sequence will have its greatest effect when a small percentage of these species fragments to give the C1-. Because the peaks due to CI- are approximately 150-200 times as intense as the grouping between 47 and 51, a small isotopic contribution from these species will produce a negligible addition to the C1- peaks. We must conclude, then, that the m/e 35 and 37 peaks used in the negative ion ratios are probably devoid of fragmentation isotope effects. Mass discrimination effects should be minimal because of the masses of the isotopes involved and the fact that a standard containing the two isotopes in approximately the same ratio as the sample is employed as the reference. In conclusion, we see several features of the negative ion ratios which recommend their use in future measurements. Background contributions to the measured peaks are virtually nonexistent and this may permit the use of less rigorously purified samples for the analysis. The second feature is that the measurement is being made directly on the two isotopes in question and this permits ease of comparison between samples of interest without extensive correction factors. Because the kinetic isotope effects reflect the change in isotopic content during the course of the reaction, the standard to which all reaction samples should be compared is the one representing complete reaction from the compound in question and not some arbitrary standard sample for which the ratios are known absolutely. In common with the positive ion ratios, the only sample
limitation appears to be the requirement that it can be converted into inorganic chloride for subsequent precipitation by silver nitrate. The lower sensitivity in the negative ion intensity does cause a loss in measurement precision, but this appears not to be a severe limitation for isotope effects over the range 1.000 to 1.010 where we have shown a standard deviation leading to an isotope effect uncertainty of 10.00022. The sample preparation time is not unduly long except for the heating period. Changing samples to the mass spectrometer involves nothing more than pumping out the previous sample and balancing the sample and reference currents. This can be done in 15 minutes or less. ACKNOWLEDGMENT The authors are indebted to Robert C. Williams for the computer programs used to perform the statistical analysis of the experimental data. RECEIVED for review November 1, 1968. Accepted March 11, 1969. This research was supported by the Wisconsin Alumni Research Foundation and the National Science Foundation through Grant GP-8369. Portions of this work were presented at the 155th Meeting of the American Chemical Society, San Francisco, April 1968. Use of the University of Wisconsin Computer Center was made possible through support of the Wisconsin Alumni Research Foundation (WARF) through the Wisconsin Research Committee.
Role of Induction Forces Interactions in Effecting Gas Chromatographic Separation of Cardiac Glycoside Trimethylsilyl Ethers W. E. Wilson’ and J. E. Ripley Biochemistry Section, Southern Research Support Center, Veteran’s Administration Hospital, Little Rock, Ark. 72206 Trimethylsilyl (TMSi) ether derivatives of digoxin, digitoxin, and gitoxin have been shown to be resolvable on a gas chromatographic column packing containing a phenyl-methyl siloxane polymeric liquid phase (OV-17). At 340 O C , separation of the glycoside derivatives was effected by permitting differing degrees of dipole-induced dipole interaction between solute and liquid phase. In the cases of digoxin TMSi ether and gitoxin TMSi ether, the sphere of influence of the dipole of the lactone ring in the aglycone was sterically hindered, resulting in varying effectiveness of induction of electron delocalization in the T electron system of the aromatic groups of the liquid phase.
THEAGLYCONES of cardiac glycosides of plant origin possess several common structural characteristics which are illustrated in Figure 1. Fusion between rings AB, BC, and C D is cis, trans, cis, respectively. A tertiary alcohol is substituted at carbon 14. The 3p alcohol is bonded to digitoxose in the glycosides used in this study. A five-membered lactone ring is substituted at the 17p position ( I ) . 1 To whom correspondence should be sent at the following address: Analytical and Synthetic Chemistry Section, National Institute of Environmental Health Sciences, National Institutes of Health, P. 0. Box 12233, Research Triangle Park, N. C. 27709.
810
ANALYTICAL CHEMISTRY
The lactone ring of the biologically important plant glycosides possesses a,p unsaturation which confers a rather extensive a-electron system to the ring. In this regard, the conjugated lactone of the plant aglycones has been shown to be capable of enolization to a substituted a-hydroxyfuran derivative (2). Consideration of geometrical configuration and of charge distribution reveals that crotonolactone differs significantly from y-butyrolactone. The respective dipole moments of crotonolactone and y-butyrolactone have been reported to be 4.62 debyes and 4.13 debyes (3). Trimethylsilyl (TMSi) ether derivatives of the cardiac glycosides were demonstrated to be eluted during gas-liquid chromatography (GLC) using a relatively nonpolar methyl siloxane polymeric liquid phase (4). That the TMSi ether derivatives were not separable under the reported conditions (1) Ch. Tamm, First International Pharmacological Meeting,
Vol. 3, “New Aspects of Cardiac Glycosides,” W. Wilbrandt, Ed., The Macmillan Company, New York (1963) pp. 11-26. (2) B. Maume, W. E. Wilson, and E. C. Horning, Anal. Letters, 1, 401 (1968). (3) K. Sukigara, Y.Hata, J. Kurta, and M. Kubo, Tetrahedron, 4, 337 (1958). (4) W. E. Wilson, S. A. Johnson, W. A. Perkins, and J. E. Ripley, ANAL.CHEM., 39,40(1967).
Table I. Thin-La yer Chromatographic Behavior of the Aglycones Thin-layer chromatographic solvent system Digitoxigenin Digoxigenin Gitoxigenin Rf‘s of sterols after chromatography Number of developments, height of development Cyclohexane, acetone, and acetic acid (65 :33 :2; v/v/v). Three developments, 15 cm high B. Ethyl acetate, methanol, and water (80:5:5; v/v/v). Two developments, 15 cm high C. Chloroform and pyridine (60: 10; v/v). One development, 15 cm high D. Ethyl acetate and chloroform (90: 10; v/v). One development, 15 cm high E. Methyl ethyl ketone, toluene, methanol, acetic acid, and water (80:6:4:2:6; v/v/v/v/v). One development, 15 cm high Type of light used to visualize spots White light A.
0.45
0.28
0.33
0.59
0.43
0.45
0.69
0.35
0.38
0.33
0.13
0.12
0.71 0.69 0.77 Color of spot after spraying the developed chromatograma Tan Blue-green Light green Light blue Brown Tan UV light, 32W-3800 A Colors of spots were determined after spraying the thin-layer plate with a modified Liebermann Burchard reagent: acetic anhydride, sulfuric acid, and absolute ethanol ( 5 : 5 :100; v/v/v). Colors appeared after heating for two minutes with a portable forced air heater.
was related to the requirements of column dimensions, operating temperatures, quantity of liquid phase, and type of liquid phase. Of the various cohesive forces which might permit separation of solutes during G L C (9,those which are generally important are dipole-dipole and dipole-induced dipole interaction forces. Dipole-dipole interactions may permit separations; however, the lack of stability of TMSi ethers in the presence of acids and alcohols at high temperatures, the extremely low vapor pressures of sterol glycoside TMSi ethers, the lack of an appropriate thermally stable liquid phase, and the temperature dependence of dipole-dipole interactions eliminated further consideration of this alternative. It was considered feasible to attempt separation of cardiac glycoside TMSi ethers as a result of varying degrees of induction of electron delocalization in an extensively T bonded liquid phase in response to the significant dipole moment of the aglycone lactone ring. Varying the degree of induced dipole would be effected by derivatizing an alcohol group in either the C or D ring to form a bulky TMSi ether. This glycoside TMSi ether in which the freedom of lactone ring movement is most hindered should demonstrate the least ability for dipole induction and, thus, the least affinity for the liquid phase. Demonstration of this point was permitted by allowing the chemical composition of the GLC liquid phase to be the only variable under conditions where vapor pressure differences among the TMSi ethers were immeasurable. The thermostable liquid phases selected were a methyl siloxane polymer (OV-1) and a phenyl-methyl siloxane polymer (OV-17). Although methyl siloxane polymers are more polar than squalane, according to the convention described by Rohrschneider (6), significant dipole-dipole interactions on corresponding GLC liquid phases should be observable only a t low temperatures. The phenylmethyl siloxane polymer would be able to participate in each of the various types of cohesive force interaction; however, the temperature-dependence of the dipole-dipole interactions should minimize ( 5 ) S.Dal Nogare and R. S.Juvet, “Gas-Liquid Chromatography,” Interscience Publ., New York, 1962, pp 104-131. (6) L. Rohrschneider, Z . Anal. Chem., 170, 256 (1959).
CH3
COMPOUND
SUBSTITUTION
H.
Digitoxigenin (0-2)
R1
G i t o x i g e n i n (G-3)
R, = H.
D i g o x i g e n i n (D-3)
R1 = OH.
R2
R1 = OH.
R2
D i h y d r o d i g o x t g e n i n (DHD-3)
3
R2 R2
=
OH. H.
=
H.
Lactone r i n g i s saturated.
Figure 1. Aglycones of cardiac glycosides from Digitalis lanata In corresponding trisaccharide glycosides, the 3P-OH group is substituted with 1-L 3 linked digitoxose
involvement of their importance at very high temperatures. The interaction forces were assumed to be directly reflected by retention times of solutes under investigation. EXPERIMENTAL
Thin-Layer Chromatography (TLC) of the Aglycones and Sterol Glycosides. Digitoxigenin (D-2), gitoxigenin (G-3), and digoxigenin (D-3) (obtained from the Boehringer Mannheim Company) were chromatographed in several different solvent systems prior to GLC. The Rfs shown in Table I were obtained using 20 X 20 cm, abrasive-resistant, TLC plates precoated with Silica Gel~254,from E. Merkog, Darmstadt, Germany (distributed by Brinkmann Instruments, Inc.). Dihydrodigoxigenin (DHD-3) was purified prior to gas chromatography by preparative TLC using 20 X 20 cm plates coated with a 250-p layer of Silica Gel G (Merck). After one development t o a height of 15 cm in solvent system A, the sterol had a n Rf of 0.25. DHD-3 was recovered from the silica gel by adding several milliliters of methanol followed by filtration of the suspension on a fine-porosity sintered glass funnel. VOL. 41, NO. 6,MAY 1969
811
Gitoxin and the mono-, di-, and tridigitoxoside derivatives of D-2 and of D-3 (obtained from Boehringer Mannheim Co.) were chromatographed using solvent system A in a previously reported protocol (4). Each glycoside and aglycone was chromatographically homogeneous prior to GLC. Preparation of Sterol Derivatives for Gas Chromatography, Although the cardiac glycoside aglycones may be chromatographed as the free sterols, as the sterol TMSi ethers, or as the sterol acetates, derivatization is preferred to permit lower solute vapor pressures and to minimize adsorption effects. Acetates were prepared by heating 1 mg of sterol and 0.5 ml of pyridine-acetic anhydride solution (10:3; v/v) in a 16/125mm culture tube lined in Teflon (Dupont) at 75 “C for 1.5 hours. Trimethylsilylation was accomplished by dissolving 1 to 6 mg of sterol in 1 ml of “silanizing mixture” which contained hexamethyldisilazane, pyridine, and trimethylchlorosilane in a volume ratio of 1O:lO:l. Reaction was allowed to occur at room temperature for at least 60 minutes. After evaporation of the “silanizing mixture” with a jet of dry nitrogen gas, the TMSi ether derivatives were dissolved in acetone. The resulting suspension was passed through a fine-porosity sintered glass funnel prior to chromatography. Acetone solutions of the sterol-TMSi ethers were quite stable when stored in plastic-stoppered vials for periods up to a month. The aglycones D-2, D-3, and G-3, the sterol acetates, and the sterol TMSi ethers were characterized by nuclear magnetic resonance spectrometry. Dimethylsulfoxide-d6 solutions of the sterols and deuterochloroform solutions of the acetates and TMSi ethers were analyzed using a Varian Model A 60 spectrometer (7). Integration of peak areas permitted the conclusions that only the secondary alcohol groups of the aglycones were derivatized and that derivatization was essentially complete. Recovery of the original sterols could be effected by adding 1 ml of water to an acetone solution of the TMSi ether derivatives, followed by heating on a steam bath until the water evaporated completely. The resulting residue was dissolved in acetone and was subjected to TLC in solvent systems A and B. The acetone solution was also subjected to gas chromatography, In each case, the parent sterol was recovered as the only hydrolysis product from the TMSi ethers. Descriptions of Gas Chromatographic Columns and Packings. All columns were U-shaped. Columns I, I-A, and I1 had internal diameters of 5 mm and lengths of 6 feet. Columns I11 and IV measured 4 mm in i.d. and 1 foot in length. All chromatographic U-tubes were treated with dimethyldichlorosilane and methanol prior to packing. Packing in columns I and 111 consisted of 2.5% OV-1 coated on Chromosorb W, HP, 80-100 mesh (lot no. 0585). Packing in columns I1 and IV consisted of 2.5% OV-17 on Chromosorb W, HP, 80-100 mesh (lot no. CA 0150). Packing in column I-A consisted of 1.0% OV-1 coated on Chromosorb W, HP, 80-100 mesh (lot no. CA 0149). Liquid phase loads on the column packings (which were obtained from Supelco, Inc., Bellefont, Pa.) were determined by exhaustive extraction with hot toluene in the case of OV-1 or chloroform in the case of OV-17. Prior to use, column packings were conditioned at their highest operation temperature for four hours with a nitrogen flow of 10 ml mind’. After it was conditioned at 290 “C, the load of liquid phase on packings in columns I and I1 was approximately 1.9%. When columns I11 and IV were conditioned at 350 “C, the liquid phase loads decreased to approximately 75% of the original loads; after 28 hours the liquid phase loads decreased to 67 % of the starting loads. (7) F. C. Chang, Univ. of Tenn. Med. Units, Memphis, Tenn.,
personal communication. 812
ANALYTICAL CHEMISTRY
Gas Chromatographic Analysis. Gas chromatography was performed on a Barber-Colman 5000 Series instrument equipped with hydrogen flame detectors. The flow rate for nitrogen carrier gas was maintained at 100 ml min-1 in all chromatographic operations. During isothermal operation, injection port temperature and column bath temperature were identical. In the cases of temperature programming, injection port temperature was identical to the lower (starting) temperature. Detector bath temperature was maintained at 340 “C. Sample injection involved administration of 1 p1 of solution directly on column. Chromatograms indicated the presence of a single major component for the parent alcohols as well as for the acetate or TMSi ether derivatives. Retention times were arbitrarily defined as the time interval occurring between the appearance of the leading edge of the solvent peak and the midpoint of the solute effluent peak in the chromatogram. This definition was permitted as acetone could not dissolve in the liquid phase under the operating conditions used in this study. The values for retention times represent values from at least three runs with maximum variations of 1.O %. Methylene unit values for the TMSi ether derivatives of the aglycones have previously been reported and provide a convenient reference concerning behavior on SE-30 and OV-17 relative to saturated hydrocarbons (2). These dimensions were not employed in the present study due to difficulties including the magnitude of the temperature interval employed in facilitating glycoside TMSi ether separations on the 1-ft columns. Mass Spectral Determinations. In order to determine the molecular weights and, thus, the extent of substitution of the sterol derivatives which were eluted from the chromatographic columns, mass spectra were obtained on a tandem gas chromatograph-mass spectrometer. The LKB Model 9000 mass spectrometer-gas chromatograph was fitted with a 9-foot by 6-mm glass column containing 1% SE-30 liquid phase coated on acid-washed, silanized Gas Chrom P (8). Operating parameters for the mass spectrometer included an accelerating voltage of 70 eV, a filament current of 60 pA, and an ion source temperature of 250 “C. The GLC column was temperature programmed from 230 “C at 1 “C min-l after injection of sample. RESULTS AND DISCUSSION
Mass Spectra of Aglycone Derivatives. Parent molecular ions of the aglycone TMSi ether derivatives eluted from the GLC column were: digitoxigenin mono-TMSi ether m/e = 446; digoxigenin di-TMSi ether m/e = 534; dihydrodigoxigenin di-TMSi ether m/e = 536; and gitoxigenin di-TMSi ether mje = 534. In no case was there an indication of derivatization of the tertiary alcohol or of the lactone ring. Prominent fragments occurred in the spectra of the aglycone TMSi ethers corresponding to the loss of methyl groups [(M-l5)?; (M-90-15)+; (M-90-90-15)+] and to the loss of trimethyl silanol groups [(M-90)+; (M-90-90)+]. Gas chromatography of G-3 acetate resulted in elution of a compound with a parent molecular ion corresponding to a m/e of 414. This result was indicative that the conditions used for GLC permitted elimination of a molecule of acetic acid from the D ring, resulting in the formation of the A15 and/or the A16 analog(s) of D-2 acetate. Gas Chromatography of the Aglycone Derivatives. Relative retention orders on columns I and I-A were essentially identical (Table 11) with the exception of the result with DHD-3(8) E. C. Homing, W. J. A. Vanden Heuval, and B. G. Creech, in “Methods of Biochemical Analysis,” Vol. 11, D. Glick, Ed., Interscience Publ., New York, 1963.
Table 11. Gas Chromatographic Retention Time Relationships of Aglycones and Their Various Derivatives 1.OX OV-1 (Column I-A)
Aglycones and derivatives Digitoxigenin (D-2) Gitoxigenin (G-3) Digoxigenin (D-3) Dihydrodigoxigenin (DHD-3) D-2-Ac G-3-Ac D-3-Ac DHD-3-Ac D-2-TMSi ether G-3-TMSi ether D-3-TMSi ether DHD-3-TMSi ether
Isothermal: 260 'C ' Retention relative to Retention derivatives time, of D-3, minutes 7.6 7.0 13.1
z
11.5 9.4 8.6 12.7 10.4 7.5 8.5 8.5 7.5
74 68 100 82 88 100 100 88
TMSi ether. These results were also quite similar to those previously reported using a 1 SE-30 liquid phase in a longer column (2). Relative retention orders of the sterol acetates were similar to those of the free sterols on both OV-1 and OV-17 (Table 11). The elimination product from G-3-acetate had a shorter retention time than that of D-2-acetate on either type of liquid phase. It appeared that elimination of a molecule of water may also have occurred upon G L C of the free sterol, as G-3 has a shorter retention time than D-2 in these systems. The observation that retention times of the sterol acetate were 5.5 times greater on Column I1 (OV-17) than on Column I (OV-I) was of interest because of the exception in the case of DHD-3-acetate where there was only a 5.0 fold greater affinity for OV-17. Relative affinities for OV-17, as compared t o OV-I, are consistent with the postulate that the more extensive T electron system of those aglycones possessing conjugated lactone rings permits a stronger interaction with the aromatic groups of the liquid phase than one could predict from consideration of group dipole moments alone. Relative retention times of the aglycones and the acetates on Columns I and I1 were correlated with: (a) assumed molecular weight differences, (b) assumed vapor pressure differences, and (c) the frequency of occurrence of various groups with an appreciable dipole moment. Despite the inability to discriminate among the varying contributions of molecular properties of the TMSi ethers in determining affinities on OV-1, one may exclude the possibility of a contribution of dipole induction in the liquid phase by the lactone ring of the aglycone derivatives to affinities in this system. On the 6-ft columns containing OV-1 liquid phase, the sterol TMSi ethers had retention orders that were predictable to a large extent from consideration of the behavior of corresponding acetates on OV-1. The relative retention of G-3-TMSi ether on OV-1 was anticipated in that its vapor pressure should more closely approximate that of D-3-TMSi ether than of D-2-TMSi ether. The retention order of the aglycone-TMSi ethers on OV-17 was obviously related to molecular characteristics other than vapor pressure differences. D-3-TMSi ether had a shorter retention time than D-2-TMSi ether or G-3-TMSi ether, and was very well resolved from the former compound. While the retention time of D-2-TMSi ether was 3.6 times
2.5X OV-1 (Column 1) Isothermal.: 290 "C ' Retention relative to Retention derivatives time, of D-3, minutes 6.0 5.6 9.8
z
8.6 7.1 6.5 9.1 7.9 5.7
6.3 6.3 5.2
78 71 100 87 90 100 100 82
2.5X OV-17 (Column III Isothermal; 290 "C ' RetenTg relative to Retention derivatives time, of D-3, minutes 18.5 16.9 39.8
z
24.0 38.4 35.1 50.4 39.3 20.9 17.7 17.1 14.0
76 70 100 78 121 103 100
82
Figure 2. A CPK model demonstrating the van der Waals radii of the 17a-epimer of diginatigenin, 38, 128, 14, 16p tetrahydroxy-50, -card-20 (22)-enolide, in which the 3p hydroxyl group is hidden greater on Column I1 (OV-17) than on Column I (OV-1), retention times of the triol TMSi ether were only about 2.7 times greater on OV-17. Relative affinities on OV-I7 are generally consistent with the hypothesis that steric hindrance of the lactone greatly influences the degree of separation of the aglycone TMSi ethers. The long range shielding effects of trimethylsilylsubstitution are obviously related to the van der Waals radii for carbon and silicon, which are 1.7 A and 2.0 A, respectively. D-2-TMSi ether, in which the lactone ring has complete freedom of rotation, had the expected highest affinity for OV-17. The observation that G-3-TMSi ether had a greater affinity VOL. 41, NO. 6, MAY 1969
813
z4 D-2-TMS; efher D-3-TMSi ether
*
t
. . . s . . . * io'. . * t
is'
' '
'id
i
. , . .5
,
...
. .+.r.5.. .
'
io'
MINUTES
MINUTES
Figure 3. Chromatograms of the D-2- and D-3-glycoside TMSi ether mixtures The left-hand arrow indicates the time of sample injection and of initiation of temperature programming at 240 "C. The right-hand arrow indicates the end of temperature programming at 340 "C. ( A ) D-2-TMSi ether and D-3-TMSi ether are resolved on Column 111, whereas the mono-, di-, and tridigitoxoside TMSi ethers overlap to form single peaks. ( B )D-3 and the corresponding mono-, di-, and tridigitoxoside TMSi ethers precede D-2 and corresponding D-2-glycoside TMSi ether derivatives on Column IV than D-3-TMSi ether for OV-17 was difficult to reconcile with presently accepted assignments of substitution of the aglycone D-ring (9). The TMSi ether group at the 16p position would not only shield the 14p-hydroxy1, but would be expected to more effectively hinder rotation of and/or shield the 17P-lactone ring than would a TMSi ether group in the 12p position. (9) L. F. Fieser and M. Fieser, Steroids, Reinhold, New York, (1959), pp 756759.
Table 111. Gas Chromatographic Retention Time Relationships of Glycoside TMSi Ether Derivatives 2 . 5 z OV-1 2.5 OV-17 (Column 111) (Column IV) Temp Temp Programmed from Programmed from 240 "C to 340 "C 240 "C to 340 "C at 7.5 "C min-1 at 7.5 "C min-1 Glycoside trimethylsilyl ether Retention time, Retention time, derivatives min min Gitoxigenin 2.2 3.6 Gitoxin 15.8 20.7 Dih ydrodigoxigenin 2.0 3.0 Digitoxigenin (D-2) 2.0 4.0 Digitoxigenin mono digitoxoside (D-2-MD) 7.0 10.0 Digitoxigenin bis digitoxoside (D-2-BD) 11 .7 14.5 Digitoxin 15.8 21.6 Digoxigenin (D-3) 2.2 3.6 Digoxigenin mono digitoxoside (D-3-MD) 7.1 9.3 Digoxigenin bis digitoxoside (D-3-BD) 11.7 13.7 Digoxin 15.8 19.5 TMSi Ether Mixtures D-2 and D-3 D-2-MD and D-3-MD D-2-BD and D-3-BD Digitoxin and digoxin
814
2.0; 7.0; 11.8; 15.9;
2.2 7.0 11.8 15.9
ANALYTICAL CHEMISTRY
4.0; 10.0; 14.5; 21.6;
Should the absolute configuration about carbon-14, -16, or -17 be a rather than p, the experimentally obtained relative retention relationships would correspond exactly with predicted results of steric hindrance of inductive interactions with the aromatic liquid phase. A CPK model of the 17aepimer of the 3 4 12p, 14, 16p aglycone tetraol (Figure 2) indicates that the TMSi ether of the 12p-01 would provide more pronounced shielding of the lactone ring than would the same derivative of the 16/3-01. Gas Chromatography of the Cardiac Glycoside TMSi Ethers. Relative retention times of the aglycone TMSi ethers on a 1-ft column, packed with OV-1, reflected almost exactly the results obtained on much longer columns (Table 111). However, G L C of the mono-, di-, and trisaccharide glycosides resulted in loss of separation ability on this column. The progressive nature of the loss in separation ability was followed by determining the number of plates resulting from chromatography of the mixture as compared with those resulting from G L C of the individual components (10). The results with glycoside TMSi ether mixtures on Column I11 indicated that, at temperatures in excess of 300 OC, neither small differences in vapor pressures or in group dipoles could be reflected by the relative retention relationship measurements. From the chromatograms in Figure 3, resolution effected by OV-17 (Column IV) was found to be reasonably satisfactory for the mixture of derivatives of D-2 and D-3; and digoxin TMSi ether was completely resolved from digitoxin TMSi ether. In this regard it is of interest to note that chromatograms of the individual glycoside TMSi ethers appeared to result in no measurable decomposition, even in the cases of the trisaccharide glycoside derivatives. Due to technical difficulties, mass spectral information was not obtained for glycoside TMSi ethers which were eluted from the gas chromatographic columns. The possibility that a rearrangement reaction occurred in the D-rings could
3.6 9.3 13.7 19.5
(10) Vapor Phase Chromatography, D. H. Desty, Ed., Academic Press, New York, 1957, p xiii.
neither be verified nor contradicted. However, the configuration about the D-ring of gitoxigenin derivatives eluted from a GLC column is apparently different from presently accepted assignments. The separations achieved for trisaccharide glycoside TMSi ethers demonstrated clearly that the affinity of gitoxin TMSi ether for OV-17 is significantly greater than that of digoxin TMSi ether and is less than that of digitoxin TMSi ether. If we assume (a) that dipole-dipole interactions between the aromatic liquid phase and the 14P-OH (as well as the lactone ring) were significant during gas chromatography of the aglycone TMSi ethers and that they became much less significant during GLC of the trisaccharide TMSi ethers and (b) that vapor pressure differences provide an immeasurably small contribution to retention time differences in the cases of the trisaccharide TMSi ethers, the relative retention time relationships observed for the trisaccharide TMSi ethers correlate with results expected from gas chromatography of the aglycone
TMSi ethers. The importance of dipole-induced dipole interactions in determining relative affinity of the various glycoside TMSi ether derivatives for OV-17 is more clearly demonstrated than was the case for the aglycone TMSi ethers at the lower temperatures. ACKNOWLEDGMENT
The authors acknowledge the generous assistance of E. C. Horning in providing accessibility to the tandem gas chromatography-mass spectrometer facilities at the Institute for Lipid Research, Baylor University School of Medicine, Houston, Texas. The authors are indebted to J. H. Doherty for a sample of dihydrodigoxigenin. The authors acknowledge the generosity of F. C. Chang in permitting utilization of the nuclear magnetic resonance spectrometer. RECEIVED for review November 15,1968. Accepted February 6, 1969.
Continual Analysis of Gas Chromatographic Effluents by Rapid Repetitive Infrared Scanning Burton Krakow The Warner & Swasey Company, Control Instrument Dioision, 32-16 Downing St., Flushing, N . Y . 11354 Rapid, on-the-fly, infrared analysis of gas chromatographic effluents was investigated using a grating spectrometer that continually produced infrared spectra from 2.7 to 9 at the rate of 1.6 spectra per second. The gas eluting from the gas chromatograph was passed continuously through a flow-through infrared cell where its spectrum was scanned repetitively by the spectrometer. The quality of the spectra obtained was adequate for functional group analyses of a number of organic compounds studied. Potential ability to detect unresolved GC peaks appeared to be a particularly valuable feature of this analytical method.
,.I
Two
TECHNIQUES have been used for obtaining infrared spectra of gas chromatograph effluents: (1) trapping isolated fractions of the effluent containing G C peaks (hopefully single pure peaks) and study of each fraction at leisure with an ordinary infrared spectrometer, (2) on-the-fly rapid-scanning of the infrared spectrum of each fraction as it elutes from the G C column. Trapping is tedious and time consuming. Moreover, ordinary spectrometers take several minutes to scan, while the gas chromatograph elutes peaks at intervals of a few seconds. Backlogs of trapped samples can develop very quickly this way. Rapid, on-the-fly scanning would be desirable. A number of attempts have been made to meet this need by accelerating the scanning speed of standard types of infrared spectrometers ( I , 2, 3). A filter wheel spectrometer ( 4 ) and an interferometer ( 5 ) have been applied to the problem. These instruments took from one second (5) to 45 seconds ( 2 ) to scan a spectrum. Up to 800 scans per second, with good spectral quality, are produced by the Warner & Swasey Model 501 rapid scanning spectrometer (6). A modification, Model 503, which has been made for analysis of gas chromatograph effluents, produces 1.6 scans per second. Such a rate represents sufficient speed to follow
variations in column effluents without producing excess spectra that would make the job of data processing unnecessarily gross. This paper will describe experiments to evaluate the performance of the modified spectrometer and the utility of on-the-fly rapid scanning. EXPERIMENTAL
A schematic diagram of the apparatus used for studying infrared spectra of gas chromatograph effluents is shown in Figure 1. As gas elutes from the gas chromatograph, it is passed continuously through a light pipe that serves as an infrared absorption cell. Radiation from the infrared source passes through the light pipe and is focused on the entrance slit of a rapid-scanning grating spectrometer. The rapid-scanning spectrometer uses a unique method of scanning with corner mirrors through the intermediate focal plane of a double-pass monochromator, keeping the grating and all other components of the dispersing train immobile (6). The spectrometer repetitively scans the infrared absorption spectrum, from 2.7 to 9 p, of the sample in the light pipe. One complete spectral scan takes '/s second, there is a pause of second during which time no spectra are observed, then the scan is repeated, and the sequence is continued indefinitely. A single case, which can be flushed with dry nitrogen, contains the entire optical system, including the source, (1) A. M. Bartz and H. D. Ruhl, ANAL.CHEM., 36,1892 (1964). (2) P. A. Wilks, Jr. and R.A. Brown, ibid., 36,1896 (1964). (3) C. W. Warren, J. J. Heigl, R. A. Brown, and J. M. Kelliher, Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 8, 1968, Paper 217. (4) G. T. Keahl, Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., February 25, 1966, Paper 209. ( 5 ) M. J. D. Low and S . K. Freeman, ANAL.CHEM., 39, 194 (1967). (6) S. A. D o h , H. A. Kruegle, and G. J. Penzias, Appl. Opt., 6, 267 (1967). VOL. 41, NO. 6, MAY 1969
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