these compounds following their addition to the sulfosalicylic acid homogenate (84.2 and 89.0%, respectively ; Table 11). Urochloralic acid, the glucuronic acid conjugate of trichloroethanol, yields trichloroethanol on hydrolysis. We have found that when mice are injected with 500 mg/kg of trichloroethanol, killed between 1 and six hours later and the trichloroethanol is estimated after subjecting tissue supernatants to hydrolysis, as described in procedure C, 97.3 i 0.7 of the injected trichloroethanol can be accounted for. If the trichloroethanol content of tissue is determined by procedure A, that is without prior hydrolysis, trichloroethanol is found to have a biological half life of 3.5 hours. We conclude, therefore, that the difference in recoveries of trichloroethanol by procedures A and C is due to a metabolite which on hydrolysis yields trichloroethanol. The only known metabolite which will do this is urochloralic acid.
procedure used. When whole body homogenates are being assayed, it is, therefore, essential to take into account tissue recovery values. Similar consideration would presumably apply to other biological materials. Furthermore, the chromophore formation is affected by minor variations in the temperatures and time intervals of the assay procedure. Accordingly calculations should always be based on contemporaneously run recoveries of known amounts of the compounds from tissue homogenates. Since the recoveries from tissue homogenates of trichloroacetic acid and chloral hydrate using procedure A are not significantly different, the necessity to run both is obviated. The considerable decrease in sensitivity which occurs for tissue recoveries of trichloroacetic acid using procedure B should be particularly stressed. Failure to take this fact into account when analyzing mixtures containing both chloral hydrate and trichloracetic acid leads to an erroneously high value for the former and low values for the latter.
CONCLUSIONS
The procedures described herein afford a sensitive, accurate, and reproducible method for the estimation of chloral hydrate and its metabolites, trichloroethanol, trichloroacetic acid, and urochloralic acid, in whole body homogenates without necessitating prior extraction or separation procedures. Other compounds possessing trihalogens substituted carbon atoms can undergo the Fujiwara reactions ( 4 ) and would be expected, if present, to interfere with the assay. When tissue recoveries are performed, there always occurs a significant loss of sensitivity, although the magnitude of this effect differs with the chromophore being formed and the
ACKNOWLEDGhIENT
We thank the Computing Center of the State University of New York at Buffalo for the use of its equipment. This Computing Center is partially supported by National Institute of Health Grant FR-00126 and National Science Foundation Grant GP-5675. RECEIVED for review May 15, 1967. Accepted June 26, 1967. Investigation supported by a grant from the Licensed Beverage Industries and by U. S. Public Health Grants 5T1 GM 107 and R01 MH 12542.
Gas Chromatographic Determination of Normal Paraffins iln Kerosine H. S . Knight Shell Decelopment Cornpanj*,Emerycille, Calif. A SUBTRACTION METHOD for the determination of normal paraffins in kerosine and gas oil was developed by Whitham ( I ) . The sample was analyzed twice by gas chromatography, once with and once without a Molecular Sieve 5A column for selectively sorbing the normal paraffins (normals). By comparison of the two independent curves, Whitham was able to calculate the normals content. This method is not very sensitive. Eggertsen and Groennings ( 2 ) developed a more elaborate gas chromatographic method in which the normals were released from the sieve by heating. Only one sample was needed and the normals and non-normals areas were observed independently, which resulted in improved sensitivity. The method, limited to gasoline, employed a specialized apparatus for programming the sieve and analytical columns independently and oxidizing the effluent to COz and water prior to detection. By modifying the apparatus and the temperature programs Blytas and Peterson were able to extend the method to the kerosine range (3).
Ponnamperuma and Pering ( 4 ) and Brunnock (5) reported on methods for recovering the normal paraffins from the sieve by destroying the sieve lattice with H F and extracting the released normals, The need for specialized gas chromatographic apparatus is thus avoided, particularly if the sorption step is also independent of the GC equipment. The sorption of the normals external to the GC is usually carried out as described by Brunnock. The sample is diluted with a non-normal solvent and an excess of molecular sieve is added. The mixture is then refluxed until the normals are all sorbed. This takes many hours. In the present work the kerosine is added directly to an excess of sieve and the free-flowing mixture is warmed to 100"120" C for half an hour. This completes the sorption step. The sieve is washed free of non-normals and is then destroyed with dilute H F and the normals are extracted with isooctane. The more complicated recovery procedure used by Brunnock for higher boiling normals is not necessary for kerosine. H F is hazardous and the usual handling precautions must
(1) B. T. Whitham, Nature, 182, 391 (1958). (2) F. T. Eggertsen and S. Groennings, ANAL.CHEM., 33, 1147
(3) G. C . Blytas and D. L. Peterson, ANAL. CHEM.,39, 1434 (1967). (4) C. Ponnamperuma and K. Pering, Nafure, 209, 979 (1966).
(1961). 1452
ANALYTICAL CHEMISTRY
be observed. However, the entire procedure is scaled for G C determination of the normals and the amount of HF required is not large, EXPERIMENTAL Apparatus. A temperature-programmed gas chromatograph with hydrogen flame detector was employed. The stainless steel column was packed with 5 2-foot X 3/16-in~h SE-30 on Chromosorb W. It was programmed from 60" to 160" C in 5 minutes. The G C conditions are not critical. An oven at 100"-120" C is required, and 1-dram glass and 2-dram plastic vials are required for the sorption and decomposition steps, respectively. Small stirring rods were cut from polyethylene rod. The diluted HF was stored in a plastic dropping bottle. Reagents. C P hydrofluoric acid was diluted 2-1 (to about 16%) with distilled water. Molecular Sieve (5A) was obtained in 80-100 mesh from Coast Engineering Laboratory, Redondo Beach, Calif. The sieve was activated in a stream of dry nitrogen for two hours at 250"-300" C. Oven drying in an evaporating dish was less satisfactory. Procedure. To about 1 ml of sample add accurately 2-5Z of a normal paraffin not present in the sample originally; nonane and hexadecane have been employed. The added paraffin serves as a marker for the G C analysis. In a 1-dram glass vial, place 0.4 gram of freshly activated sieve, add 25 p1 of the sample with marker, cap the vial, and shake to mix the contents thoroughly. Place the vial in the oven and shake to mix the contents after 5 and 15 minutes, tightening the cap if necessary. After half an hour, cool the vial and transfer the sieve to the glass tube from a small medicine dropper with a cotton wool plug in the tip, and wash the sieve with isooctane to remove unsorbed material. Draw air through the dropper to evaporate most of the solvent and transfer the free flowing sieve to a plastic vial. Add 1 ml of isooctane and then 1 ml of water, and fill the vial about half full with the dilute HF solution. Leave the vial uncapped. After the first rapid gas evolution ceases, fill the vial to about 1 cm from the top with the H F solution and let it stand until the sieve structure has disappeared. Occasional stirring hastens the process. Cap the vial and cool it in cold water or allow it to cool to room temperature. Then shake it vigorously to extract the released normals into the isooctane layer. If the vial is not cool, it may leak, and in any case rubber gloves should be worn. Analyze 2 pl of the isooctape layer by gas chromatography. Whatever conditions are used should be confirmed with known blends of normal paraffins in isooctane that approximate the extracts in composition. RESULTS AND DISCUSSION The method was first tested by analyzing known mixtures of pure normal paraffins to confirm the G C and sorption conditions. The results given in Table I show that only small errors were incurred. Next a kerosine sample was denormalized using a large excess of sieve, and 0.1 of each normal paraffin from CloCI4was added. This sample was analyzed, with the results shown in Table 11. Finally, several kerosine samples were analyzed by the present procedure and by the method of Blytas and Peterson
Table I. Analysis of Known Paraffin Blend, % w rz-Paraffin carbon number 10 11 12 13 14 Known 20 1 18 6 19 9 19 6 20 1 17 5 20 0 Found 1 20 6 20 0 20 6 2 20 6 18 2 20 6 20 2 20 8 3 19 7 18 0 20 3 20 2 21 8 4 20 1 18 3 20 0 19 9 21 1
Table 11. Analysis of Denormalized Kerosine, 10 Known Found 1 2
0.10
0.10 0.08
17-Paraffincarbon number 11 12 13 0.10 0.10 0.10 0.09 0.11 0.12 0.08 0.10 0.14
w 14 0.10 0.08 0.16
Table 111. Comparative Analysis of Kerosine, % w Sample Method A Present A Bly-tas-Peterson B Present B Blytas-Peterson
11 3.3
3.2 5.4 5.4
ri-Paraffin carbon number 12 13 14 15 6.5 6.3 4.3 0.12 6.5 6.4 4.7 0.09 5.0 4.6 3.3 0.6 5.1 4.6 3.3 0.4
(3). Data given in their paper are repeated for convenience in Table 111. It is shown that the results agree within is%. The overall recovery of kerosine from the sieve was studied by analyzing for both non-normals and normals. This was done with the aid of an additional marker, ethylcyclohexane. The isooctane wash containing the non-normals was analyzed as well as the extract containing the normal paraffins. The overall recovery was 95-100 %. Some of the loss was due to sorption of non-normals on the sieve. These non-normals are not measured during the normals determination because they are spread out and do not form discrete peaks. When the normals peaks are small, as in the case where traces of normals are to be determined, the sensitivity must be increased for accurate measurement and the non-normals may then be observed. The extent of sorption of non-normals varies with the sieve lot. With one batch of sieve there was interference with normals at the 0.1 level, and this lot was discarded. Because non-normals are sorbed, it follows that the determination of non-normals described here is not suitable for traces of non-normals in normals. The nature of the sorbed material other than normal paraffins was not studied in detail but it was not predominantly 2-methyl branched paraffins.
RECEIVED for review May 3, 1967. Accepted June 16, 1967. (5) J. V. Brunnock, ASAL.CHEM., 38, 1648 (1966).
VOL. 39, NO. 12, OCTOBER 1967
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