have been reached. Gehrke ( 5 ) has chosen 150 “C for 2.5 hr as adequate to reach equilibrium between the two TMSi glycine derivatives and we have found that at least that much time is required for equilibration of the butyl-Gly derivatives. Because of interference with the determination of Ileu by 0-butyl Gly, and since the 0-butyl derivative of Arg is chromatographically stable, the use of methylene chloride as silylating solvent is recommended. Close examination of the chromatograms shows numerous extraneous peaks along the base line. The majority of these minor peaks correspond in retention time to the fully trimethylsilylated amino acid derivatives and imply that esterification of the amino acids is incomplete. This observation and conclusion led us to examine the esterification reaction in considerable detail to determine the factors which influence ester formation. These results are to be published shortly, but our rather startling major finding is that in less than 15
min, n-BuOH/2.7M HC1 will produce more than 2M water at 150 OC and more than 0.2M water at 100 “C by the following reactions : BuOH
+ HC1+
2 BuOH
+
BuCl
Bun0
+ HzO
(1)
+ HzO
(2)
Obviously the production of such large amounts of water will have significant influence on the equilibrium position of the esterification reactions.
RECEIVED for review January 6, 1972. Accepted March 17, 1972. This paper presents the results of one phase of research carried out at the Jet Propulsion Laboratory, California Institute of Technology, under Contract No. NSA 7-100, sponsored by the National Aeronautics and Space Administration.
Analysis of Purine and Strychnos Alkaloids by High-speed Liquid Chromatography Cheng-Yi Wu and Sidney Siggia Department of Chemistry, University of Massachusetts, Amherst, Mass. 01002
PURINEAND STRYCHNOS ALKALOIDS are of pharmacological importance. Purine alkaloids are also important in the food industry. Therefore, an improved method of analyzing these compounds is of value. Some gas chromatographic analyses of these compounds have been reported (1-8), but their application has been limited by the lack of volatility and thermal instability (9). An excellent review on the analysis of these compounds by classical methods and conventional chromatographic techniques has been edited by Higuchi ( I O ) . The determination of caffeine in beverages and pharmaceuticals by anion-exchange and ligand-exchange chromatography has also been reported (11, 12). However, no study has been done using high-speed partition liquid chromatography for the separation of three major purine alkaloids which commonly occur together. Two major Strychnos alkaloids which commonly (1) H. A. Lloyd, H. M. Fales, P. H. Highet, W. J. A. VandenHeuvel, and W. C. Wildman, J. Amer. Chem. Soc.. 82. 3791 (1960). (2) K. D. Parker, C. R. Fontan, and P. L. Kirk, ANAL.CHEM:, 34,757 (1962). (3) S. L. Massingill, Jr., and S . E. Hodgkins, ibid., 37, 952 (1965). (4) T. Hattori, S . Kawai, and M. Nishiumi, Burisrki Kagaku, 14, 586(1965). (5) H,Kolb and P. W. Patt, Arzneim.-Forsch., 15,924 (1965). ( 6 ) J. Reisch and H . Walker, Pharmazie, 21,467 (1966). (7) E. Brockmann-Hanssen and A. B. Svendsen, J . Pharm. Sei., 52, 1134 (1963). (8) H. Kern, P. Schilling, and S. H. Muller, “Gas Chromatographic Analysis of Pharmaceuticals and Drugs,” Varian Aerograph, Walnut Creek, Calif., 1968. (9) P. L. Kirk, in “Advances in Chromatography,” J. C. Giddings and R . A. Keller, Ed., Vol 5, 1968, pp 79-125. (IO) “Pharmaceutical Analysis,” T. Higuchi and E. BrockmannHanssen, Ed., Interscience, New York, N. Y . , 1961. (11) R. A. Henry and J. A. Schmidt, C‘hromatographia, 3, 116 (1970). (12) J. C. Wolford, J. A. Dean, and G. Goldstein, J. Chromatogr., 62,148 (1971).
Injection Tee
-
Tn
Waste
L v a r iai
Figure 1. Schematic of liquid chromatograph occur together were also successfully separated. This approach, in addition to overcoming the limitation of gas chromatography, also offers distinct selectivity. Because all the experimental work is performed at ambient temperature, the problem of thermal instability is eliminated. Selectivity is achieved by using an appropriate column and a detector which responds only to the ultraviolet-absorbing components. EXPERIMENTAL
Apparatus. A schematic diagram of the liquid chromatograph used is shown in Figure 1. All parts of the chromatographic system other than those detailed below have been described in a previous report by Siggia and Dishman (13). The degassing apparatus, which is new, consists of a heating tape, a 1-meter column of 1.5-cm outer diameter with a cold (13) S . Siggia and R. A. Dishman, ANAL.CHEM.,42, 1223 (1970). ANALYTICAL CHEMISTRY, VOL. 44, NO. 8, JULY 1972
1499
2
x 0
u
v)
0
f a m AU
K
I
8
J;
$ U
0.008 AU
Time (minutes)
Figure 2. Separation of purine alkaloids Conditions. Column : Corasil I1 coated with 1.1% Poly G-300 stationary phase, 1-m X 1.0 -mm; mobile phase: heptane: ethanol = 100:10, v/v; flow rate: 0.27 ml/min; column input pressure 300 psi; wavelength: 270 nm; full scale: 0.08 absorbance unit; size: 2 j11 containing (1) caffeine 160 ng, (2) theophylline 250 ng, (3) theobromine 83 ng, (4) solvent peak
finger, a thermometer, and a variac to control the temperature so that the solvent can be held 4 "C below its boiling point. A tiny hole in the cold finger region is provided to prevent vapor building up in the system. With proper temperature control, the vapor will be condensed well before reaching the cold finger region; this minimizes the variation of solvent composition due to ventilation. A drying tube (not shown) is attached to the reservoir to prevent atmospheric moisture from getting into the eluent reservoir and creating a situation which may cause turbidity when the eluent is pumping through the column. This would interfere with the signal from the detector. All fittings and tubings used were Teflon (DuPont), Kel-F, or stainless steel. The column fittings and injection tee were obtained from Chromatronix Inc., Berkeley, Calif. The dimensions of the borosilicate glass columns used were 1.O f 0.25-mm, i.d. and length 50-cm or 100-cm. Reagents. Caffeine (Eastman Kodak); theophylline, theobromine, and brucine (K and K Laboratories); strychnine (Aldrich Chemical Co.); heptane (Eastman Kodak, Catalog No. 2215) were used. Solid Support and Stationary Liquid Phase. Corasil I and Corasil I1 (Waters Associates, Framingham, Mass.); Poly G-300 (Olin Corporation) were employed. Procedure, Corasil I and Corasil I1 were coated with 1.1 (by weight) Poly G-300 by dissolving the liquid phase in dichloromethane. The mixture was kept constantly stirring until nearly dry; then it was air-dried overnight. The c011500
ANALYTICAL CHEMISTRY, VOL. 44, NO. 8, JULY 1972
I
I
I
7.5
15
22.5
Time (minutes)
Figure 3. Separation of purine alkaloids Conditions. Column: Corasil I coated with 1.1%Poly (3-300 stationary phase, 100-cm X 1.0-mm; sample size 2 j11 containing (1) caffeine 130ng, theophylline 264 ng, (3) theobromine 68 ng. Refer to Figure 2 for all other conditions
T
0.008 All
_L
d
15
30
Time (min.utes)
Figure 4. Separation of purine alkaloids Conditions. Column: Corasil I coated with 1.1%Poly G-300 stationary phase, 100-cm X 1.0-mm; mobile phase: heptane :ethanol = 100:5 v/v; sample size 2 p1 containing (1) caffeine 260 ng, (2) theobromine 130 ng, (3) theophylline 528 ng. Refer to Figure 2 for all other conditions
~~
Table I.
Quantitative Analysis of Purine Alkaloids
Compound Caffeine Theobromine Theophylline
Used, ng 40
100 204
Found, ng 31 113 231
Relative error -22
+13 +11
T
0.008 AU
umns were dry-packed and then primed with eluent for 2 hours before any sample injection was made. The procedure for saturating the mobile phase has been described by Kirkland (14). A 25-cm X 2-mm i.d. glass column was packed with 25% Poly G-300 coated on Chromosorb G (lOOjl20 mesh). The eluents used were mixtures of ethanol and heptane, with the ethanol to heptane ratio ranging from 1 : 100 to 10: 100, vjv. The column effluent was monitored at 270 nm using a regular Beckman DU spectrophotometer which allows the monitoring wavelength to be selected. Samples were dissolved in ethanol or an ethanol-chloroform mixture and injected directly into the column without interrupting the solvent flow. Hamilton HP-305 5-pl syringes were used.
L
RESULTS AND DISCUSSION Time ( m i n u t e s )
All chromatograms in this paper had a full scale absorbance of 0.08. Poly G-300 was coated on Corasil I and Corasil I1 to effect separation. Although the same weight per cent of stationary liquid phase was used in all cases, the chromatograms showed that the order of separation was different for different solid supports. Figure 2 and Figure 3 demonstrate the difference. Liquid-solid adsorption may be contributing to the separation process. This is possibly due to the surface properties of Corasil I being somewhat different from those of Corasil 11, taking into account the fact that Corasil I1 has twice the surface area of Corasil 1. Better separation was obtained by lowering the solvent polarity (lowering the proportion of ethanol). This can be seen by comparing Figure 2 and Figure 4. However, the time of analysis was increased. A mixture of heptane-ethanol (100:1, vjv) was also tried. It was assumed that this mixture might improve the separation even further. However, an extended retention time for caffeine was observed; therefore, this mixture was discarded. Higher flow rate might improve the situation but the pressure limitation of 500 psi of the system prevented a study of this effect. In order to maintain a convenient speed of analysis, eluents composed of heptaneethanol ranging from 100:5 to 100:10, vjv, are recommended for this analysis at these pressures. Figure 5 shows that strychnine and brucine can be separated on Corasil I with 1.1% Poly G-300. The separation order is interesting. Brucine has two methoxy groups on the molecule which may account for longer retention on the (14) “Modern Practice of Liquid Chromatography,” J. J. Kirkland, Ed., Wiley-Interscience, New York, N. Y., 1970, p 182.
Figure 5. Separation of strychnine and brucine Conditions. Column: Corasil I coated with 1.1 Poly (3-300stationary phase, 50-cm X 1.0-mm; sample size: 2 pI containing (5) strychnine 1.48 pg, (6) brucine 0.91 pg. Refer to Figure 2 for all other conditions
column. Poly G-300 is rich in ether linkages which may favor the retention of the component with the methoxy groups. Quantitative analysis can be performed by establishing calibration curves. Mixtures of known quantities of caffeine, theobromine, and theophylline were made. To reduce the time of analysis, a mixture of heptane-ethanol (1OO:lO) v/v, had been selected for the quantitative analysis although slight overlap in theobromine and theophylline peaks are observed. The quantities recovered after comparing with the calibration curves were tabulated in Table I. These results indicate that quantities at the nanogram level can be satisfactorily analyzed with reasonable accuracy. ACKNOWLEDGMENT
The authors would like to thank James N. Little of Waters Associates for his help. We are also grateful for the technical assistance received from Daniel Keedy and many valuable discussions with Trevor Robinson.
RECEIVED for review January 26, 1972. Accepted March 30, 1972.
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