Table 11. Response Factors of Homologous Alcohols Relative to Methanol Response factor Alcohol Weight basis Mole basis Methanol 1 .o I .o Ethanol 0.8 1.1 1-Propanol 1 .o 2.0 1-Butanol 1.3 3.1 1-Hexanol 2.7 8.6 1-Octanol 3.8 15.4 ionization efficiency of 2 7 3 , and the limit of detection, Qo, was found t o be 8 X 10-6 mg/ml(6). The response factors of the members of a n homologous series of alcohols relative t o methanol were also determined, and are shown in Table 11. Although the stainless steel anode was used throughout, several types of cathode materials and shapes were evaluated to determine their effects on glow discharge stability, sensitivity, noise, and ease of starting. Cathodes of high purity silver or aluminum gave frequent arcing, and a glow discharge was difficult t o obtain. After a glow discharge was finally obtained with such cathodes, it was stable only over a narrow voltage range. Considerable sputtering was also evident with the silver cathode. With various iron or stainless steel cathodes, cold starting of the glow discharge (for example, at the beginning of the day) was a random process, and frequently required 15-20 minutes. Once started, however, the discharge was stable, reasonably quiet, and operable over a range of 360-400 V, depending upon electrode configuration, spacing, and pressure. After running for some time, the discharge could be extinguished then restarted more readily. It was eventually found that the discharge could be started immediately with such cathodes, even from a cold ~~~~
~
(6) S . Dal Nogare, and R. S . Juvet, “Gas-Liquid Chromatography, Theory and Practice,” Interscience, New York, 1962, p 181.
start, by introducing a small amount of acetone or 1,l-dimethoxyethane vapor into the cell through the air bleed line. Efforts t o obtain a glow discharge with helium carrier gas at 400 V were unsuccessful. It should, however, be possible to use helium, despite its high ionization potential (24.6 eV, compared t o 14.5 eV for nitrogen) if voltages somewhat above 400 are used. When only pure nitrogen was used as carrier, no discharge could be obtained with the stainless steel cathode. Under such conditions, however, introduction of a small sample of n-butane produced a discharge which disappeared as soon as the butane peak had passed. Continuous introduction of a small flow of moist air into the cell with the stainless steel cathode gave a stable discharge which stopped when a CaClz drying tube was inserted in the air line. Air saturated with water at room temperature produced a strong discharge, but the cell then failed to respond t o n-butane. Apparently excess moisture saturated the detector and desensitized it. When a n iron cathode (made from a common nail) was substituted for the stainless one, introduction of dry air was sufficient and necessary to produce discharge. The discharge was somewhat sensitive to pressure and voltage fluctuations, but signal output variations caused by these factors were not troublesome. Although the detector was not continuously exposed to organic vapors over a long period of time, cathode fouling was not a problem during the period of study. The stainless steel cathode did acquire a bronzed appearance o n part of its surface, but the central area directly exposed to the full discharge remained clean and bright. The introduction of (moist) air into the detector may have served t o reduce cathode fouling, but did not prevent formation of a tenacious black deposit (possibly carbon) on the face of the anode. The effect of oxygen upon the response of the detector was not studied.
RECEIVED for review December 1, 1967. Accepted March 1 1 , 1968.
Separation of Resin from Fatty Acid Methyl Esters by Gel-Permeation Chromatography Duane F. Zinkel and Lester C. Zank Forest Product Laboratory, Forest Serrice, U. S . Department of Agriculture, Madison, Wis. 53705
ANALYSIS OF COMPLEX MIXTURES of resin and fatty acids for the individual constituents by gas-liquid chromatography (GLC) is complicated by the overlap of the two solute systems. A two column analysis on diethyleneglycol succinate (DEGS) and a modified SE-30 packing was suggested as a practical approach to the problem ( I ) . The object of this study was t o achieve the quantitative group separation of fatty acid methyl esters from resin acid methyl esters by gel-permeation chromatography (GPC); this separation would simplify and improve the subsequent gas chromatographic analysis. One of the earlier reports on the application of the GPC technique t o the separation of low molecular weight materials is the paper by Cortis-Jones (2). Bartosiewicz (3) has separated fatty acid polymers into monomer, dimer, and trimer fractions. Cazes and Gaskill ( 4 ) recently investigated the G P C separations of low molecular weight, oxygen-containing 1144
ANALYTICAL CHEMISTRY
substances, including a number of saturated fatty acids. Chang ( 5 ) has developed a GPC method for the group analysis of tall oil as total resin acids, fatty acids, fatty acid dimers, and resin acid dimers. EXPERIMENTAL
A jacketed glass column cooled with tapwater (13 “C) and fitted with a reservoir was used. For most of the work, a column was gsed which contained a bed of 1 X 200 cm of Styragel, 40 A porosity, particle size of 37-70 p (Waters As(1) F. H. M. Nestler and D. F. Zinkel, ANAL.CHEM.,39, 1118 (1967). (2) B. Cortis-Jones, Nature, 191,272 (1961). (3) R. Bartosiewicz, J. Paint Technol., 39, 28 (1967). (4) J. Cazes and R. Gaskill, Separation Sci., 2,421 (1967). (5) T. Chang, ANAL.CHEM.,40, 989 (1968).
sociates, Inc., Framingham, Mass.) in anhydrous ethyl ether (fresh Mallinckrodt analytical reagent). Ethyl ether was used as the solvent and eluent. Samples of resin and fatty acid methyl esters totaling less than 50 mg were placed on the column in a 100-pl total volume; larger amounts as 50% solutions. The flow rate was 16 ml/hour under gravity flow. The column effluent was monitored with a differential refractometer (Nester-Faust model 404) and Texas Instruments Servowriter I1 recorder (0-0.8 mV span) and collected in 1-ml aliquots for G L C analysis. The tip of the effluent tube was continuously washed with ether. The G L C analysis was done using a D E G S column ( I ) in a n F & M model 5750 gas chromatograph equipped with flame ionization detectors. Temperature fluctuations at the refractometer detector head were avoided by immersion in a large tank of water at ambient temperature. The column effluent passed through 4 feet of 0.054-inch o.d., thin-wall tubing, made of Teflon (Du Pont), in the tank so that it would be at the same temperature as the detector.
Table I. Representative Elution Volumes of Resin and Fatty Acid Methyl Estersa Methyl ester ml Methyl ester ml Lignocerate (24 :0) 86 Tetrahydroabietate 111 Behenate (22:O) 87 (8P,9a,l CY-H) Arachidate (20:O) 89 Pimarate 112 Stearate (18 :0) 90 Tetrahydroabietate 112 Palmitate (16:O) 92 (8a,9a,l3a-H) Oleate (18:l) 93 Palustrate 113 Myristate (14:O) 94 Isopimarate 115 Linoleate (18:2) 94 Abietate 116 Laurate (12:O) 96 Levopimarate 117 Linolenate (18 :3) 96 Dehydroabietate 119 Caprate (1O:O) 97 Neoabietate 126 98 Nonanoate (9:O) Compilation of data from several experiments. A 3-mg sample of methyl linolenate or methyl pimarate is eluted in a total volume of ca. 12 ml.
RESULTS AND DISCUSSION Representative elution volumes for a number of resin and fatty acid methyl esters are shown in Table I. The interstitial volume of the column is 60 ml, the elution volume obtained for polyisobutylene (M.W. 12,000). The elution order for the saturated fatty acid esters agrees with that reported by both Cazes and Gaskill ( 4 ) and Chang ( 5 ) for the free acids. However, we have found a difference in the elution order of unsaturated fatty acid esters. We have also observed a partial fractionation among the resin acid esters. The table shows that fatty acid esters are eluted before the resin acid esters. On examination of molecular models of the various fatty and resin acids, it can be seen that the sphere generated by the gyration of the resin acid methyl esters and methyl nonanoate (9:0), as an extended chain, have a diameter of approximately 14A. That the elution volumes for the methyl tetrahydroabietates and the methyl nonanoate are quite different would appear t o indicate a significantly greater solvation of the primary carboxy ester of the fatty acid, and consequently a larger effective molecular size. Solvation differences, however, would not appear to be the only factor involved. The extended chain length for methyl linoleate (18 :29~12cis@) is approximately equivalent t o that for methyl stearate (18 :0) less one -CHz-group; methyl linolenate (18 :39, 1 2 , l5CfS ,Cis ,Cis) t o 18 :O less 2+ -CH2groups; whereas methyl oleate (18:19@) is only slightly smaller than 18:O. Either interaction of the unsaturation with the Styragel and/or folding of these molecules could explain the larger elution volumes than were expected on the basis of molecular size. Although there is some correlation between the dimensions of the models for the unsaturated resin acid esters and the elution order, the exceptions indicate that a small amount of interaction occurs between the unsaturation of the resin acid esters and the Styragel. The two methyl tetrahydroabietates, 8p,9a, 13a-H and 8a,9a,13a-H (6) eluted in the expected order, that is, the latter having a smaller molecular volume eluted later. The effectiveness of the separation of the resin acid esters from fatty acid esters depends both on the nature of the constituents present and on the sample size. The method described here was developed for use with extractives of pine and other conifers, and for this purpose quantitatively separates fatty from resin acid esters. The fatty acids of pine (6) J. W. Huffman, T. Kamiya, L. H. Wright, J. J. Schmid, and W. Herz, J . Org. Chem., 31, 4128 (1966).
range from 14:O through 26:O with only a trace of 12:O; the principal acids being oleic, various octadecadienoic isomers, and possibly some octadecatrienoic acids. All other reported minor components would be expected to elute before the 14: 0 and 18: 3 esters. Certain of the tetrahydro resin acid esters, particularly the 8a,9a,l3a-H tetrahydroabietate, would be expected to have the lowest elution volume of the resin acid esters. For IO-mg mixtures of resin and fatty acid methyl esters, complete resolution of 14: 0 and 18: 3 from the tetrahydroabietate was achieved. When a 125-mg sample was used, only a trace (less than 0.5%) of overlap was detected by GLC. The error caused by this overlap is significantly less than the experimental error in the gas chromatographic analysis ( I ) . This Styragel column can also be used for preparative separations; samples as large as 500 mg were separated with only a relatively small loss in resolution. This method is convenient and simple. The use of anhydrous ethyl ether as the eluting solvent avoids the problem of purification, such as with tetrahydrofuran. In addition, the ethyl ether has a lower viscosity and there is a greater differential between refractive indices for the ethyl ether (nD*O 1.3526) and the fatty and resin acid esters (nozo1.43-1.47 and 1 S 3 , respectively) than with the tetrahydrofuran ( n D Z 1 1,4076). However, the column must be cooled to prevent the disruptive effect of solvent vaporization. The differential refractometer is used to monitor the eluate and select the point at which to divide the eluate into fatty acid ester and resin acid ester fractions. It seems possible that a colored substance could also be used to define the point of division. I n this regard, we have investigated a number of colored substances, and preliminary work indicates that P-carotene elutes with the resin acid esters and can serve as a marker. It should be possible to quantitatively effect the separation of these ester fractions using the first trace of eluted @-carotene (less than 1 mg to be used, the amount depending upon the specific experimental conditions) as the dividing point. Although low flow rates are preferable, flow rates up t o about 60 ml/hour appear t o be satisfactory. Compressed nitrogen cannot be used as the pressure source when a differential refractometer is the monitor, as the dissolved nitrogen forms bubbles after elution from the column, which disrupts the refractometer. Pine wood extractives consist of about equal amounts of resin and higher fatty acids. Other systems such as heartwood extractives and tall oil distillate fractions in which the ratios are quite different may require modification in the proVOL 40, NO. 7, JUNE 1968
* 1145
cedure. This might be accomplished by a rough separation on a large scale followed by refractionation of the overlap region. Although the effect of anhydrides and polymers was not investigated in our system, the latter is considered by Chang (5). The resin and fatty acids were fractionated as the methyl esters because the subsequent G L C analysis requires the ester derivative and complications caused by any dimerization are avoided. Some preliminary experiments, and the work of Chang, indicate that a n effective separation can be achieved using the free acids. Quantitative separation of the fatty acid methyl esters from the resin acid methyl esters will simplify the G L C analysis (1) and result in more accurate data for the individual components. Only one G L C column packing (DEGS) will now be necessary. Complications caused by the greater instability of some of the constituents, particularly methyl levopimarate in the SE-30 columns, would be avoided.
The partial GPC separation of fatty acid esters can, perhaps, be used to advantage as an enrichment technique in the determination of trace esters in natural oils, in conjunction with urea fractionation (7) and silver nitrate-complex chromatography.
RECEIVED for review February 12, 1968. Accepted March 19, 1968. The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. This work was supported in part by the Pulp Chemicals Association, New York, N.Y. Mention of trade or company names is for purposes of identification only and does not imply endorsement by the US.Department of Agriculture. (7) J. L. Iverson and P. G. Harrill, J . Assoc. Ofic.Anal. Chemists., 50, 1335 (1967).
Spectrophotometric Determination of Antimony in Arsenic T. L. C. de Souza and J. D. Kerbyson Noranda Research Centre, Pointe Claire, Quebec, Canada A SPECIFIC PROBLEM required the determination of antimony a t the 1 to 50 ppm level in pure arsenic and arsenic(II1) oxide. These levels were too low for the titrimetric (I, 2) and polarographic (3, 4 ) methods which have been used for antimony in arsenic, and although a number of colorimetric procedures have been developed for antimony, the published applications to materials high in arsenic are rare. Of the seven colorimetric reagents detailed by Sandell (9,malachite green has been used by Kristaleva (6) to determine 30 to 3,000 ppm of antimony in arsenic, but arsenic interfered a t lower antimony levels. Brilliant green has been compared favorably with rhodamine-B for determining antimony in geological materials (7), but neither reagent has been reported for higharsenic applications. The separation of antimony from interfering elements prior to its determination with rhodamine-B has been studied in some detail by solvent extraction methods (8, 9). Isolation of anitmony by evolution of stibine has also been investigated by several workers (5,8, IO). The reported results are, however, conflicting with respect to the recovery of antimony, as compared with the distillation method for separating the chlorides of arsenic, antimony, and tin (11). (1) “Standard Methods of Chemical Analysis,” 6th Ed., Vol. 1, N.
H. Furman, Ed., p 131, Van Nostrand, New York, 1962. (2) L. B. Dunlop and W. D. Shults, ANAL.CHEM.,34, 499 (1962). (3) I. M. Kolthoff and R. L. Probst, ibid., 21, 753 (1949). (4) G. P. Haight, Jr., ibid., 26, 593 (1954). (5) E. B. Sandell, “Colorimetric Determination of Traces of Metals,” 3rd ed., Chap. VI, Interscience Publishers, New York, 1959. (6) L. B. Kristaleva, Industrial Laboratory, 25, (ll), 1352 (1959). (7) R. E. Stanton, and A. J. McDonald, Analyst, 87, 299 (1962). (8) S. H. Webster, and L. T. Fairhall, J . Ind. Hyg. Toxicol., 27, 184 (1945). (9) C. E. White, and H. J. Rose, ANAL.CHEM.,25, 351 (1953). (10) J. Cholak, and D. M. Hubbard, J . Ind. Hyg. Toxicol., 28, 121 (1946). (11) Hillebrand and Lundell, “Applied Inorganic Analysis,” 2nd ed., 1962, John Wiley and Sons, p 70. 1146
ANALYTICAL CHEMISTRY
We have examined the rhodamine-B procedure of Webster and Fairhall (8) for its tolerance to arsenic, and two routes for the necessary preseparation of antimony from the bulk of arsenic, or vice versa. Ethyl acetate extraction of antimony hexachloride (9) proved unsatisfactory when applied to the separation of traces of antimony from high-arsenic solutions. A simple distillation procedure (11)was successful in removing a bulk of arsenic prior to the determination of 1 to 50 ppm of antimony in high-arsenic materials. EXPERIMENTAL
Apparatus. A compact all-glass apparatus was used for the distillation stage, consisting of a 250-ml flask, entry socket for a tap-funnel containing 100 ml of HCl, exit socket for the vapor connected t o a water-cooled condenser, and a thermometer to record the vapor temperature. A Beckman D U spectrophotometer with 5-cm cells was used for the absorption measurements in the 565 mp range. Reagents. 0.1N Ceric Sulfate. Dissolve 16.5 grams of the anhydrous salt in 500 ml of 2 N HzS04. 0.1 % Hydroxylamine Hydrochloride. Dissolve 0.2 gram of the solid in 200 ml of distilled water. 0.2% Rhodamine-B Solution. Dissolve 0.4 gram of the solid in 200 ml of distilled water. Standard Antimony Stock Solution (1,000 pg per ml). Dissolve 0.500 gram of antimony metal in 12 ml of aqua 1 part H N 0 3 ) , and boil down t o about regia (3 parts HC1 5 ml to remove free chlorine and “0,. Dilute to 500 ml with 4 N HCI. Standard Antimony Working Solution (5 pg per ml). From the above stock solution, prepare a 200-fold dilution in 4 N HC1 immediately before use. Procedure. Weigh 1.OOO gram of the - 100 mesh sample into a 250-1111 beaker. Carefully pour 12 ml of aqua regia down the side of the covered beaker. The initial reaction may be quite violent. Warm geqtly until the arsenic has dissolved completely, then evaporate to a low volume, about 2 t o 5 ml, to remove free chlorine and nitric acid. Dissolve the contents by warming with about 10 ml of HCl. Transfer t o the 250-ml distillation flask containing 0.5
+
