Application of On-Column Acid-Base Reactions to the Gas Chromatographic Separation of Free Fatty Acids and Amines SIR: The use of gas chromatographic fore-columns or columns in series is well known (3-5). Although E. Brochmann-Hannsen and A. B. Svendsen (2) have chromatographed alkaloids by dissociation of their salts in a flash heater a t 325' C., a search of the literature has revealed no gas chromatographic methods for on-column acid-base reactions liberating free acids or amines from their salts prior to their separation on an analytical column. We now wish to report that fatty acid salts in aqueous solutions can be converted to the free acids in a fore-column (via on-column injection) packed with an acid coated support. I n an analogous manner, a basic packed fore-column can liberate amines from their salts just prior to their separation on an analytical column. EXPERIMENTAL
Apparatus. A flame ionization gas chromatograph ( F and M Scientific Corp., Model 810) was used with a 1-mv. Brown Electronik Recorder. Operating Conditions. Injection port temperature, 212" C.; column temperature 130' C.; detector temperature 322' C.; helium flow measured a t the inlet, 85 ml. per minute; recorder sensitivity, 1 X 256. Reagents. Except for a practical grade of butyric acid, the f a t t y acids
\ 0
4
8
1'2
20
16
24
28
TIME IN MINUTES
Figure 1. Typical chromatogram of free fatty acids obtained by injecting an aqueous solution of their corresponding potassium salts into an acid-packed fore-column followed by an analytical column 1.
2. 3. 4.
CH3COzH 5. CH3CHzCOzH 6. CHdCH2)zCOzH 7. CH~(CHZI~COZH
CHdCHz)4COzH CHdCHz)&OzH CH3(CHz)&OzH
employed were of reagent grade (Fisher Scientific Co. and Eastman Kodak Co.). Amphetamine (Smith Kline & French) was shown to be 99.9 % by conventional gas chromatographic analysis. Columns for Acid Analysis. Analytical column: l/a-inch o.d., 3/16inch i.d. X 4.3 foot stainless steel packed with 10% by weight of diethylene glycol adipate and 2% phosphoric acid (85y0) on 60- to 80-mesh, acid-washed Chromosorb-W. The forecolumn packing was prepared by coating 60- to 80-mesh, acid-washed Chromosorb-W with 20% by weight of 857, phosphoric acid using methanol as the solvent. This was packed into the injection port linear (1/4-inch o.d., inch i.d. X 4 inch stainless steel). The glass wool used for plugs mas soaked for several minutes in 85% phosphoric acid, the excess being removed by compression in an all-glass syringe. CoIumns for Amine Analysis. Analytical column: 1/8-inch o.d., 3/3*-inch i.d. X 5foot Teflon (Sparta Manufacturing Co., Dover, Ohio) packed with 10% by weight of Apiezon L on 60- to 80-mesh, acid-washed Chromosorb-W previously coated with loyo by weight of 85y0 KOH. The forecolumn packing was prepared by coating 60- to 80-mesh, acid-washed Chromosorb-W with 207, by weight of 85% KOH using methanol as a solvent. The glass wool used for plugs was soaked for a few minutes in a saturated methanolic KOH and dried in an oven a t 100' C.for one hour. I n addition to the injection port liner, a '/*-inch o.d., 3/32-in~hi.d. X 6 inch column of Teflon, inserted between the analytical column and the injection port liner, was packed with the KOH coated support. Procedure for Acid Analysis. One microliter of a n aqueous solution, 0.01M in KOH, of the potassium salts of the Cp to Cs normal fatty acids, present in a concentration equivalent to 0.01% by volume of each acid, was injected into the fore-column packing in the normal way using a 1O-pl. Hamilton syringe with a 2-inch needle. The needle penetrated about 11/4 inches of packing material before the solution was discharged. To ensure complete removal of the acid salts from the forecolumn and to prevent "ghosting" ( 1 ) from the analytical column, 2 bl. of 0.01Jf HC1 was used to "wash out" the system, 1 pl. being injected with . maximum penetration and 1 ~ 1 with a minimum penetration. Procedure for Detection of Amphetamine in Urine. Two microliters of an acidified ( p H 2 with dilute HC1) urine specimen was injected in the normal manner into the fore-column packing using a 10-p1. Hamilton syringe with a 4-inch needle. The needle penetrated about 31/4 inches of packing
+
material before the solution was discharged. After each analysis in which amphetamine was detected, the column system was cleaned out with one injection of 4 p l , of concentrated ammonium hydroxide, the first 2 pl. being liberated from the syringe with maximum penetration of the fore-column packing, followed by 2 pl, with minimum penetration. RESULTS AND DISCUSSION
Figure 1 shows a chromatogram of low molecular weight fatty acids obtained by injection of an aqueous solution of their corresponding potassium salts into an acid-packed injection port liner (the fore-column) followed by the analytical column. The general appearance of the acid peaks is essentially identical wit,h those of the free acids chromatographed (in aqueous acetone) on the same column, but without the fore-column. The chromatogram in Figure 2 showing an amphetamine peak was obtained by the direct injection of a n acidified urine specimen using a basepacked fore-column (the free base is only slightly soluble in water and less soluble in urine). The retention time for amphetamine was identical with that of an authentic sample (injected as the hydrochloride), Ghosting, the appearance of peaks with the same retention time as the various fatty acids or amines when water is injected, was not a serious problem. The height of a ghost peak was
I
I
I
0
2
4
I 1 I I I 6 8 1 0 1 2 1 4 TIME IN MINUTES
Figure 2. Typical chromatogram of amphetamine in urine obtained by injection of an acidified urine specimen into a base-packed fore-column followed by an analytical column 1.
Amphetamine
VOL. 37, NO. 12, NOVEMBER 1965
1591
never more than 4% of the corresponding peak of the previously injected sample. Contamination of a component by a ghost peak was completely eliminated by “washing out” the system prior to each injection as mentioned in the experimental. The method was rapid and simple. The fore-columns were easily packed, required little or no conditioning, and had good stability and reproducibility. There was no evidence of deterioration of the basic fore-column after fifty 2 4 injections of aqueous solutions at pH 2. The chief advantages of the method appeared to be simple sample preparation without loss of components to be analyzed, the use of water as a solvent for the analysis of water insoluble acids and amines, and the stability of standard chromatographic solutions. Although the main limitations of the method will undoubtedly be decomposi-
tions and undesirable side reactions on the fore-columns, the products from side reactions may serve equally well for the analysis of the reactants. For example, Peterson and Tao (4) analyzed trifluoroacetate esters in trifluoroacetic acid using a base-packed forecolumn which trapped out the solvent and converted the esters entirely to their corresponding alcohols. Corrosion of the metal parts of the instrument which came into contact with the acids and bases appeared to be nonexistent or negligible. Although only the qualitative aspects of the method have been studied, we are currently investigating its quantitative potential. ACKNOWLEDGMENT
The authors express appreciation to Harry D. Koster for help with the ex-
Lauric Acid-Diethanolamine Maximum Suppressor SIR: A number of communications (4-7) have discussed the importance and utility of soaps (sulfonated phenyl, xylyl, and tolyl stearic acids, dodecyl and dodecyl benzene sulfonates, dodecyl pyridinium bromide, and isothiourea dodecyl ether hydrobromide) in suppressing the polarographic maxima of simple and complex metal ions which are not ordinarily suppressed by the usual maximum suppressors. I n addition to the anionic and cationic soaps, nonionic soaps may also be used for this purpose. However, very few references describe this approach and those available refer to commercial products. Therefore, investigations on the use of these compounds in polarographic work was considered worth undertaking. This Table I.
communication describes the results from the use of a simple nonionic soap, lauric acid-diethanolamine condensate (LDC), in suppressing the polarographic maxima of Pb+2,Ni+2,C O + ~N,i + W o + 2 mixture, iodide cadmium complex, copper biuret, copper succinimide, and copper glycine complexes. Data on the influence of LDC on the electrocapillary curves of the dropping mercury electrode (d.m.e.) in suitable supporting electrolytes have been included to demonstrate the superiority of nonionic soaps in polargraphy. EXPERIMENTAL
Reagents. L D C (8) was prepared by condensing pure lauric acid (BDH) with diethanolamine. Biuret (2) and
Relative Effectiveness of Ionic and Nonionic Soaps as Maximum Suppressors
SXSA 10-6M 12.1 56.01 56.01
555.0 23.8 200.0 238.0 23.8 10.0 (%-biuret 6.0 83.3 60.5 16.9 60.05 Ci-gly cine 9.0 Cu-succinimide 12.5 ... ... LDC = Lauric acid-diethanolamine condensate SPSA Sulfonated phenyl stearic acid = Sulfonated tolyl stearic acid STSA =
32.71 23.8 4.9 6.05 ...
SXSA DPB IDEH 1592
= = =
LDC 10-5~ 2.0 7.0 9.0
SPSA 10-5M 23.5 43.9 24.0
Sulfonated xylyl stearic acid Dodecyl pyridinium bromide Isothiourea dodecyl ether hydrobromide
ANALYTICAL CHEMISTRY
LITERATURE CITED
(1) Brivin, E. S., Marco, G. J., Emery, E. M., J . Dairy Sci. 44,1768 (1961).
(2) Brochmann-Hannsen, E., Svendsen, A. B., J . Pharm. Sci. 51, 1095 (1962). (3) Decora, A. W., Dinnean, G. U., ANAL.CHEM.32, 164 (1960). (4) Peterson, P. E., Tao, E. V. P., J. Org. Chem. 29, 2322 (1964). (5) Swoboda, P. A. T., Chem. d% Ind. (London) 1960,1262. F.THOMPSON GEOFFREY KATHLEEN SMITH Department of Psychiatry Washington University School of Medicine and the Research Laboratories Malcolm Bliss Mental Health Center St. Louis, >lo. 63104 WORK supported by Grants MH 05415 and MH 5804 from the National Institutes of Mental Health, United States Public Health Service.
Condensate as a Polarographic
STSA 10-6M 23.8 56.01 107.8
Ions or complexes Pb+2 in KNOs Ni+2 in KCl Co+2 in KC1 Co+*-Ni +z in pyridine CdI,-KI complex
perimental work and to the Smith Kline & French Laboratories, Philadelphia, Pa., for a supply of amphetamine.
DPB i o - 5 ~
6.17 7.38 23.8 4.95 14.55 4.95 6.172 ...
IDEH 10-5~ 7.38 7’38
7.38
8.599 34.88 7.38
...
succinimide (9) were prepared in the laboratory. Analytical reagents and chemically pure reagents werk used in all the experiments. Double distilled water (all glass) was used in making the solutions. Triple distilled mercury was used for the dropping mercury electrode. Apparatus. The polarographic measurements were made using a Heyrovsky polarograph (No. L p 55A) in conjunction with a Pye scalamp galvanometer (No. 7903/5). All measurements u-ere carried out at 25’ =k 0.1’ C. in the water thermostat. Procedure. Polarographic measurements were made after adding a known volume of the metal salt or complex metal ion solution t o the polarographic cell, adding the supporting electrolyte, and making u p the total volume t o 20 ml. The solutions were deaerated bv bubbling purified nitrogen gas through them for about 20 t o 30 minutes. The nolarouam was taken. and the nrocess ;vas thYn repeated in’ the presence of the nonionic soap. An increasing amount of soap was added until the maxima were completely eliminated. For studying the effect of soap on the electrocapillary curves, a known volume of the ion to be reduced was added to the polarographic cell and mixed with suitable supporting electrolyte. The drop time with and without the soap was measured between the potential range -0.0 volt to - 1.0 volt. RESULTS AND DISCUSSION
Lauric acid-diethanolamine condensate is a useful maximum suppressor for a number of metal and complex ions
