four of t h e six xylenols, only t h e 2,4a n d 2,5-xylenols remaining unresolved. T h e peak shapes for phenols are not completely symmetrical but are more nearly so t h a n the elution curves obtained using 2- to 5-pl. samples of phenols on many conventional packed columns. The RRV’s, relative to mcresol, were o-cresol, 0.698; p-cresol, 0.885; 2,6-xylenol, 0.483; 2,5-xylenolJ 0.930; 2,4-sylenol, 0.930; 2,3-xylenol, 1.23; 3,5-xylenolJ 1.51; and 3,4-xylenol, 1.64.
DISCUSSION
The use of a modifying solvent with Bentone-34 in a chromatographic column undoubtedly reduces the selectivity of the organo-clay to metalpara isomers but, a t the same time it improves the shape of the elution curves to the extent that the column performance is similar to that of a conventional gas liquid partition column, even when 2- to 5-p1. samples of aromatics are introduced. Thus the effective separating power of the Bentone is enhanced and the partial loss of selectivity is, to a large extent, outweighed. This is well illustrated in Figure 3 where, although the metalpara separation factor is seen to be very much less than that obtainable on an unmodified Bentone-34 column, the
separation of the Cs alkylbenzenes is complete. The change of retention volumes of aromatics relative to paraffins associated with change of operating temperature (Figure 1) and change of ratio of modifying solvent to Bentone-34 (Figure 2 ) , provides considerable scope for varying the experimental details to give the most satisfactory chromatograms for the analysis of a particular mixture. This “tailoring” of columns can be developed further by using different modifying solvents or even mixtures of modifying solvents. For example, Uentone34 modified with a mixture of polyethylene glycol and trinitrobenzoic acid will cause n-nonane to elute before toluene and still effect the separation of cumene and pseudo-cumene from the Cs alkylbenzenes and the partial separation of m- and p-xylenes. Such separations on conventional packed columns are unknoirn to the authors. The examples cited of the separation of substituted aromatics further illustrate the use of modified Bentone-34 columns although i t should be noted that with phenols some asymmetry occurs. However, it would seem that wherever there is the need to separate a rneta,’para pair of isomers this type of column should be considered.
ACKNOWLEDGMENT
The authors thank F. W. Berk and Co., Ltd., for supplying samples of Bentone-34 and similar organo-clay compounds, and also the Directors of The British Petroleum Co., Ltd., for permission to publish this paper. LITERATURE CITED
(1) Cowan, C. T., Hartwell, J. hf., Nature 190, 712 (1961). (2) Desty, D. H., Goldup, A., Swanton, W. T., Zbid., 183, 107 (1959). (3) Hughes, M. A., White, D., Roberts, A. L., Ibid., 184, 1796 (1959). (4) Mortimer, J. V., Gent, P. L., Zbid., 197, 789 (1963). (5) Mortimer, J. V., Gent, P. L., Gas
Chromatography Discussion Group, London, April 1963; Littlewood, A. B.,
J . Gas Chromatography 1, Xo. 5, 28 (1963). (6) Spencer, S. F., ANAL. CHEM.35, 592 (1963). (7) Van Rysselberge, J., Van Der Stricht, 11..Nature 193. 1282 (1962). (8) t a n Der Stricht, M., van Rysselberge, J., J . Gas Chromatography 1, S o . 8 , 29 (1963). (9) White, D., ,Vatitre 179, 1075 (1957). (10) White, D., Cowan, C. T., “Gas Chromatography 1958,” D. H. Desty, ed., p. 116, Biitterworths, London, 1938.
RECEIVEDfor review October 14, 1963. Accepted December 23, 1963.
Gas Liquid Chromatography of Retinol (Vitamin A) Derivatives PERCY E. DUNAGIN, Jr., and JAMES A. OLSON Department of Biochemistry, Universify of Florida College of Medicine, Gainesville, Fla.
b Anhydro retinol, methyl retinyl ether, retinal, and methyl retinoate are separated by gas liquid chromatography at 150” C. on conventional columns, packed with Gas Chrom P or glass beads coated with SE-30, whereas retinol and retinyl acetate are largely converted to anhydro retinol under the same conditions. After treatment of these columns with p-carotene, however, retinol and retinyl acetate can also be separated with little destruction. These compounds were collected after chromatography by adsorption on alumina or cotton and were identified by their ultraviolet spectra.
R
[the nomenclature adopted by the International Union of Pure and Applied Chemistry has been used throughout this paper ( I ) ] and its derivatives occur in very small quantities in many living organisms and are common constituents of many pharETINOL
756
ANALYTICAL CHEMISTRY
maceutical preparations. Standard methods for their determination involve the extraction of the vitamin with organic solvents and subsequent measurement by one of several colorimetric procedures (6). At present the determination of several of these retinol derivatives in a given solution necessitates their separation on alumina or some other adsorbent prior to quantitative analysis. The high resolution and sensitivity of gas chromatography might be applicable to the rapid separation and quantitation of these compounds, providing that their thermal destruction could be eliminated. Sinomiya et al. (6) have reported that retinol, retinyl acetate, and retinyl palmitate are dehydrated to form anhydro retinol under conventional conditions of gas chromatography. I n our study, the use of short retention times and special inhibitors has enabled us virtually to eliminate the dehydration of retinol and
retinyl acetate. I n addition, conditions are defined for the successful separation of anhydro retinol, methyl retinyl ether, retinal, and methyl retinoate by gas liquid chromatography, and the feasibility of using this method for analytical purposes has been explored. EXPERIMENTAL
All-trans-retinol, alltrans-retinyl acetate, all-trans-retinal, and all-trans retinoic acid were purchased from Distillation Products Inc., and were used directly for subsequent analyses and syntheses. Xnhydro retinol was prepared by the method of Shantz, Cawley, and Norris (8). h s a final step in purification, the neutralized extract of anhydro retinol was chromatographed on activated alumina and eluted with 0.5% acetone in hexane. The ultraviolet spectrum of the product (maxima are 346, 365, and 388 mh) agreed with that reported by Shantz, Cawley, and Norris. Reagents.
NHYDRO R E T I N O L M E T H Y L IRETINYL ETHER
M E T H Y L RETINOATE
+-
'L
T I M E I N IN.)
Figure 1 . Separation of mixture of retinc I derivatives on untreated Gas-Chrom P
Methyl retinyl ether was prepared b y the method of Hanae et al. ( 3 ) . The ether was readily sept rated from retinol b y chromatography on alumina, and i t was used without further purification. Methyl retinoate was prepared by treating retinoic acid with diazomethane in ether. The diazomethane was released from a n ethereal solution of Diazald (hldrich Chemical Co., Inc., Milwaukee, Wis.) b , j treatment with ethanolic K O H and was distilled into cold ether. The resulting methyl retinoate was crystallized twice from methanol:H20 (5: 1). I t s melting point and ultraviolet and infrared spectra agree with those reported in the literature ( 2 , 7 ) . Apparatus. A Perkin-Elmer Model 4000 A ultraviolet r x o r d i n g spectrophotometer a n d Zeiss Model PMQ I1 spectrophotometer were used for ultraviolet spectrosccpy, and a PerkinElmer Model 237 Infracord was used t o determine t h e infrared spectrum of methyl retinoate. All gas chromatography was performed on a Research Specialties blodel 600 series gas chromatograph (>quipped with a n argon detector, a capillary injection system, a Minneapolit-Honeywell 5-mv. recorder, and a column oven designed to contain vertically positioned U tubes. Chromatographic Conditions. Two types of columns were used: a 35-cm. glass column (4-min. i.d.) packed with silanized 60- i o 80-mesh GasChrom P coated with 1% SE-30 (the pretested packing was purchased from .lpplied Science Lab., Inc., State College, Pa.) and a 35-cm. glass column (4-mm. i d . ) packed with 80- to 120mesh glass beads (Aprlied Science Lab., Inc.) coated with O.lyo.SE-30. For both columns the operating temperatures were: column, l!jOo C.; vaporizer,
230" to 250' C.; detector, 165' C.; outlet, 170' C. Sample size was 1 or 5 pl. To chromatograph retinol or retinyl acetate with a minimum of dehydration, 50 to 300 pg. of @-carotene in ether or 25 to 30 pl. of saturated hydroquinone in ether were injected onto both columns. Subsequent conditioning of the column at elevated temperatures (column, 250' C.; vaporizer, 300' C.) for several hours prior to usage provided the best protection against dehydration. Both reagents worked well on the glass bead column, but only @-carotene worked on the Gas-Chrom P column. The protective effect of 300 p g . of @carotene diminished somewhat after 24 hours but was adequate for 3 days, whereas hydroquinone, which sublimed quickly a t 150' C., lost its activity within 2 to 3 hours. In the absence of protective agents, destruction of retinol and retinyl acetate occurred mainly on thc column, but to a lesser degree may have occurred in the vaporizer and in the metal tubing leading to the column. The protective effect of @-carotene toward retinol was approximately the same whether @carotene was added directly to the top of the column, thereby bypassing the vaporizer, or was injected via the vaporizer and inlet tubing. Moreover, retinol injected directly on the column via a direct column injection system was completely dehydrated unless @carotene had been previously added to the column. Collection of Chromatographed Compounds for Ultraviolet Spectral Analysis. Anhydro retinol, retinal, and methyl retinoate were collected a t the conventional outlet port from t h e detector in a glass collection tube whose distal end was plugged with a small wad of cotton or, alternatively, with a small mad of glass wool covered with a 4-mm. layer of &03. T h e compounds were adsorbed at the surface of either adsorbent, were eluted with 0.5 to 2.5 ml. of absolute ethanol into a graduated absorption cell, and were analyzed spectrophotometrically.
Table 1.
Retinol, methyl retinyl ether, and retinyl acetate, on the other hand, were collected immediately after leaving the column by replacing the stainless steel exit system (about 75 cm. including the detector) with an 8-cm. long vertically positioned Teflon tube (AWG No. 22), which extended 3 cm. outside the column oven. These compounds were adsorbed onto a small cotton pad placed a t the bottom of a 20-gauge hypodermic needle which was inverted over the Teflon exit tube. Samples were eluted with 0.1 to 0.5 ml. of absolute ethanol for ultraviolet analysis. Collection b y this procedure virtually eliminated dehydration, while collection with the former procedure resulted in 25 to 100% dehydration. RESULTS
Chromatography. Anhydro retinol, methyl retinyl ether, retinal, and methyl retinoate were chromatographed with little destruction on untreated Gas-Chrom P. Figure I shows the separation of a mixture containing approximately 1 pg. of each of these four compounds. Chromatography of the individual components revealed no hidden peaks. To prevent the dehydration of methyl retinyl ether, the column was necessarily aged overnight a t an elevated temperature before use. Table I lists the distinctive column conditions for the chromatography presented in this study, as well as the approximate temperature at which each compound started to decompose when chromatographed at the given retention time. These values were determined by separately chromatographing each compound a t 150°, 162O, 175', and 200' C. while maintaining the retention times approximately the same by decreasing the flow rate, by lengthening the column, or by both. An increase in peak asymmetry or the appearance of extraneous peaks was interpreted as decomposition. Since changing the
Experimental Conditions
Column inlet pressure, Column support lb. Gas-Chrom P 17 Gas-Chrom P 17
Compound Anhydro retinol Methyl retinyl ether Retinal Gas-Chrom P Methyl retinoate Gas-Chrom P Retinol Carotene-treated Gas Chrom P Retinyl acetate Carotene-treated Gas Chrom P Retinol Carotene-treated glass beads a Theoretical plates equal the square width a t half height.
Column effi-
Flow ciency, rate, Retention theom!./ time, reticsal min. min. platesa 150 3.8 34 150 5.8 92
Decomposition temp. >200°
162"
17
150
9.0
53
17 60
150 880
11.0 3.1
74 31
175' 200 150"
60
880
5.3
32
150"
50
500
4.4
'3
150°
of the quotient of retention time divided by peak
VOL. 36, NO. 4, APRIL 1964
757
successfully at a high flow rate and low retention time on lightly loaded glass beads treated with either p-carotene or hydroquinone. Retinyl acetate, whose retention time is much greater than that of retinol, was dehydrated under these conditions. With respect to efficiency this column was greatly inferior to the Gas-Chrom P column, and also required a higher inlet pressure for equivalent retention times and flow rates. Spectra. The ultraviolet spectrum of a n authentic sample of each of the six retinol derivatives was compared with the spectrum of the corresponding material collected from the gas chromatograph in the manner previously described. T h e spectra of each of t h e six derivatives indicated t h a t 8
Figure 2. Peak height as function of amount of injected retinol derivatives 6
column length or flow rate could affect the appearance of the chromatogram, these decomposition temperatures can be considered only approximations. Separation of these four compounds is sufficient for both qualitative and quantitative determinations. Although some deviation from linearity was found when either the peak height or the peak area was used as a measure of the amount injected, Figure 2 indicates that peak height provides a simple means of quantitating small amounts of these compounds. This deviation from linearity apparently was characteristic of the detector since the amount recovered from the output port appears to be linear with concentration (Figure 3). Retinol and retinyl acetate, when chromatographed under the above conditions, were completely converted to a compound which appeared in the anhydro retinol area. The identity of this peak as anhydro retinol was confirmed by the ultraviolet spectrum of the collected material, in agreement with Kinomiya's report (6). With aged p-carotene treated columns of Gas-Chrom 1' and a flow rate of 150 ml. of argon/minute, the retention times for retinol and retinyl acetate were 8.5 and 16 minutes, respectively. I n both cases the injected compound accounted for 50 to 70% of the total peak area. Two other peaks were evident, one of which was anhydro retinol. Increasing flow rate to 880 ml. per minute greatly diminished the decomposition of retinol and retinyl acetate (Figure 4), while maintaining a good separation between them. The extraneous peaks were reduced to 6% of the total area. Retinol was also chromatographed 758
ANALYTICAL CHEMISTRY
. 3 4 2 cb
p"
2
Refinal Injected ( p p l
Figure 3. Comparison of recorder response with the amount of retinal collected. One-MI. quantities of various retinal concentrations were injected, collected, eluted with ethanol, diluted to 0.5 ml., and measured spectrophotornetricaIly a t 3 80 mk.
-0-0-0-0-
Peak height Absorbance
very little destruction took place. Figure 5 shows the spectra of the most unstable compound, retinyl acetate. The contribution of anhydro retinol to the spectrum of the collected materia1 appears to result from conversion in the exit tubing and not during injection or separation on the column. When the entire exit tubing and detector were used during collection, the anhydro component virtually obscured the injected parent compound. Recoveries. Spectrophotometric comparison of the collected material with appropriate dilutions of authentic samples of corresponding compounds indicated that typical recoveries from the conventional outlet port were: anhydro retinol, 78%; retinal, 109%; and methyl retinoate, 94%. Recoveries
izL RETINOL
RETINYL ACETATE
i
TIME ( M I N I
Figure 4. Separation of retinol and retinyl acetate on @-carotene treated Gas-Chrom P
from the improvised collection tube were considerably lower : methyl retinyl ether, 59%; retinol and retinyl acetate, less than 25Yc,. The low recoveries for the latter two compounds were caused by high flows and injection pressures. Although the yield of most of these compounds was greater than 50%, recoveries were only approximate a t the high concentrations (about 5 mg. per ml.) used for spectral analysis. DISCUSSION
This study clearly shows the feasibility of chromatographing six retinol derivatives on diatomaceous earth supports. The high molecular weight and the thermal instability of these derivatives limit the efficiency of the chromatographic column. With the exception of methyl retinoate and anhydro retinol, chromatographed a t 200' C. with little destruction, these compounds must be chromatographed below 175' C. To maintain reasonable retention times and to keep the flow of argon low enough to ensure good separation, short columns, a very small amount of liquid phase, or both must be used. d short retention time reduces the efficiency of the column while a small amount of liquid phase reduces its capacity. Compounds which were destroyed a t temperatures above 150' C. are limited to separation on packed columns similar to those used in this study. However, capillary columns might separate these compounds with higher efficiency. R E T I N Y L ACETATE
IO-
3
08-
2t
06-
%
9
04-
0 2-
0 WAVELENGTH ( m p l
Figure 5. Ultraviolet spectra of retinyl acetate before (-) and after (. ..)chromatography
...
All retinol derivatives saturated at the terminal oxygen (methyl retinyl ether, retinol, retiqyl acetate, and retinyl palmitate) tended to form anhydro retinol upon exposure to hot columns and metal tubing. A free radical-induced dehydration of this group of compounds is favored over the well known acid catalyzed reaction on the following grounds: Injection of NH, or pyridine, which should neutralize acidic sites, did not decrease the dehydration. The rehtive rates of dehydration in acid msdia are retinol > methyl retinyl ether > retinyl acetate ( 3 ) . During gas chromatography, the relative rates are esswtially invertede g , retinyl acetate >: retinol > methyl retinyl ether. Hydroquinone, a free radical inhibitor, decreased dehydration in columns packed with glass beads. Further study of the mechanism by which p-carotene and hydroquinone
inhibit the dehydration reactions would be welcome and might lead t o the introduction of other buitable protective agents for retinol and other unstable compounds. The dehydration reaction has been used in liquid medium as a n assay procedure for free retinol (4). Similarly, the formation of anhydro retinol during gas chromatography could be used as an assay for retinol, its ethers, and its esters if the procedure were carried out under conditions conducive to dehydration-i.e., high column temperatures and long retention times. This assay might be particularly applicable to the natural esters of retinol, which cannot be chromatographed without destruction a t this time. LITERATURE CITED
(1) Commission on Somenclature of Rio-
logical Chemistry, International Union
of Pure and Applied Chemistry, J. Am. Chem. SOC.8 2 , 5575 (1960). (2) Farrar, K. R., Hamlet, J. C., Henbest, H. B., Jones, E. R. H., J . Chem. SOC. 1952, p. 2657. (3) Hanze, A. R., Conger, T. W., Wise, E. C., Weisblat, D. I.,J . Am. Chem. SOC.
70,1253 (1948). (4) Harashima, K., Okazaki, H., Aoki, H., J . Vitaminol. 7, 150 (1961),., (5) Moore, T., "Vitamin A, pp. 43-66, Elsevier, Amsterdam, 1957. (6) Ninomiya, T., Kidokoro, K., Horiguchi, M., Higosaki, N., Bitamin 27, 349 (1863'). \ - - - - , -
(7)Robeson, C. D., Cawley, J. D., Weisler, I,., Stern, M. H., Eddinger, C. C., Chechak, A. J., J . Am. Chem. SOC. 77, 4111 (1955). (8) Shantz, E. hf., Cawley, J. D., Norris, D. E., Tbid., 65, 901 (1843).
RECEIVEDfor review October 23, 1963. Accepted December 30, 1963. This investigation was supported by reeearch grant AM-1278 from the National Institute of Arthritis and Metabolic Iliseases, V.S.P.H.S., and by research grant G-16327 from the Nntional Science Foundation.
Use of a Mass Spectrometer as a Detector and Analyzer for Effluents Emerging from High Temperature Gas Liquid Chromatography Columns RAGNAR RYHAGE laboratory for Mass Specfrometry, Karolinska Instifufet, Stockholm, Sweden
b A modified Atlas CH4 mass spectrometer has been coupled to a gas liquid chromatograpliy (GLC) column. As the compounds (emerge from the column they are ionized in the ion source of the mass spectrometer and about 10% of the i,otaI ion current is used for continuous registration of the effluent. The temperature of the column can be regula+ed from 50" to 350" C. by using the temperature programmer. Two molecule separators are coupled iri series between the column and the gas inlet line of the mass spectrometer. With this technique the sample-to-helium ratio is increased at least 100 times. Less than 1 pg. of matericil introduced onto the column suffices for a good mass spectrum. The mass range m/e 12 to 500 can be scanned and recorded in 1 or 2 seconds. Examples are given of the sepai'ation and mass spectrometric identification of 27 components from 200 pg. of methylated fatty acids from butter fat and the separation of a mixiure of C19 to hydrocarbons.
0
A FEW papers have been published which describe compound instruments in which a mass spectrometer connected directly to a gas
NLY
chromatograph has been used for the continuous registration of components from separated organic compounds. The separation and identification of mixtures of organic compounds using 8, combination gas chromatograph and time-of-flight mass spectrometer of the Bendix type was performed for the first time a few years ago (5, 7). Details were given for acetone and some benzene derivatives and for some halogenated hydrocarbons. Another type of instrument, the analytical mass spectrometer Model 21-103 €3 (Consolidated Electrodynamics Corp.), has been used with a capillary column for the separation of the known hydrocarbons up to Cll (11). The cycloidal focusing mass spectrometer, C E C Model 21-620, has been used for the same purpose and a misture of 16 C S hydrocarbon isomers were identified from the mass spectra (4). For a similar application, a n Atlas CH4 mass spectrometer has been connected to a capillary column ( 3 ) . A thermionic ionization gauge in combination with a mass analyzer system has been used as a quantitative and qualitative (QQ) detector for a gas chromatograph (18. 1.9). This type of detector is very sensitive but not suitable for qualitative work with compounds having molecular weights higher
than 50, because of insufficient mass resolution. The above-mentioned compound instruments have usually been used for the analysis of low molecular weight compounds. I n studies of compounds with higher molecular weight, techniques whereby samples collected from a gas chromatograph could be subsequently analyzed in the mass spectrometer have h e m used for several years ( I S ) . Usually the samples are collected in a cold trap. This system is time consuming and suffers from many disadvantages particularly those which are associated with the collection, handling, and introduction of the sample into the mass spectrometer. I n an attempt to overcome these difficulties, work was started 4 years ago to connect a mass spectrometer with the output of a gas Chromatograph. T o make possible the analysis of most types of compounds t h a t can presently he separated by gas chromatography i t is necessary to have a high-mass instrument with a relatively high ion-current intensity-for H mass unit resolution a t m/e 600. An Atlas CH4 mass spectrometer equipped with a n electron multiplier and a magnet current regulator with the possibility of scanning the magnetic field to cover t h e masq range m / e 12 to 400 in 3 seconds VOL. 36, NO. 4, APRIL 1964
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