~~
Table 11.Relative Retention as a Function of Electrolyte Concentration (NaC1)a t l e c t r o l y t e concentmtion/solute
1.oo 1.oo 1- Pentene 1.oo 2.23 2.47 2.51 1-Hexene 6.22 5.97 1-Heptene 14.60 15.80 1-0ctene 11.70 16.40 16.90 2-Octene 12.80 1.oo 1.oo 1.oo Benzene 2.46 2.97 1.93 Toluene 5.51 7.36 Ethylbenzene 3.76 1.oo 1.oo 1.oo Carbon tetrachloride 3.88 2.81 4.84 Chloroform 3.84 2.24 5.74 Dichloromethane Temp: 12.5 "C. 39.52% loading. 38.8670 loading. 39.737~ loading.
partition into the aqueous electrolyte solutions. Here, the net retention volumes increase with salt addition for each member of the homologous series; however, solutes of higher carbon number are selectively retained to a greater degree. This trend is also exhibited for the same homologous series on Ca(N03)Z columns (6). Similar retention patterns are also displayed for alkylated aromatic solutes in Table 11. Such a trend seems to confirm the hypothesis that the introduction of a hydrophobic moiety into the solute will increase the retention of that solute relative to its parent structure. The retention trends for the chlorinated hydrocarbons as a function of electrolyte concentration are interesting. At 2.34m NaCl, as noted previously, the retention of these two solutes is approximately equal, while a t the highest electrolyte concentration, dichloromethane elutes before chloroform. This is a direct result of their relative adsorption and partition coefficients, the K A of chloroform being greater than that for dichloromethane over the entire range of NaCl concentrations examined while the reverse situation holds true with regard to the relative trends in KL. Earlier workers (7-1 I ) have also shown the advantages gained by the addition of electrolyte to the stationary
phase on the separation of solutes in GLC. Unfortunately, these studies have attributed the bulk of the retention behavior and, hence, the selectivity exhibited, to the bulk retention mechanism term, KLVLO. Where these workers have acknowledged the possibility of adsorption a t the gasliquid interface (8, l o ) ,their experiments do not permit the ascertainment of its importance to the retention mechanism. If the data presented in this study are typical, then we feel that some of the assumptions of Wasik and coworkers (10, 1 1 ) and Bighi et al. (8, 9) are unwarranted. CONCLUSIONS The stationary phase systems discussed above show high and variable selectivity characteristics for different types of solutes. Whereas the thermal stability of the aqueous electrolyte stationary phases is limited, the principles illustrated in this study would not preclude the use of less volatile solvents ( 7 ) . Further, the increase in V N O of a solute with electrolyte addition to the stationary phase shows that the increase in K A with additive concentration must be taken into account in studies of bulk distribution processes via chromatographic methods. ACKNOWLEDGMENT The author gratefully acknowledges helpful discussions with Barry L. Karger, Department of Chemistry, Northeastern University, Boston, MA 02115. LITERATURE C I T E D (1) J. W. King, Ph.D. Thesis, Northeastern University, Boston, MA, 1973. (2) C. Devillez, C. Eon, and G. Guiochon. J. Colloid Interface Sci., 49, 232 (1974). (3) B. L. Karger, P. A. Sewell, R. C. Castells, and A. Hartkopf, J. Colloid lnterface Sci., 35, 328 (1971). (4) B. L. Karger, R. C. Castells, P. A. Sewell, and A. Hartkopf, J. Pbys. Cbem., 75, 3870 (1971). (5) A. K. Chatterjee, J. W. King, and B. L. Karger, J. Colloid Interface Sci., 41, 71 (1972). (6) W. J. Andrade, Ph.D. Thesis, Northeastern University, Boston, MA, 1971. (7) C. Bighi, A. Betti, G. Saglietto, and F. Dondi, J. Cbromatogr., 34, 389 (1968). (8) C. Bighi. A. Betti, and F. Dondi, J. Cbromatogr., 39, 125 (1969). (9) A. Betti. F. Dondi. G. Lodi, and C. Bighi, J. Cbromatogr., 68, 59 (1972). (10) S. P. Wasik and W. Tsang. Anal. Cbem., 42, 1648 (1970). (11) S. P. Wasik and W. Tsang, J. Phys. Cbem., 74, 2970 (1970).
RECEIVEDfor review January 9, 1975. Accepted March 13, 1975.
High Performance Liquid Chromatography of Fatty Methyl Esters: Preparative Separations C. R. Scholfield Northern Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 6 1604
Various liquid column chromatographic methods have been used over the years for small preparative separations and analysis of fatty acid and fatty ester mixtures. In the past few years, new developments of commercial high-performance equipment using high pressure and small particle-size column packing with more rapid solvent flow are being applied to give greater speed for such separations. Although these developments have been described many
times and in many places, a 1971 book edited by Kirkland ( 1 ) contains the best references. Pei et al. ( 2 ) separated fatty methyl esters using a stationary phase of hydrocarbon bonded to a solid support and a liquid phase of aqueous methanol. We obtained similar separations on an analytical scale with aqueous acetonitrile in place of methanol ( 3 ) . We have since modified our analytical procedure to prepare up to ca. 200 mg samples of pure esters or fractions ANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1417
i ,
i
Methvl
I
Methyl
1
Methyl Palmitate
1
60
Time
300
Retention Volume ml
120 600
90
min
450
I 0 0
Time
min
20 IO0
10
50
30
40
50
150
200
250
Methyl c i s 9 a n d c i s 1 2
R e t e n t i o n V o l u m e ml
Figure I.Chromatogram of 95 pl linseed oil methyl esters; min. 100% acetonitrile
5.0ml/
- Refractive index; - - - ultraviolet. Peaks are identified by number of carbon atoms in acid and number of double bonds. Ultraviolet detector peaks are unidentified oxidized or polymerized material. Refractometer attenuation 32,UV nonlinear range
150
200 Retention Volume,
ml
250
t
i
\
Methyl c s I5 Octa
Figure 3. ( A ) Chromatogram of 95 pI methyl oleate, elaidate, palmitate, stearate mixture. Stearate was eluted at 1080 ml; 5.0 ml/min, 85 wt % acetonitrile. ( B ) Chromatogram of 95 pl methyl esters of a cis-9,12-, and 15-octadecenoate mixture: 5.0ml/min, 85 wt % acetonitrile. Refractometer attenuation 16. UV 16
300
Figure 2. Chromatogram of 100 pi equal parts methyl linolenate, methyl linoleate: 5.0 ml/min, 85 wt % acetonitrile. Refractometer attenuation 32. UV 32 200
f r o m h y d r o g e n a t e d f a t s for s u b s e q u e n t analyses b y inf r a r e d and ultraviolet m e a s u r e m e n t s and d e t e r m i n a t i o n of d o u b l e b o n d positions.
EXPERIMENTAL Apparatus. Chromatograms were run on a Waters Associates ALC-202 instrument with differential refractometer to detect the methyl esters. A 254-nm ultraviolet detector in tandem with the refractometer detected conjugated esters, including oxidized and polymerized material. Two 2-foot X ye-inch 0.d. (Yls-inch i.d.) stainless steel columns in series were packed with Bondapak C18/ Porasil. a commercial packing with CIShydrocarbon bonded to silica. Solvent was either acetonitrile or an acetonitrile-water mixture. The used solvent was recovered by distillation, and the acetonitrile-water azeotrope with 85 wt % acetonitrile ( 4 ) was a convenient mixture and one which gave a good separation. Water content was determined by comparing the difference in density between the solvent mixture and pure acetonitrile with a standard curve on which this difference was plotted vs. water percentage. This standard curve did not change over the normal range of room temperature. When necessary, fractions were identified by comparison with known esters on an 11%EGSS-X gas chromatographic column at 174 "C. Capillary gas chromatograms on some fractions were run a t 190 'C on a 150-foot X 0.01-inch i.d. polyphenyl ether column with Radium D argon detector, and the peaks were identified by comparison of equivalent chain lengths with previous values ( 5 , 6 ) . Samples. Methyl esters were prepared by transesterification of triglycerides with sodium methoxide catalyst. Methyl linolenate, linoleate, and oleate were isolated by counter double current distribution (7). Methyl elaidate was obtained by nitrous acid isomerization of oleate and purification on a silver resin column (8).The mixture of cis-g-, -12-, and -15-octadecenoates was prepared by hydrazine reduction of linolenic acid and countercurrent distribution (9). Conjugated esters were isolated from dehydrated ricinoleic acid by argentation countercurrent distribution ( I O ) and chromatography (11). Methyl lauroleate, myristoleate, palmitoleate, and eicosenoate were concentrates from chromatography of fish oil esters (12). Saturated esters were purchased from The Hormel Institute. General Procedure. Samples of 10 to 400 11 were introduced by syringe injection through a septum with solvent flow rates of 5.0 1418
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
150
200
250
300
200
250
300
Ratsntlon Volume m l
Figure 4. Chromatogram of 5 @I methyl linoleate and 2 pl conjugated methyl linoleate isomers: 5.0 ml/min, 85 wt % acetonitrile
- Refractive index; - - - ultraviolet Refractometer attenuation 8, UV 64
to 9.9 ml/min. Liquid pressure from the pump was approximately 330 psi and 500 psi a t these flow rates. Refractometer and ultraviolet absorption signals were recorded on a two-pen strip chart recorder a t 30 inchedhour. Fractions were collected in an automatic fraction collector and combined as indicated by the recorder chart t o give samples for gas chromatographic measurements of purity.
RESULTS AND DISCUSSION T h e u s e of acetonitrile as a solvent w a s suggested b y o u r previous experience with acetonitrile and h e x a n e as solv e n t s for c o u n t e r c u r r e n t d i s t r i b u t i o n of m e t h y l esters (9). However w h e n C18/Corasil was used in the analytical colu m n , because of the s m a l l a m o u n t of h y d r o c a r b o n o n the s u p p o r t , i t was necessary t o add w a t e r to t h e acetonitrile t o g e t useful s e p a r a t i o n s (3). When C18/Porasil is used f o r p r e p a r a t i v e work, capacity ratios a r e a p p r o x i m a t e l y 10 t i m e s g r e a t e r because t h e r e is a greater a m o u n t of h y d r o c a r b o n o n the s u p p o r t a n d so less w a t e r is necessary. Capacity ratios for m e t h y l linolenate a r e a p p r o x i m a t e l y 0.1 f o r C18/Corasil and 1.0 for C18/Porasil w i t h 100% acetonit r i l e and 0.3 f o r C18/Corasil and 3.3 for C18/Porasil with 85 wt % acetonitrile. As s h o w n i n F i g u r e 1, linseed m e t h y l est e r s s e p a r a t e well with 100% acetonitrile. W i t h a solvent flow of 5 ml/min, resolution between linolenate and linol e a t e is 1.35 and s t e a r a t e is e l u t e d i n 45 min. M a x i m u m s a m p l e size varies w i t h t h e n u m b e r of c o m p o n e n t s i n the m i x t u r e and the a m o u n t of t h e largest c o m p o n e n t . W i t h an e q u i m i x t u r e of linolenate a n d linoleate, s e p a r a t i o n s a r e good w i t h 100-pl samples. T h e r e is little loss i n resolution w h e n flow r a t e is increased t o 9.9 ml/min, the m a x i m u m f o r t h e apparatus.
-Refractive Index _ _ _ _ Ultraviolet Absorption
9 < 12< I,O"S
60
70
Minutes
w 50
60
Minutes
.h 60
55
65
M in u t e I
Figure 5. Chromatogram of 190 pI methyl esters from a commercial hydrogenated vegetable oil shortening; 5.0 ml/min, 85 wt % acetonitrile
- Refractive index: - - - ultraviolet. Refractometer attenuation 32, UV 32. Fractions are (1) octadecatrienoate.(2) octadecadienoate, (3) cis-xtadecenoate,
(4)
trans-octadecenoate with cis-15-octadecenoate, and ( 5 ) palmitate. Stearate off at 785 ml. Capillary gas chromatograms of fractions are shown above the respective liquid chromatogram peaks. Ultraviolet detector peaks are unidentified oxidized materials and conjugated triene and diene
Resolution is greater with 85 wt % acetonitrile (Figure 2) and this solvent is advantageous when isomers of the esters are present. However, peaks tend to be more skewed; retention times vary more with sample size and are less reproducible. These effects may be caused by column overloading with larger samples. Also longer retention times are associated with lower room temperatures. Resolution decreases more with greater flow rates and most work with 85 wt % acetonitrile was a t 5 ml/min. With the 100-p1 sample of methyl linoleate-linolenate equimixture in Figure 2 resolution is 1.6. Separation decreases to approximately base line as sample size is increased to 400 p1. Under these conditions, methyl stearate would require 3.5 hours for elution, but flow rate may be increased after other components are off. With 85% acetonitrile, partial separations of methyl oleate from palmitate, elaidate, and cis-15-octadecenoate achieved with the analytical column ( 3 ) improve. Figure 3 illustrates separation of oleate, elaidate, and palmitate and of oleate and cis- 15-octadecenoate. There is some fractionation of other cis positional isomers; however, a mixture of oleate and cis-12-octadecenoate recycled through the column five times still did not separate to produce two peaks. Samples containing methyl linolenate or linoleate stored some time in a freezer contain ultraviolet-absorbing materials that may be in too small amounts to give appreciable signals on the differential refractometer. Both esters show a large peak in the ultraviolet detector eluting soon after the position for an unretarded component. With methyl linolenate and 85% acetonitrile, the peak is resolved into three. Conjugated linoleate isomers are eluted later than the unconjugated cis-9,cis-12-linoleate, and the difference is greater with 85% than with 100% acetonitrile. As shown in Figure 4, the conjugated cis,trans isomer is only slightly separated from unconjugated linoleate; the conjugated trans,trans isomer is well separated and the conjugated cis,cis isomer lies between the other conjugated isomers. A mixture of the three conjugated isomers has been separated with 85% acetonitrile by recycling four times through the instrument to separate the trans,trans isomer, which was
Table I. Average Retention Volumes for Methyl Esters on a &Foot C18/Porasil Column lstcr
Urire tarded component. / o 20: 1 18:3 9 ~ 1 2 ~ 1 5 ~ 18:2 9c12c 18:2 9/12/ 18:2 9 ~ 1 1 / 18:2 9c11c 18:2 9/11/ 18:l 9c 1 8 : l 9/ 18:l 15c 18:O 16:l c 16:O 14:l c 14:O 12:l c 12:o
38.5 588 167 233 295 236 257 288 326 428 432 1046 211 525 137 267 94 132
38.5
75 92
125
253 166 112
82
then removed; recycling was continued four times more to separate the cis,cis and cis,trans isomers. Average retention volumes were calculated from a number of runs (Table I). In many solvent partition separations, the effect of adding a double bond to a fatty ester is approximately the same as that of removing two methylene groups. For example in countercurrent distribution between acetonitrile and hexane, methyl oleate and palmitate are found in the same band although palmitate moves slightly faster than oleate (9). In the chromatographic system discussed here, the effect of adding a double bond is generally greater than removing two methylene groups, especially where saturated esters are concerned, and the difference increases with chain length of the ester. Palmitate is separated from oleate; and stearate, from eicoseANALYTICALCHEMISTRY, VOL. 47, NO. 8, JULY 1975
1419
noate. Myristate is separated from palmitoleate, but linoleate appears in the same peak as palmitoleate and is concentrated in the slower part of the band. Laurate, myristoleate, and linolenate occur in the same band, but myristoleate is higher in the leading part of the band with little difference between laurate and linolenate. Methyl esters were separated from a commercial hydrogenated vegetable oil shortening (Figure 5). Six fractions were obtained: (1) octadecatrienoate, (2) octadecadienoate, (3) cis-octadecenoate, (4) trans-octadecenoate with cis15-0ctadecenoate, ( 5 ) palmitate, and (6) stearate. Capillary gas chromatograms indicate the following isomers in addition to the naturally occurring all-cis esters: Fraction 1, mono-trans-linolenate isomer; fraction 2, cis-9,trans- 12-, and trans-9,cis- 12-linoleate; fraction 3, cis-monoene with double bond near the 12 position; fraction 4, cis-monoene like fraction 3, trans-monoenes and cis- 15-monoene; fraction 5 , monoene and palmitate. With 100% acetonitrile, separations were less complete with trans- monoenes appearing as a shoulder on the monoene peak.
ACKNOWLEDGMENT J. M. Snyder prepared many methyl ester samples. Fish oil esters were provided by L. L. Wallen.
LiTERATURE CITED (1) J. J. Kirkland, "Modern Practice of Liquid Chromatography", John Wiley 8 Sons, New York, 1971. (2) P. T-S. Pei, R. S. Henly, and S. Ramachandran, Lipids, 10 152 (1975). (3) C. R. Scholfield. J. Am. Oil Chem. SOC.,52, 36 (1975). (4) L. H. Horsley, Anal. Chem., 19, 508 (1947). (5) C. R. Scholfield and H. J. Dutton, J. Am. Oil Chem. SOC.,47, 1 (1970). (6) C. R . Scholfield and H. J. Dutton, J. Am. Oil Chem. SOC., 48, 228 (1971). (7) C. R. Scholfield, R . 0. Butterfield, and H. J. Dutton, Lipids, 1, 163 (1966). (8) E. A. Emken. C. R. Scholfield. and H. J. Dutton, J. Am. OilChem. SOC., 41, 388 (1964). (9) C. R. Scholfield, J. Nowakowska, and H. J. Dutton, J. Am. Oil Chem. SOC.,37, 27 (1960). (10) C. R. Scholfield, E. P. Jones, R . 0. Butterfield, and H. J. Dutton. Anal. Chem., 35, 1588 (1963). (11) E. A. Emken, C. R. Scholfield. V. L. Davison, and E. N. Frankel, J. Am. Oil Chem. SOC.,44, 373 (1967). (12) J. Hirsch, J. Coloq. Intern. Centre Nat. Rech. Sci. (Paris), 99, 11 (1961).
RECEIVEDfor review January 3, 1975. Accepted March 17, 1975. Presented before the Division of Analytical Chemistry, American Chemical Society Meeting, Atlantic City, NJ, September 8-13, 1974. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U S . Department of Agriculture over other firms or similar products not mentioned.
Detection of Unstable Lactones by Means of Thin Layer Chromatography and the Ferric Hydroxamate Test Randi Kringstad Department of Pharmacognosy, Institute of Pharmacy, University of Oslo, P.O. Box 1068, Oslo 3,Norway
The ferric hydroxamate test (the hydroxamic acid reaction) has been used in connection with TLC and PC for the detection of lactones ( I , 2 ) , and for the detection of esters of carbohydrates (2) and choline ( 3 ) . The spray reagents used in the above papers, all consist of an alkaline solution of hydroxylamine and an acidic solution of ferric chloride. Goddu et al. ( 4 ) found that lactones and acid anhydrides form hydroxamic acids in neutral solutions, contrary to most esters. During an investigation on lactone forming acids in succulents where the ferric hydroxamate test described by Krogh was used in combination with TLC ( I ) , it was found that the different types of lactones showed a different behavior towards the test, and that the developing solvent had a marked influence on the results. The reproducibility of the test was also found unsatisfactory. The aim of the present investigation was t o work out a modification of the ferric hydroxamate test that could be used for detection of organic acids which are easily converted into lactones (unstable lactones).
EXPERIMENTAL Spraying Apparatus. A two-part sprayer for atomizing of the reagents by compressed nitrogen was used ( 5 ) . Substances Tested. Most of the substances listed and classified in Table I are commercially available and of analytical reagent grade. Some of the substances were previously prepared in this laboratory. Dilactophorbic acid and quinide were isolated from the latex of Euphorbia resinifera Berg (6, 7) and D-glucaric acid from calcium D-saccharate by means of Dowex 50 [H+].The methyl and 1420
ANALYTICAL CHEMISTRY, VOL. 47, NO. 8, JULY 1975
ethyl esters were prepared from the corresponding acids by means of diazomethane and diazoethane. Test Solutions. The unstable lactones (cp. Table I) and acid anhydrides were used in concentrations of 0.1M in water. Of the other substances, 1M solutions were prepared with water or ethanol as a solvent. Of the slightly soluble substances, saturated solutions were applied. For determination of the detection limits, 0.005M solutions were used. TLC-Plates. Only Polygram cel 300 NM (0.1 mm, 20 X 20 cm), pretreated with the solvent system, was used. Solvent Systems. I. Ethanol-chloroform-ammonia-water (55: 30:15:1.5 v/v). 11. Ammonia-propanol (30:70 viv). 111. Ammoniaethanol (6535 v/v). IV. Ethanol-chloroform-acetone-water (23: 37:37:3 v/v). V. Benzene-methanol (99:l v/v). VI. Pyridine-ethyl acetate-acetic acid-water (5:5:1:3 v/v). VII. n- Propanol-formic acid-water (8:l:l v/v). The Ferric Hydroxamate Test. For this test, spray reagents 1 and 2 are needed. Spray reagent 1 was prepared by mixing varying amounts of a 1.5M solution of hydroxylamine hydrochloride and a 1.5 M solution of KOH in methanol till pH values of 6.0, 7.0, 8.0, 9.0, 11.0, and 13.0 were obtained in the mixture. Spray reagent 2 consisted of 0.037M ferric chloride in 2.7M HC1 ( I ) . Procedure. Five and 10 p1 of the test solutions were applied on the plates; for the saturated solutions, correspondingly more. Following chromatography, the TLC-plates were heated for 1 hour a t 105 " C and then sprayed with 10 ml of spray reagent 1. After drying in air at room temperature for 10 min, the plates were sprayed with 3.5 ml of spray reagent 2. The test was regarded as positive when a purple-brown color appeared immediately after spraying. The minimum amount of the unstable lactones and of salicyl acetate giving a positive test, was determined after chromatography in solvent system VI. For lactones which after spraying gave more than one purple-brown spot, the major spot was used for determination of the detection limits. To maintain a steady
