Determination of Gamma-Bienzene Hexachloride by Partition Chrornatography OTTO T. AEPLI, PAUL A. MUNTER, AND JOHN F. GALL Whitemarsh Research Laboratories, Pennsyluania Salt Manufacturing Company, Wyndmoor, Pa.
A partition ohromatographio method for determining the g a m m a isomer con-, tent oT benzene hexachloride products is described. The partition solvents are nitromethane and n-hexane and the supporting medium is silieie acid. The method is sufficiently oonvenient and rapid for routine produotion analysis, and requires no expensive equipment or specially trained personnel. The accuracy is ahout 29%based upon the aotual gamma isomer content of synthetic mixtures of the isomers. The procedure appears to be adaptable to the determination of s o m e of the other isomers and related constituents.
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analysis of the _-______ ..?e usual formulations. OMMERCIAL benzene hemchloride, produced hy the The method is an absolute one, requiring no empirical calibration. chlorination of benzene, is 8 complex mixture containing a t least five space isomers of 1,2,3,4,5,6hexaehlorocy~lohe~~ne,It does not require expensive equipment and materials or specially trained personnel. together with other closely related chlorinated materials (6, 6). It is known that the insecticidal activity of benzene hexachloride REAGENTS products resides principally in the gamma isomer of hexachloro%Hexane, commercial grade. Nitromethane, commercial cyolohexane (6). Heretofore, no completely satisfactory method grade. has been available for the determination of this constituent. Mobile solvent (n-hexane saturated with nitromethane). Direct methods for determining the hiologicd activity of inAdd approximately 900 ml. of the n-hexme to 50 ml. of the secticidal preparations are, of course, applicable to this material, nitromcthane contained in a 1-liter separatory funnel and agitate vigorously for 5 minutes. Allow the excess nitromethane to hut their accuracy is subject to statistical limitation, and the Beparate out, draw off from the mobile solvent, and store for methods are time-consuming and require special laboratory future use. facilities and stringent control of technique and environmental conditions. Methods based an infrared absorption have been described by Daasch and by Kauer, DuVall, and Alquist (8). The infrared methods require expensive equipment, considerable preliminary calibration, and a detailed qualitative knowledge of the composition of the material to he analyzed. The only other method which has been reported for the analysis of benzene hexachloride products is the cryoscopic method of Bowen and Pogarelskin ( I ) . This method, as described, requires substantial quantities of highly purified gamma isomer, and involves the methods of precision thermometry. The method described below involves gamma isomer from the other constituent tography. A column .of a solid (such as siiieic acmj 1s mea EO support a solvent phase (nonmabile solvent), through which is flowed a second immiscible solvent (mobile solvent) in which the sample is initially dissolved. A continuous partition between the two solvents takes place 6.a the sample is washed down through the oolumn, and a progressive separation of the constituents of the sample occurs. Partition chromatography was first employed for the separawho also tion of acetylamino acids by Martin and Synge (4), proposed a theory for the method. Ot.her applications of the method and the theory were recently reviewed by Synge (7). Ramsey and Patterson (6) applied the procedure to the separs, tion and identification of the constituents in commercial benzene hexachloride; they employed nitromethane and %hexane as the partition solvents and silicic acid as the supporting medium, The present authors have established the conditions and detailed procedures required for the quantitative determination of the gamma isomer content and have demonstrated that the method e m he made of good precision and accuracy, and is sufficiently convenient and rapid for routine production analysis. The partition chromatographic method is also applicable to the determination of the other constituents present in commercial benzene hexachloride and can he conveniently adapted to the Figure 1. Partition Chromatographic Apparatus ~I
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(e
610
I __.____I
611
V O L U M E 20, NO. 7, J U L Y 1 9 4 8
2rl closed and v6 and u4 open. Apply pressure to the reservoir by closing vl, us, u4, and 06, adjusting the pressurereducing valve on the nitrogen gas cylinder to about 8 pounds' gage pressure, and carefully opening vI. Preparation of Partition Chromatographic Column (Figure 2). Keigh out 100 * 0.5 grams of silicic acid and transfer it to a large mortar. Measure out 55 ml. of nitroniethane and add it in successive 10-ml. portions to the silicic acid, thoroughly mixing after each addition by grinding with the pestle. A4fter all the nitromethane has been added and mixed, add 300 ml. of the mobile solvent in 100-ml. portions, mixing after each addition. Transfer the resultant slurry to a 600-ml. beaker. Carefully disconnect and remove the clean and dry glass partition column, a, from its support and insert the glass wool plug and the porous plate support, d, in the bottom of the column. The amount of glass wool used should be just sufficient to support the silicic acid column, as excessively large plugs tend to decrease the sharpness of separation. Stopper the bottom of the partition column with a small cork. Stir the slurry of silicic acid and the mobile solvent and pour it from the beaker into the glass column, holding down the porous plate and glass wool plug with a glass rod. When about half of the slurry has been added, remove the glass rod and pour in the remainder of the slurry, Replace the charged partition column in its support and connect the top of column b to the reagent and pressure lines, k , by means of the flanged glass pipe fitting. Remove the cork stopper from the bottom of the column and drain the excess mobile solvent under pressure from the column by opening 214. During the removal of the excess solvent, the silicic acid tends to pack down in the column ahead of the solvent level. When the level of the mobile solvent just reaches the top of the silicic acid layer, release the pressure in the column by closing v4 and opening Q. Care must be taken to prevent the splitting, cracking, or drying of the silicic acid column which may result if the solvent is allowed to drain below the level of the silicic acid layer. Such defects seriously impair the efficiency of the column. The partition column is now ready for use.
v6, keeping
d
F. k
A r
Figure 2.
Schematic Diagram of Apparatus
Partition chromatographic column, Pyrex pipe, 32 inches long X 1 inch inside diameter b. Flanged glass pipe fitting (Corning) on upper end of a with Koroseal gasket C. Pyrex ball and socket joint, size 18/7 d . Porous i h t e supported by glass wool e. Pyrex fraction collector, 10-ml. capacity in each side arm 0 . Pressure gage, 0 t o 30 pounds, 3.5-inch, single bronze tube h . Safety valve, 50 pounds, side outlet IC. Connecting lines, 1 1 4 inch copper tubing r . Mobile solvent reservoir, 8-liter capacity s. Stoucocks, 2 - m m . P y x c. Valves, Hoke type, 1 r-inch a.
Preparation of Sample (Figure 3). Rendne hexachloride products are convenicntly classified for analysis on the basis of the gamma iSomer content as follows:
Low gamma gamples, 5% 15% gamma isomer content The sample preparation involves the complete extraction of the gamma isomer from the bulk of the other constituents present in the sample. By variation of the sample weight, the optimum amount of gamma isomer is secured for addition to the chromatographic column.
Ilisceilaneous I p p a r a t u . Pyrex flasks, round-bottomed. long-necked, 1OO-ni1. capacity for collection and evaporation of partition f ractions Pyrex flasks round-bottomed. lone-necked 200-ml. capacity for colI k t i o n and evaporation:f the gamma isomer cut Wood rack for storing flasks used in collection of partition fractions Gas pressure regulator and reducing valve, Hoke-Phoenix type, for nitrogen Nitrogen, water pumped
Silicic acid (precipitated), analytical reagent grade. A suitable grade may be obtained, for example, from Mallinckrodt Chemical Work:. APPARATUS AND PROCEDURE
The apparatus developed for the partition chromatographic analysis of benzene hexachloride samples is shown in Figures 1 and 2. The enclosed wooden column support with a removable Plexiglas plate on the front side is desirable, considering the length of the column and the fact that it is operated under pressure. For the routine application of the method, the pressure reservoir for the partition solvent is of great advantage in permitting the uninterrupted addition of the solvent to the top of the silicic acid column. As shown in Figures 1 and 2, multiple connections may be provided, so that more than one column can be operated from the same solvent supply. The multiple solvent evaporator (Figure 5 ) permits the removal of the solvent concurrent with the collection of successive fractions from the column, so that immediate observation of solids content is obtained. A wooden rack is a convenience for holding in order the many flasks used in each determination. Charging the Solvent Reservoir (Figure 2). Charge the solvent reservoir, r , by adding about 6 liters of the mobile solvent through
Figure 3. S a m p l e P r e p a r ation Apparatus
Crush and thoroughly mix the sample by means of a mortar and pestle. n'eigh into a tared 125-nil. Erlenmeyer flask an amount of the ground sample sufficient to provide approximately 0.2 to 0.3 gram of the gamma isomer after extracting and aliquoting. Add a. Kohlrausch 25 ml. of the mobile solvent to the flask and type flask, to contain 100 rnl. heat just to boiling on the electric hot a t 20' C. plate. (Caution-fire.) Stopper the flask h. Buchner with a cork and agitate for 5 minutes. Cool funnel, fritted disk type, meto room temperature and decant the superdium porosity, natant solution through the Buchner funnel Pyfex, 34-mm. into the Kohlrausch flask, employing gentle inside diameter c. Vacuum consuction. Repeat the hot extraction step on nection the residue in the flask employing this time 10 ml. of the mobile solvent. (This second hot extraction mav be omitted on low gamma samples, where the quantity of gamins isomer to be extracted is small; and on high gamma material, where the sample may be almost completely soluble.) Wash the residue and flask with five 10-ml. portions of the cold mobile solvent, decanting each wash through the Buchner funnel into the Kohlrausch flask. Remove the Buchner funnel and vacuum connection from the Kohlrausch flask and dilute the contents to the 100-ml. mark with the mobile solvent. Mix thoroughly and transfer a 25-1111. aliquot of the solution to the prepared partition chromatographic column.
ANALYTICAL CHEMISTRY
612 Operation of Column (Figurp 2). Disconnect thc flanged glass fitting carrying the solvent and pressure hnes from the top of column b. Measure out and carefully allor?- a 25-ml. aliquot of the sample solution to flow onto the top of the silicic acid column by means of a volumetric pipet, being careful that the flow of sample solution does not disturb the silicic acid layer. Re connect the solvent and uressure lines to the tou of the column &d drive the sample solution into the columh by closing " 2 and v3 and opening v4. When the level of the smnple solution reaches t,he top of the Silicic acid layer, release the gas pressure by closing o4 and opening v l . Place a 100-ml. graduated cylinder a t the bottom of column at, c, to collect the effluent. Wash down the x~allsof the glass column once with approximately 10 ml. of the mobile solvent by opening R and UI. Drive the wash solution into the column hy closing 02 and va and opening v1 until the level of the wash solution reaches the top of the ailicic acid layer. Release the pressure by closing vd and opening ul, aqd add about 50 ml. of the mobile solvent to the column by opening vZ. Close v3 and start partition separation with a continuous flow of solvent to the column from the reservoir. Cont,rol the rate of solvent flow by adjusting the gas pressure in'thc reservoir so that t,he effluent, flow from t,be column is 4 t,o 5 ml. per minute. Allow 100 ml. of the mobile solvent to flow through the column into the graduated cylinder. Stop the flow by closing vn, remove the graduated cylinder, and attach the fraction collector, e (see also Figure 4j, to the ball joint, c. Close stopcocks SY and sa, open s, to the left vessel, open US, and continue the partition separation. Collect approximately 10 ml. (to mark) in the left, vessel, then turn s, to the right. vesscl. While the right vessel is filling, drain the left vessel through si into a 100-ml. roundbottomed long-necked flask labeled f r a h o n 1. When the right vessel is filled, turn s, to the left vessel and drain the right vessel through sa into a. second 100-ml. flaflk, labeled fraction 2. Continue the collection of effluent fractions, labeling each in SUCCBSsion until the separation of the gamma isomer is completed. ( U s u d y 30 to 40 10-ml. fractions a.re required.) Removal of Mobile Solvent from EWuent Fractions. .While the partition column is still operating, begin the recovery of t.he gamma isomer by transferring the round-bottomed flasks eonmining the effluent fractions from the rack to the solvent evaporator (Figure 5). Conncet each flask by means of a cork sto pel to the tvo-mv StODcock. b. on the vacuum line, place the &ks in the water'bst6 (60" C.), and apply vac&in. When the solvent evaporation is complete, carefully release the vacuum by turning stopcock b to open the line, remove the flasks, and replace them in their proper positions in the rack. IDENTIFICATION OF GAMMA ISOMER FRA'CTIONS
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The residues in the flasks are examined for the glO m m a ,enm er fraction. Recognition of these fractions is not difficult when once observed. Under ideal conditions each component of the benzene hexachloride sample partitioned occurs in a distinct and successive series of effluent fractions, separated from other components by two or more flasks which contain no solid matter. Frequently, a small amount of oily material which crystallizes slowly on prolonged standing occurs in some of the intermcdiata I
1
2 3 4
6
flasks. The first solids that appear in the series of effluent fractions are the higher chlorinated-produotsi.e., hepta- and octitchlorocyclohexanes. These are followed by more or less oily residuos and then by a series of fractions containing the alpha. isomer, ehamcterieed by its flaky or fluffy appearance. The amount of the alpha isomer then diminishes until two or three fractions occur which contain very little solid material and perhaps some oily substance. These indicate the end of the alpha isomer cuts. , . . i n e nex%cut wnicn IOL~OWSM ~ C Tm e alpna momer IS me gamma isomer, which increases in amount with succeeding fractions and is characterized by the thin rosette or fanlike crystal forms spreading out from the larger central masses. The amount of gamma. isomer in each cut diminishes rapidly in the latter fractions until several empty flasks are secured. These indicate thu end of the gamma isomer cut. The material which is identified as the gamma. isomer is recovered by dissolving the solids in each flask in n-hexane and quantitatively transferring the solutions to a weighed 200-ml. round-bottomed long-necked flask. The ,*hexane is evaporated and the weight of the combined gamma isomer fractions obtained.
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yogamma. isomer =
weight of gaiunr&found in 25-ml. aliquot weight of total sample taken for analysis
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RECOVERY OF SOLVENTS
A. oonsiderable proportion of t.he solvents employed in these determinations may he collected and purified by distillation for rcuso by saving all the ertract,ion and wash solutions and by placing a cold trap in the vacuum line from the water bath evaporator omployed for &ripping tho solvent from tho various fract,ions. RESULTS AND DISCUSSYO nm
+ha nroliminsxri rlo.n.lnrnnont o f t
partition fractions of the benzene hexachloride samples were identified by recrystalhation m d measurement of the meking
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V O L U M E 20, NO. 7, J U L Y 1 9 4 8 Table 11. Partition Chromatographic Analysis of Synthetic Samples for Gamma Isomer Content Fnrluulated Composition in Weight Per CentOctaHeptaJVt. "$ c.hloroBenzene Hexacliloride G~~~~~ vyloIsomers Isomer d:riiiidr cycloSo. tit,xan? a p 7 Foiintl hexane Y-1 o 0 0 0 100 100 d-2 0 5.0 15 u 15 2 0 80.0 d-3 65.0 0 5.0 30 0 30 2 5-4 67.8 4.8 16.1 10 1 10.3 6-5 14.1 15.0 14.2 ,& . 1 65.9 d-6 20 2 (i5 58.2 13.0 20.: si 47,9 30 4 8.8 9.7 JO., S-8 IO 2 6.1 88.8 40 i 40 4 S-!j 14 I 29 6 % I 48 1 1 48 2
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Heiati\-e Error
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1.33 0.66 1.98
0.71 o.99
0.99 0.74 i 43
~ w i i iHt a> d(wribet1 by llaniscy and 1';t on ( 6 ) . 111 the final .*ragt.sof developmcnt, the identity and ty of the alpha, beta, : r i d gitiiinia ieonier fract'ions w r e determined by thv mclt inp Iwirits arid mixed melting points Lvith the pure isomer?, a n d b\. ~ ~ i m p a r i s oofn the x-ray diffraction pattern? n-ith thaw reported l)y Kauei,, DuVall, and Slquist ( 3 ) . Because of the coniples physicwl nature of surh isomer niisturcs, it was recognized that ~ ~ t w1t s itre 1 not iiwf rily sufficicynt criteria oi t h r purit? c i f t h e ganiniit fractions. Honeuei, assuniing no great ahnoi~iiialiI i(>scBsirt, the melting point data for sonie typical conibined g t i i i i i i a fractions secured from the partition column and prtstxted i t i l ' a h l ~I are an indication of the probable purity of thr.se frariions. The mixed melting point data wcre secured on inisturr> %)i'equal parts of the gamma fractiolis n i t h the purc gamma i
