Oscillating Mirror Rapid Scanning Ultraviolet-Visible Spectrometer as a Detector for Liquid Chromatography Mark S. Denton, Thomas P. DeAngelis, Alexander M. Yacynych, William
R. Heineman,*
and T. W. Gilbert”
Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 4522 1
An oscillating mirror rapid scanning spectrometer is demonstrated as a uv-visible detector for liquid chromatography. The spectrometer can scan the 200-930 nm spectral range at a scanning rate of up to 4.25 kHz with a repetition rate of up to 218 Hr. Three-dimensional chromatograms in which the 200-328 nm range was scanned each second during the separation of uracil, cytosine, and adenine were obtained. Recording complete spectra at short time intervals during the entire chromatogram provided qualitative information about components as they eluted and enabled maximum sensitivity for all components by obtaining chromatographic data at the optimum wavelengths from the threedimensional chromatogram.
The most popular detector for liquid chromatography is presently the uv-absorption detector ( I , 2). The applicability of the uv detector is quite broad because of the significant number of compounds which absorb light in this spectral region. A limitation of the detector as it is commonly available is the restriction of monitoring a single wavelength a t a time. Most instruments are committed entirely to one wavelength (usually 254 nm). This is a significant disadvantage when some components of the sample are nonabsorbing or only weakly absorbing a t the monitored wavelength. A few commercially available detectors operate independently a t two fixed wavelengths. Also available are variable wavelength detectors which partially eliminate this problem by enabling the wavelength to be changed during the chromatogram so that optimum sensitivity can be obtained as individual components elute from the column ( 3 ) .However, some advance knowledge of the spectra and retention times of individual components is necessary in order to properly select wavelengths during a chromatogram. Some of the more recent detectors are uv-visible scanning spectrometers. Since the wavelength scan is slow on these instruments, a “stop-flow spectrum scanning” technique is necessary ( 3 ) .These flow interruptions can be a source of band broadening as well as being time consuming. The optimum spectrometric detector for liquid chromatography would monitor the entire uv-visible range throughout a chromatogram. Complete spectra would (a) facilitate qualitative identification of components as they elute and (b) enable quantitative measurement of each component to be made a t its most sensitive wavelength. A variety of rapid scanning spectrometers have been developed during the past several years (4). Successful applications of these instruments have been made to molecular absorption spectrometry ( 5 , 6), stop-flow kinetics measurements (5-7), spectroelectrochemistry ( & I O ) , flame/plasma emission spectroscopy (5, 11-14), and atomic absorption spectrometry (15, 16). The availability of rapid scanning spectrometers (RSS) makes the concept of recording complete spectra throughout a chromatogram feasible. Rogers has suggested the vidicon RSS as a detector for liquid chromatography (17). A linear photodiode array (18) and a 20
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
cathode ray tube (19) as RSS detectors for liquid chromatography have been reported. This report describes the application of a Harrick Rapid Scan Spectrometer as a detector for liquid chromatography. This instrument is an oscillating mirror RSS of the type developed by Kuwana ( 8 ) . The rapid scanning capability (up to 4.25 kHz scanning rate and 218 Hz repetition rate) combined with the wide spectral range (200-930 nm) make this instrument an obvious candidate for a liquid chromatography detector.
EXPERIMENTAL Rapid Scan Spectrometer. The Harrick Rapid Scan Spectrometer (008) with RSS Signal Processing Module (Harrick Scientific Co., Ossining, N.Y.) is an oscillating mirror RSS (8). The particular instrument used in this investigation was a RSS-B, a second generation instrument with a different optical design from that previously reported for the Harrick RSS ( 7 ) . Figure 1 shows the new optical layout. A significant difference in the new instrument is the all-reflective optics within the monochromator which gives uniform resolution over the entire optical range of the instrument. The use of GaAs photomultiplier tubes has extended the wavelength range of the RSS to 200-930 nm. When used in conjunction with the RSS Signal Processing Module, the instrument is capable of scanning rates of up to 4.25 kHz at a repetition rate of up to 218 Hz. An important feature of the Harrick RSS-B was found to be the very large sample compartment (13’h X 6 in.) which enabled easy placement of the chromatographic detector cell into the light path of the sample beam. This sample-reference compartment is removable from the T-shaped optics compartment which can be used separately. A Raytheon 704 computer was used in conjunction with the RSS. During a chromatogram, the computer triggered a scan each second. The duration of each scan was 0.94 second. The trigger signal from the computer was a +lo-V pulse which caused a 2N5225 transistor to short the trigger input of the RSS Signal Processing Module to ground. Spectra were recorded using the absorbance mode of the RSS. The absorbance signal from the Signal Processing Module was amplified -5X by standard circuitry using a Teledyne Philbrick 1009 operational amplifier before being sent along a -100-ft BNC cable to the analog to digital converter of the Raytheon. Spectra were continuously monitored during a chromatogram with a Tektronix 5103N D13 dual beam storage oscilloscope and simultaneously stored on a Raytheon Magnetic Tape System direct memory access peripheral of the Raytheon. Immediately prior to running a chromatogram, a base-line spectrum for the solvent system was recorded and stored on tape. This base line was subtracted from each spectrum recorded during the chromatogram before plotting the spectra on an x-y recorder. Software for the data acquisition and manipulation has been previously described (10, 20). Chromatographic System. Delivery of the eluting solvent was made with a Chromatronix Model CMP-2 chemically inert piston pump. Samples were introduced into the flowing solvent stream with a Chromatronix Model R6OSV rotary injection valve having a sample loop of 0.276-m1 capacity. The microbore column was a Chromatronix MB-2-75 ( 7 5 mm long, 1.9-mm i.d., 6.6-mm 0.d.). All components were interconnected with 0.79-mm i d . Teflon tubing and Chromatronix tube-end fittings. The system was capable of operation up to a maximum pressure of 500 psi. The ion exchange resins were equilibrated batch-wise three times with the eluting solvent and then slurry packed. When the column was filled, the resin bed was compressed by pumping solvent through the column a t maximum flow rate. Additional resin was added to fill the resulting void volume, and the process repeated.
€OAR
Optical Cell. The flow cell used for all optical measurements in the RSS has been described previously (21). It had a volume of 87 pl and a path length of 1.27 cm. The optical cell was located a t the focal point of the light beam which was in the center of the sample compartment. In this position, no masking of the optical beam was necessary .
RESULTS AND DISCUSSION
- PMTS Figure 1. Optical diagram for Harrick RSS-B (S)Source (75-watt xenon arc lamp): (L1, L2S, L2R) Quartz lenses; ( M l , M2)
Flat mirrors: (SM1, SM2) Spherical mirrors: (SI,52)Entrance and exit slits: (GM) Galvanometer mirror: (GR) diffraction grating (Bausch and Lomb Type 35-53-04-090, 300 grooves/mm, blazed for 300 nm); (BS) Variable angle gridded beam splitter: (OAS, OAR) Optical attenuators for sample and reference beams: (PMTS, PMTR) Sample and reference photomultiplier tubes (GaAs photocathodes, side viewing, RCA): (FC) Flow cell for liquid chromatograph. All mirrors and lenses are uv grade
Two resin packings were used: Aminex A-4 cation exchange resin (Bio-Rad Laboratories) is supplied with a narrow range of particle diameters (16-24 pm); Dowex 50 W-X8 (-400 mesh), a cation exchanger with similar chemical properties, has a larger average particle size and a wider range of particle diameters. The former resin gave the higher resolution but also the higher pressure drop. With this resin, the maximum flow rate obtainable was 0.4 ml/min. On the other hand, flow rates up to 2 ml/min could be obtained with the Dowex resin. By varying the flow rate and using these two resins, a wide variation in the chromatographic resolution of the sample components could be achieved. This permitted the investigation of the capabilities of the RSS detector under conditions of good and poor chromatographic resolution. T h e eluting solvent was 1.0 M ammonium chloride-0.10 M hydrochloric acid made up in boiled and cooled distilled water. The solutes were uracil ( L ) ,cytosine (C), and adenine (A) from Nutritional Biochemicals Corp. and were used as received. The concentrations of each in the mixed samples were: 6.24, 4.17, and 4.78 X IO-' respectively, for L', C, and A. At the p H of the solvent, C and A are in their cationic forms, whereas U remains neutral.
The purpose of this preliminary study is to demonstrate the feasibility of using a rapid scanning spectrometer of the oscillating mirror type as a detector for liquid chromatography and to illustrate advantages of recording entire spectra during a chromatogram. Consequently, a relatively simple system with known spectroscopic and chromatographic properties was sought. The system which was chosen consisted of the nucleotide bases, uracil, cytosine, and adenine. A three-dimensional presentation of the chromatogram obtained for uracil, cytosine, and adenine with the RSS detector is shown in Figure 2. The horizontal axis is the wavelength scanned by the RSS (200-328 nm), the vertical axis is absorbance, and the time axis is displaced into the paper a t a 45' angle. Although spectra were recorded every second during the elution, the spectra are plotted a t 20-second intervals (with a few exceptions) for clarity in the drawing. As shown in the 3-D chromatogram, the separation of the three nucleic acid derivatives is very good under these conditions. The components were qualitatively identified as they eluted by the positions of the absorbance peak maxima. The order of elution was uracil (A,, = 260 nm), cytosine (A, = 275 nm), and adenine (A,, = 262 nm). This assignment was verified by retention times of the pure components. Although the instrument is capable of scanning the entire 200-930 nm range, in this case the scan was restricted to the uv range since the nucleotide bases do not absorb in the visible and near infrared region. Once the 3-D chromatogram was obtained, chromatograms a t any wavelength or combination of wavelengths could be obtained from the 3-D data and displayed in the conventional manner. Figure 3A shows a plane through the 3-D chromatogram at 280 nm. The slice illustrates the chromatogram which would be obtained if a set wavelength detector a t 280 nm had been used. I t is apparent from the
Flgure 2. 3-Dchromatogram with RSS detector for separation of uracil, cytosine, and adenine Spectra displayed for 20-second intervals. Aminex A-4 cation exchange resin. Flow rate, 0.4 ml min-' ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
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980
W V
2 4.
m a
0 v)
m a
I \ I \
T
1
I
so0
280
200 Wavelength, nrn
1000
t, sac
Figure 3. Chromatograms for separation of uracil, cytosine, and adenine. Aminex A-4 cation exchange resin. Flow rate, 0.4 ml min-’ ( A ) 3-0 chromatogram with plane drawn at 280 nm. (B) Two-dimensional chromatograms drawn from 3-13 chromatogram for detection at the following wavelengths: ( a )254 nm, ( b ) 280 nm, (c)260, 275, and 262 nm
drawing that sensitivity for all three components is lost since the monitored wavelength is not a t the peak maxima. Figure 3B shows chromatograms obtained from the 3-D chromatogram a t two wavelengths commonly available in
set wavelength detectors, 254 and 280 nm. In both cases, the separation is good. However, a t both wavelengths, sensitivity for one or more components is compromised since the set wavelength is not monitoring a t A., This is most
2 8 0 nm
m a 0
m
) ‘ I , ,
,
I
, , ,
200
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,
I
,
L
300 WAVELENGTH,
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0
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1000
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Flgure 4. Chromatograms for separation of uracil, cytosine, and adenirne. Dowex 50 W-X8 cation exchanger. Flow rate, 0.4 ml min-‘ (A) 3-D chromatogram (B) Set wavelength clhromatograms drawn from the 3-D chromatogram. ( a )254 nm, ( b ) 280 nm, ( c )260, 275, an 262 nm
22
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
a 254
nm
42 0
b 2 8 0 nm
w u 2 U
m a 0 v)
a t U 2601275 2 6 2
C
t , sec
Figure 5. Chromatograms for separation of uracil, cytosine, and adenine. Dowex 50 W-X8 cation exchanger. Flow rate, 1.0 ml min-‘ ( A ) 3-D chromatogram. ( B ) Set wavelength chromatograms drawn from the 3-D chromatogram, (a) 254 nm. ( b )280 nm, (c)260, 275, and 262 nm
obvious for uracil a t 280 nm where substantial sensitivity is lost because the monitored wavelength is 21 nm displaced from the A,., The forte of the RSS detector is illustrated by curve c in Figure 3B which is a chromatogram a t the optimum wavelengths for each component. Since the 3-D chromatogram contains information a t all wavelengths, it is an easy matter for the computer to switch wavelengths a t any time so that each component is displayed a t its A,, for maximum sensitivity. Chromatogram c in Figure 3B is clearly the best of the three. The ability to obtain a multiwavelength chromatogram can be more beneficial in situations where peaks are poorly resolved. T o illustrate this situation, the separation of the three bases was purposely degraded by switching to Dowex 50 W-X8 cation exchanger and increasing the flow rate. The 3-D chromatograms in Figures 4A and 5 A show the loss of resolution among the three peaks as the flow rate was increased from 0.4 ml/min. to 1.0 ml/min. Figures 4B and 5B show the effect on set wavelength chromatograms a t 254 and 280 nm. Resolution a t these wavelengths diminished so that the cytosine and adenine peaks are indistinguishable a t 280 nm in Figure 5B. The analogous multiwavelength chromatograms a t the A,, for each component are also shown in Figures 4B and 5B. The improvement in the sensitivity for each component is apparent. This example illustrates how a poor separation can be tolerated when the complete flexibility of choice of wavelength offered by the RSS is available. Even though this capability can be useful in the simple three-component system shown here, its real utility would be in complicated multicomponent systems where a few of the components are difficult to resolve. In such a case, proper wavelength selection from a 3-D chromatogram could compensate for a lack of chromatographic resolution. In fact, two components could elute a t exactly the same time, yet be quantitatively evaluated if their absorption spectra were sufficiently different. Thus, the capability of obtaining 3-D chro-
matograms has the very real potential of minimizing the time-consuming process of finding chromatographic conditions (optimum column, solvent, flow rate, etc.) for a complete separation of a complicated mixture if some optical differences exist, as is often the case. In the example shown here, even a rather small difference in A,, values gives a noticeable differentiation. Although the 3-D chromatograms shown here were drawn as “viewed from the right”, peaks a t lower wavelengths can be obscured by peaks at higher wavelengths. Thus, a better perspective can sometimes be obtained by “viewing from the left”. Computer software is presently being developed so that chromatograms can be rapidly displayed on an oscilloscope for any wavelength or combination of wavelengths from the spectra stored on tape. This will enable the chromatographer to rapidly determine optimum wavelength conditions before a final chromatogram is recorded on paper and peak areas are integrated by the computer for quantitative information. The sensitivity of the unit a t different wavelengths and conditions of optical resolution is also being evaluated. Here the possibility of enhanced sensitivity by signal averaging exists. Rather than a single spectrum being recorded every second, 10 or more spectra a t a higher repetition rate could be recorded each second and signal averaged. For example, a great improvement in spectrum quality should be possible especially a t short wavelengths where photomultiplier noise was high (see Figure 4A) because of strong absorption by the solvent, loss of source intensity, and the use of a wide bandpass setting (10 kHz) on the RSS Signal Processing Module.
CONCLUSIONS The results of this initial investigation, show that the Harrick Rapid Scan Spectrometer has considerable potential utility as a detector for liquid chromatography. The advantages are (a) much qualitative information about the ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
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components in a mixture is contained in the complete spectra and (b) the ability to maximize the sensitivity for all components by obtaining chromatographic data a t the optimum wavelengths from a 3-D chromatogram. An implication of the second advantage is that the selection of optical conditions can compensate for poor chromatographic resolution.
ACKNOWLEDGMENT The authors gratefully acknowledge B. J. Norris for recording spectra of the nucleotide bases on a Cary 14.
LITERATURE CITED (1) L. R. Snyder and J. J. Kirkland, "introduction to Modern Liquid Chromatography", J. Wiley and Sons, New York, N.Y.. 1974, p 142ff. (2) G. Zweig and J. Sherma, Anal. Chem., 46, 73R (1974). (3) C. D. Carr, Anal. Chem., 46, 743 (1974). (4) R. E. Santini, M. J. Miiano. and H. L. Pardue, Anal. Chem., 45, 915A (1973). (5) M. J. Milano, H. L. Pardue, T. E. Cook, R. E. Santini, D. W. Margerum. and J. M. T. Raycheba, Anal. Chem., 46,374 (1974). (6) M. J. Milano and H. L. Pardue. Anal. Chem., 47, 25 (1975). (7) R. M. Wightman, R. L. Scott, C. N. Reilley, R. W. Murray, and J. N. Burnett, Anal. Chem., 46, 1492 (1974). (8) J. W. Strojek, G. A. Gruver, and T. Kuwana, Anal. Chem., 41, 481 (1969). (9) E. E. Wells, Jr.. Anal. Chem., 45, 2022 (1973). (10) H. B. Mark, Jr., R. M. Wilson, T. L. Miller, T. V. Atkinson, H. Wood, and
(11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21)
A. M. Yacynych, "The On-Line Computer in New Problems in Spectroscopy: Applications to Rapid Scanning Spectroelectrochemical Experiments and Time Resolved Phosphorescence Studies" in "Information Chemistry: Computer Assisted Chemical Research Design", S. Fujiwara and H. B. Mark, Jr.. Ed.. University of Tokyo Press, Tokyo, Japan, 1975, pp 3-28. K. W. BuschandG. H. Morrison, Anal. Chem., 45, 712A(1973). K. W. Busch, N. G. Howell, and G. H. Morrison, Anal. Chem., 46, 575 (1974). K. W. Busch, N. G. Howell, and G. H. Morrison, Anal. Chem., 46, 2074 (1974). F. L. Fricke, 0. Rose, Jr., and J. A. Caruso, Anal. Chem., 47, 2018 (1975). D. G. Mitchell, K. W. Jackson, and K. M. Aldous, Anal. Chem., 45, 1215A (1973). K. M. Aidous, D. G. Mitchell, and K. W. Jackson, Anal. Chem., 47, 1034 (1975). L. B. Rogers, CBEN, April 15, 1974, p 18. K. M. Aldous and J. S.Garden, "Abstracts of Papers", 26th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1975, Abstract No. 434. A. Bylina. D. Sybilska. 2 . R. Grabowski, and J. Koszewski, J. Chromafogr., 83, 357 (1973). A. M. Yacynych, Ph.D. Thesis, University of Cincinnati, Cincinnati, Ohio, 1975. L. C. Hansen and T. W. Gilbert, J. Chromafogr. Sci., 12, 458 (1974).
RECEIVEDfor review July 21, 1975. Accepted October 2, 1975. The authors gratefully acknowledge the financial support provided by National Science Foundation Grants GP-41981X for the RSS and GP-35979 for departmental computer equipment.
Analysis of Marijuana Samples from Different Origins by HighResolution Gas-Liquid Chromatography for Forensic Application Milos Novotny," M. L. Lee, Chow-Eng Low, and Alain Raymond' Department of Chemistry, Indiana University, Bloomington, Ind. 4740 7
A highly specific procedure for "fingerprinting" marijuana samples has been developed. The method consists of extraction of a marijuana sample, a single partition step, and the use of a precoiumn concentration technique prior to gas chromatography. The high resolving power of glass capillary columns is essential for developing complex chromatographic profiles that are unique for a given sample. Characteristic profiles of nonpolar marijuana constituents are shown for selected samples from different geographical orlgins. Considerably higher specificity of this profile method over the conventional measurement of the relative concentrations of major cannabinoids is demonstrated. Thirty-eight profile constituents have been identified by combined gas chromatography-mass spectrometry.
Chromatographic methods have been implicated in a t least three forensic applications concerning abuse of marijuana: (a) determination of whether an unknown sample of plant material contains marijuana; (b) determination of whether cannabis samples confiscated a t different locations originate from a common lot; and (c) tracing of illicit marijuana samples to their geographical origin. Generally, the first case is the most straightforward one and does not require sophisticated separation methods. Even when marijuana is blended with non-cannabis plants used as adulterants, combination of a simple histological Present address, Intersmat Instruments, 5 and 7 Allee Jean de la Fontaine, Pavillons-sous-Bois 93320, France. 24
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
technique with thin-layer chromatography appears to be sufficient ( I ) for positive identification. Tracing of illicit marijuana samples to their origin has been a considerably more complicated task; the literature concerning this problem is indeed abundant with many attempts to correlate sample composition with its origin. It has been well-established that the various types of cultivated and wild marijuana or hemp differ considerably in their respective cannabinoid content. Cannabis plants from different parts of the world may vary from those producing almost exclusively cannabidiol to those producing predominantly A9-tetrahydrocannabinoL Consequently, several workers (2-6) used the measurement of the relative concentrations of cannabidiol, A9-tetrahydrocannabinol,and cannabinol (so-called "main cannabinoids") with the objective in mind of determining from which country each sample originates. In the course of such studies, several problems have become apparent that seriously limit this analytical approach: 1) I t has been determined that, a t least for the first several generations, the content of major cannabinoids produced by the plant is dependent upon the inherited properties of the seed, and that the genotype appears to be far more important than the influence of immediate geographical location and climate. Consequently, it has been suggested ( 3 )that if cannabis seeds are shipped from one country to another for illegal cultivation, there is little valid basis for attempts to correlate the cannabinoid content with the place of origin. 2 ) Phillips et al. ( 7 ) have further observed a cyclic variation of cannabidiol and A9-tetrahydrocannabinol during the growing season of an Indiana variety. The variation in cannabidiol content ranged
