analyses. Figure 4 shows that a 3-foot column provides more resolution in the elution peaks than is actually required. Thus, shorter column lengths should make it possible to subtract as much as a minute from the elapsed time. If a relative standard deviation of *5 to f10% can be tolerated in the analytical results, the 60-second gas-cooling period can be eliminated. These two modifications should make it possible to obtain analytical results approximately 3 minutes after a sample of appropriate geometry is received in the laboratory. The ultimate sensitivity of this technique can be extended considerably. Since only one fifth of the signal generated by the chromatographic detector was used in the calibration experiments, a fivefold increase in sensitivity is readily available on operating the instrument a t its maximum output. The noise level under these conditions is less than 1 0 . 3 mm. and therefore does not constitute a serious limitation. Another 100-fold increase in sensitivity can, in principle, be achieved if all of the extracted gases are subjected to chromatographic examination. Preconcentration procedures similar to those described by Meadows, Hubbard,
and Busey (16) and Lewis and Melnick (14) appear to be directly applicable. ACKNOWLEDGMENT
The authors are indebted to C. Clifton Hill for performing the vacuum fusion determination reported in Table
111.
(1962). \___-,-
(14) Lewis, L. L., Melnick, L. M., ANAL. CHEM.34, 868 (1962). (15) Masson, C. R., Pearce, M. L., Trans. Met. SOC.A I M E 224, 1134 (1962). (16) Meadows, G. E., Hubbard, G. L.,
LITERATURE CITED
(1) Colclough, T. P., J. Iron Steel Inst. 192, 201 (1959). (2) C o v i y p , L. C., Bennett, S. J., ANAL. HEM. 32. 1334 (1960). (3) Cyscoleca, O.,‘ Rosier, K.,J. Iron Steel Inst. 192, 147 (1959). (4) Evens, F. M., Fassel, V. A., ANAL. CHEM.33, 1056 (1961). (5) Evens, F. M., Fassel, V. A., Hill,
c. c.. Iowa State Univ.. unmblished data, July 31, 1962. (6) Fassel, V. A., in “Determination of Gases in Metals,’’ Iron and Steel Institute, Spec. Rept. 68, 103-21 (1960). (7) Fassel, V. A., Altpeter, L. L., Spectrochim. Acta 16,443 (1960). (8) Fassel, V. A., Tabeling, R. W., Ibid., I
(10) Gaines, J. M., J . Iron Steel Inst. 192, ‘ 55 (1959); (11) Guernsey, D. L., Franklin, R. H., m “Symposium on Determination of Gases in Metals,” American Society for Testing and Materials, Tech. Publ. 222, 3-13 (1962). (12) Ihida. M., Japan Analyst 8, 786 ’ (1959).‘ ’ (13) Karp, H. S., Lewis, L. L., Melnick, L. M., J. Iron Steel Inst. 200, 1032
.
8,210 (1966). (9) Feichtinger,
H., Bachtold, H., Schuhknecht, W., Schweiz. Arch. Angew. Wiss. Tech. 25,426 (1959).
Busey, H. M., U. S. At. Energy Comm., Rept. LA-2540 (June 16, 1961). (17) Schults, F. C. Pittsburgh Conference on Analytical dhemistr and Ap lied Spectroscopy, Pittsburgg, Pa., d r c h
1962. (18) Thompson, J. G., Vacher, H. C., Bright, H. A., J. Res. Nall. Bur. Std. 18, 259 (1937). (19) Turovsteva, Z . M., Kunin, 1,. L., “Analysis of Gases in Metals,” Academy of Sciences of USSR, MOSCOW, (transl. Consultanta Bureau, New York,) pp. 29-107 (1959).
RECEIVEDfor review March 18,. 1963. Accepted June 10, 1963. Contrlbutlon 1298. Work performed in the Ames Laboratory of the U. S. Atomic Energy Commission. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 6, 1962.
Application of Gas Chromatography to Analytical Toxicology LEO KAZYAK and EDWARD C. KNOBLOCK Division of Biochemistry, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington 7 2, D. C. b The remarkable versatility of gas chromatography has been amply demonstrated by its many applications to complex analyses. Toxicologists in particular have recognized the usefulness of gas chromatography for the analysis of toxic gases and volatile substances. The development of silicone polymer substrates and high sensitivity detectors have extended these analyses to the separation and detection of microquantities of barbiturates and of many of the alkaloids and tranquilizers, as well as to antihistamines and sympathomimetic amines, to mention a few in general categories. Utilizing SE 30 and QF-1 (FS-1265) columns and an argon @-ionizationdetector, the authors present gas chromatographic data on compounds of general toxicological interest with a discussion on the problems of application of this technique to routine analyses. Included are examples of drug separation and identification in solvent extracts of urine. 1448
a
ANALYTICAL CHEMISTRY
A
LTHOUGH gas chromatographic de-
terminations of many compounds of interest to toxicologists have been described, the applicability of gas chromatography to routine analyses is complicated by a variety of column substrate materials and different operating conditions reported (2,4, 6-9). This paper presents data obtained from a 1% SE 30 (on Chromosorb W) column a t a series of different temperatures and inlet pressures for comparison with other available data. The column performance is compared with 5% SE 30 and 3% QF-1 (FS-1265) columns to demonstrate the effect of column substrate concentration and polarity on retention time and operating parameters. EXPERIMENTAL
A Barber-Colman Model 15 gas chromatograph with a strontium40 argon ionization detector was used with columns of U-shaped borosilicate glass, 4 mm. i.d., 6 feet long, and packed with 100/120 mesh Anakrom ABS (silanized, acid- and base-washed processed di-
atomaceous earth from Analytical Engineering Laboratories, Inc., Hamden 18, Conn.) coated with a liquid phase of siloxane polymer-i.e., SE 30 or QF-1.
The technique for the preparation of a 1% SE 30 column packing was essentially as described by Homing, Moscatelli, and Sweeley ( 5 ) and involves mixing the Anakrom ABS (25 grams) for 10 minutes in a solvent such as chloroform (125 ml.) containing the dissolved substrate (0.5 gram SE 30). The excess solvent was removed by filtration and the coated support dried for 1hour a t 100” C. The dried support was subsequently packed with the aid of a vibrator into the glass columns. The 25-gram Anakrom ABS of this mesh cut absorbed about 60 ml. of the solvent to give a 1% coating. This quantity of coated support will pack a t least three columns of the given dimensions. Although the columns used in this study mere conditioned for 8 hours a t 300” C., other investigators (4) have reported good results with SE 30 columns conditioned as little as 2 or 3 hours. Detector sensitivity was optimum
'ERIDINE
LUTETHlMlDE (DOAIDEN)
'RWOXYPHENE
5
IO
MINUTES
Figure 1.
1% SE 30 column at 200° c.
The solvent extract is then evaporated to dryness at 55" C. in a stream of air, and the residue is retained for subsequent gas chromatographic analysis. Re-extraction of the same biological specimen at a pH below 3.0 and again a t a p H of 9.0 or above is performed in the manner just described except for the substitution of a n appropriate buffer wash for the phosphate buffer pH 7.4. The residues will contain most of the remaining acidic or basic compounds after evaporation of the solvent. An acid hydrolysis of another portion of the specimen, followed by an extraction of the hydrolyzate a t pH 8.5 to 9.0 with 20% isobutanol in chloroform, completes the preparation. Conjugated basic and amphoteric compounds such as the morphine analogs can be chromatographed from the residue obtained from the evaporated extract. Depending on the amount of residue, 50 to 100 pl. of solvent are added to each fraction, and one-fifth aliquots are injected into a chromatographic column containing the 1% SE 30. A column temperature of 180" C. is usually selected for the first set of injections, and successive one-fifth aliquots are then chromatographed a t 130' and 225' C. to complete the screening of the extracts.
tinuously for several days. Some variation may exist as well in the performance of similarly prepared columns, and this equilibration period is necessary for optimal efficiency of any column. However, when continuous operation is neither practical nor possible, some means must be sought to compensate for these variations. Attempts to compute specific retention volumes involve difficulties in estimation of gas holdup or total gas volume of thc column. Since the Srwargon ionization detector is not responsive to air, the apparent retention volume, as suggested by Ambrose, Keulemans, and Purnell ( I ) , cannot be determined easily. With substitution of the initial response or "blip" of the solvent for the air peak, the absorptivity of the solvents causes this response to vary with each solvent or solvent mixture. Hence, this is not an accurate nieasurement of gas holdup volume. The simplest method of obtaining reproducible results, consists of adjusting inlet pressure and column flow rate, and, if necessary, temperature until the known retention time of a selected compound can be approximated. Subsequent column performance with respect to retention time and resolution can then be reproduced to within 10 seconds for compounds which come off the column in 10 minutes or less. I n general, the variation diminishes with the shorter retention times, and correspondingly increases with the longer ones. Above 20 minutes reproducibility may be no better than 1 to 2 minutes. This could be due in part to a tendency to overload the column, since sensitivity decreases markedly for those substances with very long retention times (Figure
Flash heater a t 260' C., detector a t 230" C., 70 ml./min. flow rate, 33 p.si.g. inlet pressure
DISCUSSION
when operated at 17bO volts and electronic "noise" was not significant a t a relative gain of 3 x IO-* ampere. Injection port (flash heater) and detector were maintained a t temperatures higher than t h a t of the column by 50" and 25" C., respectively. One per cent solutions, 10 fig./fil. of the compound expressed as free acid or base, were prepared in chloroform or ethanol-chlcroform mixtures. These solutions generally produced a response of 30% or more of full scale deflection on a 50-mv. recorder a t the lowest gain of 1 x 10- ampere with injections of 1 to 3 pl. (10 to 30 fig.). This sensitivity was found to be largely dependent on retention time since the time and sensitivity changed inversely to each other-Le., with the shorter retention times, the sensitivity was greater. For the analysis of drugs in biological specimens, the materia1 sought -was extracted a t the appropriate H with chloroform. To begirt with, t e specimen should have a p l l not lower than 4.0 nor higher than 7.5. These conditions are adequate to isolate barbiturates, neutral coinpounds, and a few of the less basic (alkaline) drugs which are solvent so uble in this p H range. The chloroform, after extracting the specimen, is filtered through Whatman No. 41 filter paper, washed with phosphate buffw p H 7.4, and refiltered through Whatman No. 41 paper again. (This filtration is a rapid, convenient device for the removal, from the solvent, of aqueous traces which usually contain interferences.)
The main difficulty of reporting gas chromatographic data entails inclusion of those pertinent details which will enable other analysts to duplicate the results. Yet under constant pressure and flow rate conditions, day to day variations may occur in retention data which are minimal only when the apparatus is allowed to operate con-
3ARBITAL
R
PHENOBARBITAL
0
5
IO
w 5
20
25
MINUTES
Figure 2.
Separation of barbiturates in a urine extract
5% SE 30 column a t 195' C.; flash heater a t 260' C.; detector a t 225' C.; 1 15 ml./min. flow rate; 35 p d g . inlet pressure VOL. 35, NO. 10, SEPTEMBER 1963
1449
2). Usually an increase in column temperature will reduce the retention time and reproducibility will be better. Other investigators have suggested multiphase (two or more different substrates) columns and special column support treatment, other than the usual silanization, to minimize retention time variation and reduce peak asymmetry ($8). However, the accompanying increase in column polarity has some disadvantages when the relatively crude
Table I.
Temperature, O C. Inlet pressure, p.s.i.g. Flow rate, ml./min.
115 19 50
130 20 50
1% SE 30 Column 150 21 60
Ethchlorvynol (Placidyl) Phenethylamine Amphetamine Desoxyephedrine (Methamphetamine) Nicotine Ephedrine Ethynylcyclohexyl carbamate (Valmid) Warfarin Methyprylon (Noludar) Barbital Probarbital (Ipral) Acetophenetidin (Phenacetin) Meperidine (Demerol) Amobarbital (Amytal) Hydroxyphenamate (Listica) Pentobarbital Pheniramine (Trimeton) Caffeine Secobarbital (Seconal) Diphenhydramine (Benadryl) Glutethimide (Doriden) Meprobamate (Miltown) Lidocaine (Xylocaine) Prominal (Mebaral) Antipyrine Aminopyrine Tripelennamine (Pyribenzamine) Methapyrilene (Histadyl) Chlorpheniramine ( Chlor-Trimeton) Phenobarbital Procaine Methadone (Dolophin) Bromodiphenhydramine (Ambodryl) Propoxyphene (Darvon) Atropine Thonzylamine (Anahist) Chlorcyclizine (Perazil) a-Cyclohexyl-a-phenyl-1-piperidine-propanol
1450
ANALYTICAL CHEMISTRY
165 21 60
180 26 65
200 33 65
210 36 70
225 39 80
250 42 80
Retention Time, Min.
Compound
(Artane) Tetracaine (Pontocaine) Promazine (Sparine) Librium Scopolamine DDT ( Dichlorodiphenyltrichloroethane) Antazoline (Antistine) Codeine Ethylmorphine (Dionin) Diphenylhydantoin (Dilantin) Chlorpromazine (Thorazine) Mor hine cincionine Cinchonidine Diacetylmorphine (Heroin) Chloroquin Dibucaine (Nupercaine) Quinidine Quinine Anileridine (Leritine) Meclizine (Bonamine) Strychnine
are approximate as these were varied when necessary to standardize the column as discussed above. Periodic recheck of these data during a 10month period shoITed reproducibility to be as claimed. Since resolution is dependent on the column temperature and polarity of the material injected, most compounds could be separated from each other if retention times differed by 30 seconds or more. Figure 1 illustrates the resolu-
extracts of biological specimens are chromatographed. Polar interferences which tend to emerge with the solvent on the SE 30 column will be retained on a more polar column long enough to obscure those peaks from drugs with shorter retention times. Table I provides data of the performance of the 1% SE 30 column. Except for temperature, which was carefully maintained at the values in the table, inlet pressure and flow rate
1.4 2.1 2.4 3.0 6.7 7.5 9.5
1.2 1.7 1.9 2.1 4.1 4.2 4.9 10.8
1.3
2.2 2.3 2.5
4.3 4.3 5.6 8.6 9.7 10.9 11.3 13.2 13.0 14.7 16.8 16.9 18.0 23.1 20.8
1.2 1.7 2.9 2.8 3.5 5.7 5.9 6.0 6.5 6.5 7.5
8.1
8.2 9.3 9.1 10.4 9.9 11.2
11.8
12.0 14.7 14.8 16.3 19.2 18.6
1.2
0.8
1.8
1.6 1.9 2.6 3.0 3.0 3.2 3.2 3.6
1.6 1.6
3.8
3.8 4.2 4.2 4.4 4.5 5.2 5.3 5.7 6.7 6.8 6.8 7.8 7.9 12.1 12.3 13.8 14.0 14.7 15.5 16.4 17.2 26.0 20.3
1.9 2.0
1.
1.8
2.1
2.3 2.6 3.0 3.0 3.0 3.3 3.4 4.9 4.7 5.5 5.6 5.7 6.3
1.9
6.5 6.4
4.3 4.2 5.5 5.6 5.7 5.5 6.0 6.8 7.4 9.0 9.7 9.9 13.6 14.6 14.5 14.7 20.9
8.1 8.5
8.6 9.2 10.7
3.3 3.3 4.0 4.0 4.0 4.4
32.2
2.0 2.3 2.3 2.4
1.3
3.0 3.0 3.1
1.6 1.6 1.6
3.2 3.5
1.6
3.8
3.9 4.6 4.7 6.2 6.4 6.9 6.9 9.1 11.0 11.2 13.9 20.3 23.7
1.8
2.0 1.9 2.2 2.3 2.8 2.8 3.0 2.6 3.1 4.4 4.4 4.4 6.2 8.7
Table II.
Comparative Retention Data
Hydrolyzed Urine
I % S E 30 225'
Temperature, O C. Inlet pressure, p.6.i.g. Flow rate, ml./min.
M "
Figure 3. Solvent aliquot equivalent to 5 mi. of urine which contained 25 pg. of morphine (5 pg./rnl.)
tion possible on the 1% SE 30 column in the separation of four compounds with similar ultraviolet Fpectral characteristics. The meperidine and glutethimide retention t mes differ by 25 seconds. -1comparison of barbiturate retention times on 5y0 SE 30 (Table 11) with those obtained on the 1% SE 30 column at 150' C. shows conLiderable similarity. For the most part the lower substrate concentration enablei comparable performance at a signifi cantly lower temperature. This is not to infer t h a t retention data of these columns are interchangeable, but cwtain predictions are possible which render multiple columns unnecessary for routine analyses. Some of the comliounds in Table I quite obviously cannot be separated on the 1% SE 30 column. Methyprylon and barbital, antipyrine and aminopyrine, and several other pairs of conipounds can be selected R-hich have very similar retention times. Separations of some of these coripounds on a 37, QF-1-0065 (FS 1265) fluorosilosane polymer column are recorded in Table 11. Comparison of t i e results given in t lie t n n tables will sh x-v substantial differences in the retention data which are tiot due to increased substrate concentration but rather to the relatively greater polarity of thi. QF-1-0065. T o date most of. our applications of gas chromatography to specimen analysis have been qual tative. Placidyl, meprobamate, glute.,himide, librium, warfarin, and t h r more coinmon bartiiturates have siicc~es.ifullybern isolattd Diid identified by mesns of the SC 30
60
200 25 80
Compound
Retention Time, Xin.
Hydroxyphenamate (Listica) Warfarin Propoxyphene (Darvon) Pheniramine (Trimeton) Diphenhydramine (Benadryl) Barbital hlethyprylon (Soludar) Acetophenetidin (Phenacetin) Caffeine Probarbital (Ipral) Amobarbital Pentobarbital
1.G 2.2
a-Cyclohexyl-a-phenyl-I-
93
195 35
piperidinepropanol (Artane) Chlorcyclizine (Perazil) Secobarbital Thonzylamine (Anahist) hminop yrine Procaine Glutethimide (Doriden) Atropine Lidocaine (Xylocaine ) Prominal Phenobarbital Librium Promazine (Sparine) Tetracaine (Pontocaine) Antipyrine Antazoline (Antistine) Scopolamine Chlorpromazine (Thorazine) Morphine Cinchonidine Cinchonine Chloroquin Diacetylmorphine (Heroin)
240 30
95
2.6 3.6 2.8
5.4
3.4 4.0
7.3 11.6 12.6 15.0
21.8 27.7
columns w e d in our laboratory. Figure 2 is a n example of the separation of
barbiturates obtained from a 10-nil. urine aliquot of a n individual mho had ingested a barbiturate mixture. The peak at 20 minutes is possibly a metabolite of phenobarbital. Total concentration of the mixture as determined by ultraviolet spectrophotometric analysis was 5.5 mg. barbiturate/100 ml. urine. This example of multiple drug separation on the 5% SE 30 column has been included to show the effectiveness of gas chromatographic techniques to resolve, simply and quickly, compleu problems of identification. Large amounts of interferencr~ hich attend the breakdonn of urinary constituenta during acid hydrolysis may present some difficulty in certain gas chromatographic analyses. However, Figure 3 shows the separation of morphine from such an extract obtained from the urine of an individual who had received therapeutic doses of the drug. Although the identification and quantitation of niorphine may he effectively performed by ultra\ iolct spcctro-
4.4 5.3 5 ,G
5.8 5.9 6.6 6.7 6.8 8.5 9.3 9.3 10.8 11.0 13.3 13.5 13.6 14.2 17.3 18.6 30.1
4.9 7.0 8.6 8.7 10.7 12.2
photometric analysis, the value of gas chromatography for screening purposes or confirmatory analyses is obvious. The application of gas chromatographic techniques to specimen analyses does not involve any unusual consideration-. If retention times of standard Tolutions can be duplicated and checked n ith available data, subsequent identification of chromatographed drugs presm t in an eLtract of biological tissue or fluid can readily be made. Retention data a t various temperatures and flow rates have been provided to allon a greater choice of operating parameter>. as n-rll as to aid the analyqt in the interpretation of hi> chromatogram5 a t whatever teniprratme he may have selected to qcreen the material in question. I n addition. the effect of temperature change on retention time may be utilized to confirm the identification or to help ascertain the effect of different substrate concentrations on resolution. To qome extent vnsitivity may be IncrrazPd in raiiing column temperatures to decrwv! retention time and t o increase peak height. Finally, the iizc VOL 35, NO. 10, SEPTEMBER 1963
1451
of a second column of different polarity to separate compounds with similar retention characteristics may be used for final confirmation of an analysis. ACKNOWLEDGMENT
The authors gratefully acknowledge the technical assistance of Margaret Saville, E. A. RIeigs, and Gerald Hou li han
LITERATURE CITED
(1) Ambrose, D. A., Keulemans, A. I. M., Purnell, J. H., ANAL. CHEM.30, 1582 (1958). (2) Anders, M. W., Mannering, G. J., Ibid., 34, 730 (1962). (3) Cieplinski, E. W., Ibid., 35,256 (1963). (4) Fales, H. M., Pisano, J. J., Anal. Biochem. 3,337 (1962). (5) Horning E. C., hloscatelli, E. A., Sweeley, 6. C., Chem. & Ind. (London) 751 (1959).
(6) Lloyd, H. A., Fales, H. M., Highet,
P. F., VandenHeuvel, W. J. A., Wildman. W. C.. J. Am. Chem. SOC.82.
379f(1960). ’ (7) Parker, K. D., Fontan, C. R., Kirk, P. L., ANAL.CHEM.34, 757 (1962). (8) Ibid., 35,356 (1963). (9) Parker, K. D., Kirk, P. L., Ibid., 33, 1378 (1961).
RECEIVED for review December 19, 1962. Accepted May 29, 1963.
Gas Chromatographic Determination of Traces of Ethanol in Methanol KARL J. BOMBAUGH and WILLIAM E. THOMASON Research and Developmenf Division, Spencer Chemical Co., Merriam, Kan.
V A
method to determine ethanol in methanol at the 10-p.p.m. level is described. By using a column containing d-sorbitol on acetylated White Chromosorb at 100’ C., ethanol is eluted before methanol into a clearly resolved peak. Solid support treatment and proper column temperature are critical factors in obtaining resolution at the parts-per-millionlevel.
M
than 20 p.p.m. ethanol is desired in certain chemical syntheses, such as in the synthesis of dimethyl isophthalate where ethanol produces undesirable side reactions which result in an off-colored product. A method was needed, therefore, to determine ethanol in methanol a t the 10 p.p.m. level. The determination of ethanol in methanol a t trace levels is much more d s c u l t than would be inferred from the relative ease with which ethanol may be determined a t the 1% level. The limitation in this determination is not only the sensitivity of the detector but also the separation ability of the column. What appeared to be complete separation a t detector sensitivities suitable for analysis a t the 1%level was only a partial separation when monitored by a high sensitivity ionization detector. A wide variety of polar substrates such as polyglycoh, polyesters, and polyfunctional surfactants suitable for alcohol separations at higher ethanol concentrations were not suitable at the 10 p.p.m. level because ethanol eluted on the tail of the methanol peak. This work showed that by using dsorbitol as the stationary phase, a t the proper column temperature, ethanol was eluted before methanol. When the solid support was pretreated with glacial acetic acid (1, %’),base-line resolution was attained even a t the 10 p.p.m. level. 1452
ETHANOL CONTAINING less
ANALYTICAL CHEMISTRY
Table 1.
Retention of Ethanol Relative to Methanol
Source Fisher Scientific Co. Fisher Scientific Co. Fisher Scientific Co. Fisher Scientific Co. Fisher Scientific Co. Fisher Scientific Co. Fisher Scientific Co. Fisher Scientific Co. Fisher Scientific Co. Fisher Scientific Co. Fisher Scientific Co. Dow Chemical Co. Union Carbide Chemical Co. Armour Industrial Chemicals Co. Armour Industrial Chemicals Co. Nonylphenoxypolyoxyethylene Rohm & Haas Co. ethanol Polyethylene glycol 6000 Union Carbide Chemical Co. Substrate d-Sorbitol d-Sorbitol &Sorbitol d-Sorbitol &Sorbitol d-Sorbitol d-Sorbitol d-Sorbitol 2% HaPo, Glycerol Glycerol Glycerol Polypropylene glycol UCON HB 660 Armeen SD Ethofat 60/25
+
EXPERIMENTAL
The work was done on a BarberColman Model 20 gas chromatograph equipped with an ionization detector using a tritium foil source. The signal was read out on a 5-mv. Wheelco recorder. The Barber-Colman split type sample injector was replaced with a direct sample injector prepared from a I/8-inch Swagelok Bulkhead fitting. The body of the fitting was drilled with a no. 52 drill and a piece of l/s-inch 0.d. tubing was silver-soldered into it. The sharp leading edge of the Swagelok fitting was ground flat to provide a seat for the septum. A resistance heater was mounted on the column side of the assembly and thermally insulated. Liquid phases investigated are shown in Table I. All liquids were loaded on the solid support to 20 weight yo. Solid supports investigated were White Chromosorb (Johns-Manville Products Corp.), acid-washed White Chromosorb, sold under the trade name Gas-Chrom P. (Applied Science Laboratory, Inc.),
Temperature, O C. 42 50 59 74 90 100 107 100 43 66 78 85 70 85 80 67 100
Relative retention 1.36 1.23 1.00 0.80 0.78 0.76 0.78
Dehydrated 1.02 0.92 0.91 1.55 1.50 1.43 1.47 1.40
1.24
and White Chromosorb acetylated in this laboratory. The acetylated solid support was prepared by digesting 60- to 80-mesh White Chromosorb for 1 hour in glacial acetic acid. After digestion, the excess acid was removed by decantation and by repeated washings with distilled water. The support was dried at 100’ C., coated and screened to select the 60to 80-mesh material. The analytical column was prepared from a 35-foot length of ‘/*-inch copper tubing, coiled after packing. It was packed with acetylated White Chromosorb which had been loaded to 20 weight yowith d-sorbitol. Samples were injected with a Hamilton microliter syringe equipped with a Chaney adapter. Ethanol-free methanol was prepared by distillation on a 30-plate Oldershaw column operated at total reflux for several hours and followed by a brief collection period a t a 30 to 1 reflux ratio. Standard calibration solutions containing known amounts of ethanol were prepared from the ethanol-free base stock.
