowax 1039 No. ol chlorines Moitculir weight IV 1 0.52 2 0.57 3 0.77 4 0.11 $ 0.35 6 0.33 IV 1 0.46 2 0.50 3 0.53 4 o. 9 0.76 6 0.11 7 0 15 IV 1 0.35 2 0.44 3 0.56 4 0.C1 S 0.31 11 6.7, 12.13 a 4.9 10.5 2 1,97 0.83,1.03, 2.48.2.82 1.18 0-44.1.28 1.53.0.62 0.23 Halowex 1013 11,15 16.17 5B.12 9.L 13.14 9 10.4 6A 1.67,4.64 4.98.5.70 0.70.1.97 0.32.1.02. 2.43,2.32 1.27 1.52.0.43 0.60 Halowax 1014 15.16,20 11.12. 17.13 5,13 6.13.14 9,4 1.60,1.89.4.21 0.72,0.84. 1.38,7.28 0.30,2.53 0.38.1.08.1.19 0.23.(7 58 5 4 5 4 1.4 4.3 2 S 6 4,5 4 5.6 4 4.3 3 6.7 5 6 3.6 4.5 4.3 298 264 298 264 230.264 264.230 196 2)1 332 264.298 264 298.332 264 264.230 230 332,366 298 332 230.332 264,298 264.230 * Condition* for RP1LC and GLC aro thus* described in text. * So* F lg. 3 for spot number* and FIrs. 4 and 6 for peak numbers. ' Rf of p.p'-DDC -0.92. *h column temporature of )C0*C was used, except for GLC ol Halowax 1014 which was resolved at 19Q*C. * Except for Halowax 1014; R% value* lor this compound are reported relative topj>'DDE. -1' > i i.iNii * ir fiC.--J. Revere* ant ndem IV) t i i i SwSwrrar FIO. 1--Reverse phat* thin layer chromateyrem ef PCS fraction af yaldllsh extract (F012), etandard ODE, and landard Aroctor 1141, TLC plala was prepared as described In text and deveteped I timet In solvent system II (Table 1). 05% petroleum ether. The resulting solutions were nnnlyzod by GLC. Temporal nre-proj'.nimmed OLC-MS tvits used to clmntctvrize I lalowux stnmlnrds ns to chlorine content of individual CLC peaks. In u previous PIG. 2--Reverse phase Ihin hyir chramataframs ltd* ent system II) af Arocfor* 1232, 1241, and 1294. p,p'*OOf. p.p'-DOE. aldrin, dieldrln, and technical chlerdan*. *--Gas chi- mn. * ,, * lUI I. Sr* |, '''aili'ii ......... * *'i Arn -..In. ; '.I.C V. HONS 082763 \ .. 2,1073) 1 r:Jr {>1 n ;) h* J.IM l.W :h > m j.m *m Mil ;u i.m m ill M 4.JU i.;a IM.LINO * HUCKINS; TLC OK AROCLOIU. H ALOWAXKS, AND PliS'l ICIUF.S riO.---3. Raven* pfcaaa thin layar chromatogram* (olv. *n< lyttam IV) of Areclor 1760, Htiowasa* 1013, 1014, nit 1099, n4 p^'-DDE. 371 03 * !- *a f * i f.i, in> >>r<am (lllv ^{i.mp'ODT. >1 iMinitM. FIO. Mlat chromatogram* of Aroclor ttandardti 6 ng 1233. 9 ng 1248, and 4 ng 12*4. Saa Fig. 4 far OLC condition* and numbaiing of ptak*. rift. Mill tkrm*l*|rimi of Htlawn Kin4r4i: I I 1031, ( ng 1000, f n| aoeh of 1031 ami 1099, and 4 ng 1013. 9a* tail far tnatrumantat condition!. Column tamimatiMO--160*6. Numktfi abavo paafc* arc > tlgnad In ardar of Incraaalng ratontlan tima. study, wc used C5I.C-MS lo determine the rhWim* content of Gl.C peeks representing eom- |Hi*ent.H of Aroclor standards (ft). ' The It/ values of KPTJ.C s|K>ls and their corrc- M'>*t<ding Ol.C retention time* {!{>) were detvr- mined for Ute. various Arodors and Holownxcs resolved by UPTLC (Tables 2 and 3). R/ and Rt values were based on >2 determinations and were reproducible in subsequent separations. From MS data, the number of chlorines and component, molecular weight of corresponding GLC peaks and lU'TI.C s|xits were determined. Components of the Aroclors were resolved bet ter by Hl'TI.C than were Hnlowax components. In some cases, RPTLC separated Aroclor compo nents which eluted ns a single GLC |ienk. How ever, HPTJ.C separations of Nalownxes resulted in several jw>ts which were resolved by GLC into X '! i !; i i I d i i SONS 082764 i l ' 372 journal or Tin: aoac (Vol. 5C, No. 2, 1973) wtxmt* a CUM' FIO. i--On f Ar&tl** ncf HiIwm l*nd*rd; 8 ng IDS), 6 ng ArMlor 1760. and $ ng - Htlows* 1014. QIC condition* wrr* th* i*m as In Fig. 4, oscopt column tomporaiur* wit 190'C. as many as 4 peaks. Wc also found that B|>ots with lower 77/ values generally correspond to I'CD or Halownx components substituted with more chlorine atoms and that GLC peaks with longer retention times correspond to spots with lower R/ values (4). Sjiot patterns of individual Aroclors or Halowtxcs were reproducible and characteristic of the material they represented. Arodor 1232 consists of n mixture of Aroctors 1221 and 1242 and it is not a well defined TCB formulation (7). Thus, Rl'TLC sjwt pattern in tensities mnv vary somewhat for different samples of this Aroelor. Separation of BCD residues in fish with HPTI.C provides effective resolution of PCD homologs. The technique in combination with GLC can pro vide information on the isomer and homolog composition oi PCD residues when MS is not available for confirmation. RPTLC separation of Aroctors, Hidowavcs, and chlorinated jiesticides July 19. 19??. offers a complementary method to GLC in the analysis of organochlorine compounds in environ mental samples. ItRlUnRSCLS (1) Peakall, B. I)., A Lincer, J. L. (1970) fiioScitnet 20.O.Vi-964 (2) llalowax Chlorinated Xaphthalmcs, Hoppers Co., Inc., Tar Product* Division, Pittsburgh, Pa. (3) Fchringer, N. V., A Westfall, J. E. (1971) J ChromaJoyr. 57, 307-400 ** (4) DeVos, R. H., A Pen, K. W. (1971) Bull. En viron. Contain. Torieol. 6, 164-170 (.r>) Stalling, D. I.., A Kurkins, J. N. (1971) JA OAC 54, 801-S07 (0) Stalling, D. 1.., ft Mayer, F. L., Jr. (1972) J. Environ. Health Paspeet., in press (7) Armour, J., McMahon, 11., Koach, J., Wiencko, W., Young, S., Mnamnoto, II., A buke, M., in PCll XrwBlettrr (March 30, 1972), V. Snarski (ed.), Environmental Protection Agency, Nationnl Water (Jualily Laboratory, Duluth, . Minn., pp. 3-4 Drlcrmiim I'reu with i T. C. ttnOIJI.< >' c/Chn A rreenfly (nt is shown t, |o> s umnioniuenl nit The resoliM m* the reduced | Standard ttevioi and the reduce* Tor iniinonliirnl phospliMte are ft The AOAC cd titration method minntion of mnii uium nitrutc or u which nmy be tit nation of aiumm nium phosphate phalc is more tlih i lie buffering rffe. cal nitrogen cun presence of urea b ncthcwl (1,2), bin lime consuming. An ammonia eh by Orion Kcsrm* by (he niamifaetn. lenninatirm of an ions in the Mni|de by ndditinn of n : diffuses through i membrane of tli<> i mini with the hit The hydroxyl inn bv (.miration of t ined by the inntvir |Miten1ial that I'ontent of the xmio nlMiut 1 min .. e--entially Xern-ti content of the sum "f the suitability m determination of lac-cnee of urea. HONS 082765