ANALYTICAL CHEMISTRY, VOL. 51,
Q& T
o
/ ,
CCOH 'BHil
Cannabinolic A c i d (354)
I Heat OH
Connabinol
'gHll
(310)
A - 6 - T H C (3141
A-I-THC (314)
NO 11,
SEPTEMBER 1979
1877
varied; ( 2 ) changes of C terms (Equation 11as a result of fluctuations in the vaporization of cannabinoids during the period spectra of various source conditions were obtained; and (3) experimental error and/or deviation from the model chosen for regression (20). Further study on the change of relative sensitivity factors at different source condit,ions is in progress. Spectra obtained from mixture other than cannabinoids may not contain all of those mass units listed in Table 11, or at least with different relative intensities. Correlation of these spectra with controlled cannabinoid data should result in significant deviation of constant term from zero, low value of coefficient of determination, or even negative correlation coefficient for some terms. To test this hypothesis, the relative intensities of mass units 314 and 310 of all three samples (Table 11) were exchanged and the same regression analyses were performed. Regression results tabulated in Table IV indicated that good correlation cannot be achieved even with this relatively minor manipulation.
ACKNOWLEDGMENT
C a n n a b ~ d ~ o (314) l
A-I-THC
COCH
A c i d A (358)
The authors express their gratitude to Broderick E. Reischl and Joseph D. Nicol for helpful discussion.
A-I-THC Acid B ( 3 5 8 )
COOH
Cannabidiolic
A c i d (358)
Figure 1. Chemical structure, molecular weight, and possible interconversions of major cannabinoids ( 74- 76)
-
Table IV.
M u l t i p l e Regression o f M o d i f i e d
Canabis sativa L. Mass Spectra
sample 1-L
regression coefficients CBN A-6-THC R Z b ctnC
Ea A-THC CBD
20
0.27 0.40 16 0.23 14 0.18 20 0.22 1 8 0.28 16 0.24 14 0.16 20 0.077 18 0.066 16 0.046 14 0.28
0.86 0.26 0.94 0.25 0.99 0.23 1 . 0 0.15 0.88 0.94
-0.51 0.30 28 -0.66 0.39 30 -0.43 0.63 20 -0.44 0.68 21 1-F -0.58 0.21 30 -0.68 0.29 33 1.1 -0.60 0 . 5 4 26 1.1 0.043 -0.49 0.72 21 2-L 0.85 0.28 -0.12 0.79 14 0.96 0.23 -0.21 0.89 15 0.97 0.21 -0.13 0.93 9.5 0.95 0.21 -0.092 0.95 8.5 a Source ionization energy. Coefficient of determination. Constant term of the regression equation. 18
concluded that the samples under examination are Cannabis satiua L. Variations of regression coefficients a t different source voltages are contributed by the following factors: (1) The relative sensitivity factors (r's in Equation 1) may have
LITERATURE CITED J. I. Thornton and G. R. Nakamura, J . Forensic Sci. Soc., 12, 461 (1972). P. S. Fetterman, E. S.Keith, C. W. Waller, 0. Guerrero, N. J. Doorenbos, and M. W. Quimby. J . Pharm. Sci., 60, 1246 (1971). N. J. Doorenbos, P. S. Fetterman, M. W. Quimby, and C. E. Turner, Ann. N . Y . Acad. Sci., 191, 3 (1971). E. Small and H. D. Beckstead, Nature (London),245, 147 (1973). L Stromberg, J . Chromatogr., 68. 153 (1972). B. B. Wheals and R. N. Smith, J . Chromatogr., 105, 396 (1975). J. Manno, B. Manno, D. Walsworth, and R. Herd, J . Forensic Sci., 19, 884 (1974). N. H. Nie, C. H. Hull, J. G. Jenkins, K. Steinbrenner, and D. H. Bent, "Statistical Package for the Social Science", 2nd ed., McGraw-Hill, New York, 1975, p 320. R. Saferstein and J. J. Manura, J. Forensic Sci., 22, 740 (1977). T. H. Risby and A. L. Yergey, Anal. Chem., 50, 316A (1978). J. A. Kelly and K. R. Arnold, J . Forensic Sci., 21, 252 (1976). H. Budzikiewicz, R. T. Alpin, P. A. Lightner, C. Djerassi, R. Mechoulam. and Y. Gaoni, Tetrahedron, 21. 1881 11965). P. C. Burgers, G. Dikstra, W. Heerma, J. K. Tedouw, A. Boon, H. F. Kramer. F. J. E. M. Kuppers, R. J. J. C. Lousberg. and C. A. Salemink in "Advances in Mass Spectrometry in Biochemistry and Medicine", Vol. 1. A. Frigerio and N. Castagnoli, Eds.. Spectrum, New York, 1976, p 391. C. E. Turner, H. W. Hadley, P. S. Fetterman, N. J. Doorenbos, M. W . Quimby, and C. Waller, J , Pharm. Sci., 62, 1601 (1973). G. W. Kinzer, R. L. Foitz, R. I. Mitchell, and E. 8. Truitt, Bull. Narc., 26, 41 (1974). M. R. Paris, and R. R. Paris, 6ull. SOC.Chim. f r . , Part 1, 118 (1973). T. B. Vree, D. D. Breimer. C.A.M. van Ginneken, and J. M. van Rossum, J . Pharm. Pharmacol., 24, 7 (1972). T. B. Vree, D. D. Breimer, C. A. M. van Ginneken, .I. M. van Rossum, R. A. Zeeuw. and A. H. Witte, Clin. Chim. Acta, 34, 365 (1971). C. W . Waller, K. W . Hadley, and C. E.Turner, "Marihuana: Chemistry, Biochemistry and Cellular Effect", G. A. Nahas Ed., Springer-Verbg, New York, 1976, p 15. J. B. Gayle and H. D. Bennett, Anal. Chem., 50, 2085 (1978).
RECEIVED for review February 5,1979. Accepted May 31,1979. This research program was supported by the Center for Research in Criminal Justice, University of Illinois at Chicago Circle.
Degassed-Solvent Reservoir for High Performance Liquid Chromatography Koichi Saitoh and Nobuo Suzuki' Department of Chemistry, Faculty of Science, Tohoku University, Sendai, 980,Japan
Degrassing is one of the effective preliminary treatments of mobile phases required for successful experiments of high performance liquid chromatography (HPLC). Solvent de0003-2700/79/0351-1877$01.00/0
gassing is effective in preventing bubbles from forming in a detector. Especially in cases where dissolved gases are reactive to mobile or stationary substances, or to some particular C 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
reservoir system. A glass syringe ( S ) serves as a variable volume solvent container. Most of the glass syringes commercially available for medical use are accessible for this purpose; a syringe with a capacity of about 500 mL may be good enough for usual analytical HPLC; if necessary, two or more syringes are connected in parallel. The outlet of the syringe is fitted to Teflon tubing (1.5-mm i.d.) with a Durrum ~ 2 4 0 4 4luer adaptor (A). A three-way valve (V) is used to control the delivery of solvent. A filter (F) is attached at the solvent inlet in this system. Prior to being stored in the syringe, the solvent to be applied must be degassed by an appropriate procedure: for example, several minutes stirring of the solvent in a flask under reduced pressure. The degassed solvent is then transferred into the syringe by pulling the plunger of the syringe manually. If air remains in the tubing or syringe, it has to be expelled from the system by pushing the plunger. When the syringe is filled with the solvent, the three-way valve is turned to deliver the solvent from the syringe to a pump. The syringe is then set upside down as shown in Figure 1, and a stand is used only to keep the syringe from upsetting; the cylinder of the syringe must be freely movable in this arrangement. The cylinder moves spontaneously and continuously downward because of its weight while the solvent is delivered from the syringe to the pump. The replenishment of degassed solvent into the syringe during a chromatographic experiment is performed, if necessary, by turning the three-way valve to deliver the solvent from the solvent inlet to both syringe and pump. The degassed solvent which has been once charged in the syringe never comes into contact with atmosphere. The present solvent reservoir is thus effective to keep the degassed solvent for a long period. According to our experience, no bubble formation in a detector was observed with the 9O:lO methanol-water mixture stored in this reservoir over a week a t room temperature.
6-3 I'
J
U
L
Figure 1. Schematic diagram of degassed solvent reservoir. (S)Glass syringe; (A) syringe-tube adaptor: (V) three-way valve; (F) filter
components of te3t samples, the solvent degassing has to be done to keep a column from damage, or to avoid undesirable transformation of the components. Some expensive commercially available instruments for HPLC include built-in degassers which enable continuous delivery of degassed solvent to the column. However, in an experiment with a relatively simple instrument, the solvents which have been degassed previously are stored in simple solvent reservoirs such as glass flasks and bottles. It is not easy to use such a simple solvent reservoir because of redissolution of gases, moistening, and vaporization of the solvent during storage. The solvent reservoir designed in our laboratory is free from the above problems because of its exposure-proof structure to atmosphere. Figure 1 is a schematic representation of the
RECEIVED for review January 3,1979. Accepted May 23,1979.
Application of the Window Analysis Optimization Method to Lanthanide Shift Nuclear Magnetic Resonance Spectra R. J. Laub Department of Chemistry, Ohio State University. Columbus, Ohio 432 10
A. Pelter and J. H. Purnell" University college of Swansea, Singleton Park, Swansea, SA2 8PP Wales The window analysis method of optimizing chromatographic analyses introduced by Laub and Purnell ( I , 2), has proved extremely successful. In chromatography the important separation parameter is the relative retention ( a ) ,and optimization involves ascertaining means to maximize this quantity for the two components presenting the greatest difficulty in separation. Laub and Purnell have shown how cy can be derived, as a function of several relevant chromatographic variables, from readily obtainable experimental information (3, 4). Plotting derived values of cy against the other variable, arranging that cy > 1 a t all times, then yields a diagram normally consisting of overlapping inverted triangles. The areas of the diagrams where overlap does not occur (windows) define a range of conditions in which complete separation of the mixture of interest is possible, the optimum choice, in terms of column length, corresponding to the peak of the highest window, Le., highest minimum cy. 0003-2700/79/0351-1878$01.00/0
The method is, in fact, widely applicable in analysis in general since its use requires only identification of the parameter to be optimized and a knowledge of its dependence upon various relevant variables. We illustrate here its application to lanthanide shift reagent NMR, now widely used for spectral clarification. It is normal for the associated shifts to be linearly dependent upon the concentration ratio [M]/[S] (where M represents the metal chelate and S the substrate) when [M]/[S] lies in the approximate range 0.1 to 0.7, although linearity sometimes extends outside this range. The data are then normally interpreted by visual inspection, often aided by graphical representation as plots of T (or 6) against [M]/[S]. There is no particular difficulty in locating an optimum value of [MI / [SI when the spectrum is simple, but in complex cases, such diagrams may be difficult even to construct, let alone interpret, because of superimposition of lines, and a value of 0 1979 American Chemical Society
