Pestic. Sci. 1914, 5, 293-304

Some Factors in the Separation of Polychlorobiphenyls (PCB’s) from Organochlorine Pesticides by Column Chromatography Combined with Gas-Liquid Chromatography”

Roy Edwards

H. J. Heinz Co. Ltd, Hayes Park, Hayes, Middlesex (Amended manuscript received 21 January 1974 and accepted 29 January 1974)

Retention volumes for polychlorobiphenyls (PCB’s) and organochlorine pesti- cides have been measured on “Florisil”* and silicic acid in various activity states using petroleum spirit eluant. The majority of organochlorine pesticides can be separated from PCBs by column chromatography. The exceptions are heptachlor, aldrin and BHC (apart from the gamma-isomer), and the separation of DDE from PCB’s was only achieved with highly active silicic acid. With PCB residues normally found, the separations not secured by column chromatography were obtained by gas chromatography.

1. Introduction

The polychlorobiphenyls (PCB’s) present both toxological and analytical problems. One major human disaster in Japan’V2 and another episode in the U.S.A. in which large numbers of poultry were lost3-’ illustrate the hazards of food contamination. The whole subject has been reviewed.6-10 The analytical problems arise because the commercial products are complex mixtures of chlorinated aromatic compounds the toxicities of which and the residues commonly found in living organisms have physical and chemical properties similar to those of some organochlorine pesti- cides, notably the DDT group. Hence earlier standard analytical procedures failed to separate PCB’s from organochlorine pesticides. Attempts to solve the problem include : chemical alteration14-19 of the pesticides and/or PCBs’ so that gas-liquid chromato- graphy (g.1.c.) retention times are drastically changed; or differential detection in favour of the pesticides.20 However, neither approach is entirely satisfactory and a separation technique seems more desirable. While it is now possible to separate the higher chlorinated PCB’s from the DDT group, in particular DDE, by thin-layer2I or column c h r o m a t ~ g r a p h y ’ ~ , ~ ~ ~ ~ ~ there are no reports of the separation of lower chlorinated members by these means. This paper attempts to clarify some of the remaining separa- tion problems in respect of column chromatography using “Florisil” or silicic acid in conjunction with g.l.c., and to indicate some of the limitations of this approach.

a Presented at a symposium Multi-residire detection systems on 1 November 1972, organised jointly by the Pesticides Group, Society of Chemical Industry, and the Society for Analytical Chemistry/ Analytical Division of the Chemical Society. For other papers from this symposium see Pestic. Sci. 1973,4, 399-430.

“Florisil” is a registered trade mark of the Floridin Co. 293

294 R. Edwards

Initial interest in PCB’s in this laboratory stemmed from their occurrence as contami- nants of many analytical materials, including “Florisil”. This is an exceptional adsor- bentz4 which when fully activated can rapidly be de-activated by adsorption of water and other compounds (including PCB’s) from the atmosphere. “Florisil” shows a markedly different response to water content compared with alumina and silica, and higher activity material may give better separation in some instances. Considering earlier use in residue ~ o r k ~ ~ * ~ ~ and a reporti6 that relatively low activity material afforded partial separation of PCB’s from organochlorine pesticides, it seemed worth investigating activation conditions. More recently Armour and Burke22 claimed that an effective separation of “Aroclor” 1254 and 1260“ PCB’s could be obtained using silicic acid.

2. Experimental

2.1. Materials

All solvents were redistilled and tested by electron-capture g.l.c., after twenty-fold con- centration, for freedom from interference by PCB or other compounds.

Petroleum spirit (b.p. 30-60”C)-Mallinckrodt “Nanograde” or Fisons “Distol”. Diethyl ether-A.R. Adsorbents : “Florisil” (Floridin Company), “regular” grade; silicic acid [( 1) Mal-

linckrodt 100 mesh “Suitable for chromatographic analysis by the method of Ramsey and Patterson”, and (2) Mallinckrodt CC-71; and silica gel (Davison 923,100-200 mesh). These adsorbents were activated (and in some cases subsequently partly de-activated) as described in Sections 2.3.1 and 2.3.2 “Celite”b 545 was usedas supplied.

Sodium sulphate A.R. was heated (600°C) for 0.5-1 .Oh to remove organic impurities. PCB’s-Monsanto “Aroclor” 1242 and “Aroclor” 1254. Pesticides-commercial samples of aldrin, BHC, DDE, DDT, dieldrin, heptachlor,

heptachlor epoxide, methoxychlor, and TDE were recrystallised.

2.2. Equipment

2.2.1. Gas chromatography

A Varian 1520B model was used, fitted with stainless steel columns (2 m x 3.2 mm o.D.) packed with 3 % OV-1 on Chromosorb G (60/80 mesh). These and the injectors fitted with glass inserts were operated at 180°C and the electron capture detector (tritium source) at 190°C. Carrier gas was nitrogen (40 ml/min).

2.2.2. Column chromatography

A glass column (100 x 10 mm) expanded into a reservoir (100 ml) above and a vacuum take-off below (with “Quickfit” joints), was used in conjunction with a dropping funnel fitted with a Springham valve having a PTFE-lined diaphragm. A larger column (300 x 20 mm) was also used with the dropping funnel and other standard “Quickfit” components.

a “Aroclor” is a registered trade mark of the Monsanto Company. “Celite” is a registered trade mark of the Johns-Manville Company.

295 Polychlorobiphenyls and organochlorine pesticides

2.3. Methods 2.3.1. “Florisil”

2.3.1.1. Activation

The adsorbent was either heated to 600°C for up to 22 h and rapidly transferred to an- other oven (I 15 “C) to cool, or PCB-free material was heated (1 15 “C) for 17 h. Heated columns were charged (approx. 3.5 g), topped with anhydrous sodium sulphate (10 mm) and cooled in a vacuum desiccator.

2.3.1.2. Preliminary determination of activity

Retention volumes for dieldrin were used as an index of activity. After removal from the vacuum desiccator, dieldrin in petroleum spirit (5 ml) was immediately applied to the column and, when drained to sulphate level, followed by a first eluant [50 ml; diethyl ether-petroleum spirit (3 : 47)] and then by a second [diethyl ether-petroleum spirit (3 : 17)], the latter being collected in fractions (1 0 ml). Retention volumes were calcu- lated, in terms of the second eluate, from g.1.c. of these fractions. The behaviour of acti- vated “Florisil” (24 h at 600 “C) on exposure to atmosphere for periods of up to 20 min was monitored in a similar way.

2.3.1.3. Retention volumes

Retention volumes of organochlorine pesticides (0.2-1 .O pg) and PCB’s [“Aroclor” 1254 (350 ng)] were determined on columns pre-washed with petroleum spirit (25 ml) by collection of successive non-polar and polar eluants (50 ml each) in fractions (2 ml), aliquots (10 pl) of which were analysed by g.1.c.

2.3.2. Silicic acid or silica gel 2.3.2. I . Activation

Charged (3.0 g) columns topped by anhydrous sodium sulphate (10 mm) were heat activated (130, 150, 170 and 185 “C) for various periods and cooled under vacuum with a dropping funnel attached above so that solvent wash (85 ml) could be admitted directly into the evacuated system after cooling.

2.3.2.2. Activity Preliminary determination of activity was carried out using a mixture of heptachlor (45 ng), aldrin (180 ng) and DDE (706 ng) which was eluted by four or five volumes (50 ml) of petroleum spirit. The collected fractions were concentrated (10 ml), aliquots (10 pl) gas chromatographed, and the pesticide (usually DDE) in each was used as an activity index.

2.3.2.3. Retention volumes Retention volumes were determined using the same pesticide loading as in Section 2.3.2.2 and in some cases with the addition of “Aroclor” 1254 (4.9 pg) by collecting the eluate in fractions (2 ml) and recording peak heights in g.1.c. In the case of “Aroclor” 1242, which has a much lower electron capture response than “Aroclor” 1254, it was

296 R. Edwards

necessary to use a larger column (20 g) and to omit heptachlor and aldrin because of possible interference with PCB’s on g.1.c. “Aroclor” 1242 ( I 57 pg) and DDE (4.7 pg) were eluted with petroleum spirit (1250 ml) collecting fractions (20 ml), aliquots (10 pl) of which were gas chromatographed. In some cases the fraction was concentrated four- fold prior to analysis by g.1.c. De-activated silicic acid was prepared and used as d e ~ c r i b e d . ~ ~ , ’ ~ In all cases calculation of retention volumes included correction for sol- vent evaporation loss during fraction collection.

3. Routine method for the separation of PCB from DDE

A column is charged with an intimate mixture of silicic acid (2 g; Mallinckrodt No. 1 in Section 2.1) and “Celite” 545 (0.5 g) and activated (185 “C) for 3 h. The adsorbent mix- ture is rapidly tapped down, topped with anhydrous sodium sulphate (10 mm), and placed under vacuum to cool as in Section 2.3.2.1. The vacuum tap is closed, petroleum spirit (25 ml) admitted to the evacuated column, the vacuum connection removed, and slight pressure (approx. 0.5 kg/cm2) applied to elute at about 5 ml/min. When the wash has drained down to the sulphate level the sample is applied and washed in with a minimum volume of petroleum spirit. Eluant is added to give a total volume of 47 ml. This i s col- lected as fraction a. A further elution with petroleum spirit (50 ml) gives fraction b ; finally a polar eluant [50 ml; diethyl ether-petroleum spirit (3:47)] gives fraction c. The fractions are concentrated as necessary. Fraction b will contain pp’-DDE (if the column is overloaded fractions a and c may contain some also). PCB’s, alpha- and beta-BHC, aldrin and heptachlor are contained in fraction a ; DDT and more polar pesticides in fraction c.

4. Results 4.1. “Florisil” “Florisil” was used routinely in this laboratory in residue analysis, and as received contained PCB’s (about 3 mg/kg as “Aroclor” 1242). Extensive washing was necessary to remove them prior to low temperature (1 15 “C) activation. A more economic and convenient method of removing the PCB’s is to heat the adsorbent at 600 “C. The opti- mum activation time at this temperature was 2.5 to 3 h (Figure 1) as determined by re- tention volumes for dieldrin. De-activation of fully activated material (2+ h at 600 “C) on exposure to atmosphere for up to 20 min was also followed using the same monitor. A linear de-activation rate of about 2.5 % /min was found, and clearly demonstrated the difficulty in handling the material. This led to the adoption of the technique described in Section 2.3.2.1. rather than the usual slurrying method.

Retention volumes of organochlorine pesticides on “Florisil” were compared (Table 1) using high (2+ h at 600 “C) and lower (1 7 h at 11 5 “C) activity adsorbent. While reten- tion volumes for the less polar compounds remained essentially the same the figures for pp’-DDE and methoxychlor are higher, and for dieldrin lower, on the “lower” activity material. This indicated that a similar comparison on “Florisil” should be made using PCB compounds (“Aroclor” 1254) and selected pesticides (heptachlor and DDE) (Table 2). The separation of the PCB compounds in “Aroclor” 1254 from all the pesticides listed in Table I , except for heptachlor, aldrin, andpp’-DDE is facile. In g.1.c.

Polychlorobiphenyls and organochlorine pesticides 297

0 ~ 3

Time ( h 1

Figure 1. Activation of “Florisil” at 600°C. Heating time versusactivity of adsorbent, thelatter being expressed as retention volumes for dieldrin eluted by diethyl ether-petroleum spirit (3 : 17) from a column containing about 3.5 g adsorbent. See Section 2.3.1.1. for further details.

TABLE I . Retention volumes and fractionation of organochlorine pesticides on “Florisil”

Retention volumes”

Fraction Activated Activated number Compound 2fh/600”C 17h/l15”C

1 aldrin heptachlor pp’-DDE

2 alpha- and beta-BHC op’-DDT pp’-DDT

pp’-TDE gamma-BHC

heptachlor epoxide methoxychlor

3 methoxychlor dieldrin

15 16 22 32 32 33 34 34 36 42 - 70

16 17

27/31b 32 32 32 33 33 36

58 60

-

~

“ ml eluate/3.5g adsorbent. Divided between two fractions and giving two peaks.

with the non-polar stationary phase used here PCB interference with heptachlor and aldrin is negligible, but PCB peaks (Nos. 13 and 14 in Figure 2) have almost identical retention times to that forpp‘-DDE. The behaviour of these PCB peaks relative to the latter in column chromatography is thus of key interest. With fully activated ‘‘Florid”

298 R. Edwards

TABLE 2. Retention of heptachlor, pp’-DDE and PCB’s on “Florisil” and in gas chromatography

“Florisil” retention volumes Pesticide or ml petroleum spiritb PCB peak , , Gas chromatography number” 2)h at 600°C 17h at 115°C RRT‘

17 1 1 10 4.02 12 11.5 1 1 2.17 15 15 14 3.12 16 22 26 3.27 It 15 16 1.80 18 15 15 4.78

heptachlor 15 16 1 .oo 13/14 1 8d 1 gd 2.65

pp’-DDE - 23‘ 2.65

a See Figure 2. Per 3.5 g adsorbent approximately. G.1.c. conditions as above, relative retention time, heptachlor = 1 .OO (6.0 min), Compounds poorly separated.

I I I I I I ,

Time (min)

Figure 2. Gas chromatograms of PCB mixtures and organochlorine pesticides. (a) Pesticides, in order of elution BHC (two peaks), heptachlor, aldrin, op’- and pp’-DDE, op’- and pp’-DDT; (b) “Aroclor” 1242 (23.5 ng); (c) “Aroclor” 1254 (4.9 ng).

Polychlorobiphenyls and organochlorine pesticides 299

no separation is detectable and the compounds fuse as a single peak, but on the less active material some separation is evidenced by the presence of a shoulder on the peak plus increased bandwidth. Deliberate de-activation of “Florisil” with water renders any separation of the above compounds impossible.

4.2. Silicic acid

In an initial trial of the method of Armour and Burkez2 retention volumes of heptachlor, aldrin and DDE were determined but the poor result (Table 3 , l ) did not indicate that a good PCB/DDE separation would be secured. Some of the 3% water de-activated material was re-activated and gave an improved result (Table 3, 2). This led to the use of silicic acid, without the addition of “Celite” 545, under the conditions describedZZ and combined with the handling technique (Section 2.3.2.1 .) used previously for “Florisil”. This gave considerably better separations in terms of retention volumes

TABLE 3. Pesticide retention volumes on silicic acid

Weight Activation Retention volume RRVd adsorbent Time ml/g adsorbent A‘ = 1 .OO

Code“ g T”C (h) Dh A“ H‘ DDE‘ H‘ DDE‘ ~~

I 20.0+5‘ 130 150 f 8.6 12.7 14.0 1.47 1.62 2 3.2+0.8‘ 130 21.0 0 9.7 18.3 32.0 1.80 3.30 3 3.0 130 17.3 0 8.8 21.6 39.8 2.46 4.51 4 3.0 170 1 .o 0 10.1 21.7 37.5 2.15 3.72 5 3 .O I70 9.0 0 11.0 25.8 54.1 2.35 4.92 6 3.0 170 18.0 0 11.4 25.8 45.9 2.26 4.11 7 3 .O 170 9.0 f 8.3 13.3 17.1 1.60 2.06 8 3.0 170 9.0 II 6.3 11.5 15.9 1.81 2.50

See text, Section 4.2. De-activation. A = aldrin, H = heptachlor, DDE =pp’-DDE. Relative retention volumes to aldrin. Weight of “Celite” 545 present.

De-activation with diethyl ether-benzene (I : 199 by volume). f De-activation with 3 % water.

relative to aldrin (Table 3,3). With silicic acid activated overnight (130 “C) the retention volumes for “Aroclor” 1242 and 1254 PCB’s as compared with the three pesticides were determined (Figure 3). The key PCB peaks (13, 14), interfering withpp’-DDE on g.l.c., were very largely separated from the latter.

Silicic acid/silica gel can usefully be activated at higher temperat~res’~-~l but time dependence must be considered in seeking optimum activation conditions. Preliminary work (1 30,150,170, 185 “C) indicated that, as with “Florisil”, optimum times of activa- tion exist (Figure 4). Then retention volumes for three pesticides were determined accurately for three activation periods at 170 “C (1, 9 and 18 h). The results (Table 3, 4,5,6) clearly establish the existence of an optimum activation time, in this case about 9 h. Optimally activated material was then de-activated with water (3 %)zz or diethyi ether in benzene3z and pesticide retention volumes measured (Table 3,7,8). Separation

300 R. Edwards

( a ) Aroclor 1242

4 a

( b ) Aroclor 1254 plus pesticides

*-a

‘*,. PP’ -DDE I I \a-

10 20 30 40 50 60 Retention volume ml/g odsorbent

Figure3. ElutionofPCBs andsomepesticidesfrom silicicacid.(a) “Aroclor” 1242(157pg)elutedfrom a 20 g column. (b) Mixture of “Aroclor” 1254 (4.9 pg), aldrin (0.18 pg), heptachlor(0.75pg),andDDE (0.75 pg) eluted from a 3.0 g column. In both cases adsorbent activated overnight at 130°C and eluant petroleum spirit. Response, as g.1.c. peakheight, at same scale within each experiment except for peak 11 (broken line) in (a) which is attenuated x5. Data points omitted, except for pesticide curves, to secure clarity. Peaks numbered in g.1.c. elution order.

I I I I I

Temperature (“C)

Figure 4. Optimum activation conditions for silicic acid. See Section 2.3.2 for details.

and resolution were poor in both cases. Finally a mixture of “Aroclor” 1242 and DDE was eluted from an optimally activated (9 h at 170 “C) column (20 g). The data (Figure 5) demonstrated that PCB peak 13 was completely separated from pp’-DDE, and peak 14 partly so.

Polychlorobiphenyls and organochlorine pesticides 301

‘%

:: P b

C

” 0) D

u u

+

+

-

0 10 20 30 40 50 “ 1,

Figure 5 . Elution of “Aroclor” 1242 and DDE from silicic acid. “Aroclor” 1242 (157 pg) and DDE (4.7 pg) eluted from silicic acid (20 g) activated for 9 ha t 170°C. Peaks numbered in g.1.c. elution order. Peaks 5 and 11 attenuated x4. Only DDE data points shown, for clarity. Eluant petroleum spirit.

Because 9 h was not a convenient activation period for routine use 3 h at 185 “C was finally adopted. Subsequently two other adsorbents were evaluated using the latter activation conditions. The second Mallinckrodt silicic acid gave a very poor result (comparable to Table 3, l), but the Davison silica gel appeared to have properties similar to those of the original silicic acid used. The separation of PCB from DDE obtained is shown in Figure 6. However further modifications (Section 3) were made because of the rather high eluant volumes needed for the 3 g columns and the time taken, in excess of 1.5 h, tocompleteelution. By theuseofa2gchargeinthecolumns(10mmo.d.)anddilu- tion with “Celite” 545 (0.5 g) six columns could be eluted in 1 h with about half the volume of eluants.

5. Discussion

Though effective separation of pp’-DDE from PCB does not appear possible with “Florisil”, the handling technique described in Section 2.3.2.1. combined with high temperature activation does afford a more useful separation of organochlorine pesti- cides than with the low temperature activated material usually employed. On a non- polar g.1.c. column such as that used here pp‘-DDE, op’-TDE and dieldrin have very similar retention times, but using high temperature activated “Florisil” in column chromatography these compounds can readily be separated from one another prior to g.1.c. There being no merit in low temperature activation from the PCB/pesticide separation viewpoint, high temperature activation simplifies the removal of any organic impurities in the adsorbent and is more convenient. The above handling technique gives

3 02 R. Edwards

\ I I

Minutes

Figure6. Separation of DDE from PCBs on silicic acid. Gas chromatograms of fractions from dried egg (5 g) extract. (a) PCBs (derived from analytical contamination). (b) Peak 1 heptachlor internal standard (0.15 ng) and peak 2pp’-DDE (0.1 ng). Silicicacid (3.0 g) activated overnight at 130°C. Petro- leum spirit eluant-90 ml to elute fraction a and a further 95 ml to elute fraction 6. Fraction a twice the concentration of b.

0 12 24 36 40

reproducible results in routine analysis, and high activity material gives mostly good recoveries. The only penalty is perhaps a reduced linear capacity.

A more effective separation of pp’-DDE from PCBs can be achieved with some silicic acids/silica gels provided that the adsorbent is fully activated. The activation conditions used by Armour and Burke” were not sufficiently well defined and this was later pointed out by M a s ~ m o t o ~ ~ who used water loss from the adsorbent as an index of activity. He found that at 130 “C heating beyond 24 h did not significantly increase water loss. However, in the present work it has been determined that the optimum acti- vation time at this temperature is about 50 h, and that longer heating than this will reduce activity. The optimum time becomes more critical the higher the activation temperature. The only reliable criterion of activity here is the retention volumes and separations of the compounds of interest.

Using Armour and Burke’s method M a ~ u m o t o ~ ~ has shown that the lower chlorin- ated PCB’s elute later than the higher chlorinated members, for example all the PCBs

Polychlorobiphenyls and organochlorine pesticides 303

in “Aroclor” 1221, were found to elute in the polar eluate. This elution order in adsorption column chromatography with silicic acid is here confirmed in general terms. Obviously in partition gas chromatography the elution order is the reverse so that the overall separation problem rests with the few compounds that have similar retention properties in both chromatographic separations. With a non-polar g.1.c. column such as OV-1 the separation problem focuses on PCB peaks 13 and 14 and with fully activated silicic acid a substantial separation frompp’-DDE can be effected in column chromato- graphy. If a more polar g.1.c. column (for example diethylene glycol succinate) were used where the retention volume ofpp’-DDE relative to these PCB peaks was higher the separation problem would be further eased.

From a fully activated silicic acid column a first fraction from a non-polar eluant can be taken to contain all the higher chlorinated PCB’s, heptachlor, aldrin and BHC (except the gamma-isomer which is only eluted by a polar eluant). While some small interference by PCB with heptachlor and aldrin is possible on g.1.c. the last two residues are unlikely to be found. No PCB interference with alpha- and beta-BHC occurs. A second non-polar eluate fraction can be taken to contain pp’-DDE and a few lower chlorinated PCB’s which cause no interference in g.1.c. While with a fully activated pure silicic acid column (3 g) the eluant volumes needed for these fractions are 30 and 32 ml/g silicic acid, if the silicic acid (2 g) is diluted with “Celite” 545 (0.5 g) these eluant volumes/g silicic acid are cut by about a fifth. To obtain the best separation the column must not be over-loaded;22*27s34 in the case of DDE the maximum permissible load is about 0.1 pgg.

6. Conclusions

The key to the separation of PCB’s from DDE lies in the correct activation of silicic acid for column chromatography, avoidance of overload and accidental de-activation during the analysis, the use of pure solvents, and the use of the right g.1.c. column. The penalty paid for an effective separation is low linear capacity in column chromatography and the need for large eluant volumes. The latter imposes the use of small columns for routine use, and the need for very high purity solvents because of the degree of concen- tration required. Fully activated adsorbent must be employed to obtain an effective separation and the use of deliberately de-activated material is precluded.

Acknowledgements

The assistance of Mrs E. A. Christmas in part of the experimental work, gifts of “Aroclors” by Monsanto, and the permission of H. J. Heinz Co. Ltd to publish this paper, are gratefully acknowledged.

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