525
Anal. Chem. 1987, 59, 525-527
total preasure of the sample and the line width of the dye laser The sensitivity be for further of pulsed thermal lens spectrophotometry is reported to be pressure Of the independent to the (I3)* Therefore, we expect that the present method is very promising for high-resolution spectrometry of trace molecules in the infrared region. Registry No. NH3, 7664-41-7.
(9) Hanna. D. C.; Pacheco, M. T. T.; Wong, K. H. Opt. Commun. 1985. 55, 188-192. (10) Herzberg, G. Molecular Spectra and Molecular Structure I I Infrared and Raman Spectra of Po&atomic Molecules; Van Nostrand Reinhokl: New York, 1945; pp 294-297. (11) Sheldon, J. S.; Knight, V. L.; Thorne, M. J. Appl. Opt. 1982, 27, 1663-1 669. (12) Specification for Nd:YAG laser (YG581C) pumped dye laser (TDLBO), Ouantel International, Santa Clara, CA, June, 1986. (13) Mori, K.; Irnasaka, T.; Ishibashi, N. Anal. Chem. 1983, 5 5 , 1075- 1079. ~
Shuichi Kawasaki Totaro Imasaka Nobuhiko Ishibashi*
LITERATURE CITED (1) Harrls, J. M.; Dovichi, N. J. Anal. Chem. 1980, 52, 695A-706A. (2) Imasaka. T.; Ishibashi, N. Trends Anal. Chem. 1982, 7 , 273-277. (3) Hlgashl. T.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1984, 56, 2010-2013. (4) Hlgashi, T.; Imasaka, T.; Ishibashi, N. Bunseki Kagaku 1982, 31, 680-68 1. ( 5 ) Long, G. R.; Bialkowski, S. E. Anal. Chem. 1984, 56, 2808-2811. (6) Nickolaisen, S. L.; Bialkowski, S. E. Ana. Chem. 1985. 57, 758-762. (7) Long. G. R.; Blaikowskl, S. E. Anal. Chem. 1986, 58, 80-86. (8) Carter, C. A.; Brady, J. M.;Harris, J. M. Appl. Speclrosc. 1982, 36. 309-314.
Faculty of Engineering Kyushu University Hakozaki, Fukuoka 812, Japan RECEIVED for review August 5, 1986. Accepted October 1, 1986.
Direct Analysis of High-Performance Thin-Layer Chromatography Spots of Nucleic Purine Derivatives by Surface-Enhanced Raman Scattering Spectrometry Sir: Direct photometric identification of high-performance thin-layer chromatography (HPTLC) spots is usually achieved by plotting absorption spectra in reflectance and fluorescence spectra ( I ) . Indeed methods for vibrational spectra such as infrared and Raman spectrometries are limited by the required high quantities of substances and spectral properties of the adsorbent (2,3). However, recent findings of an enhanced Raman scattering for various compounds adsorbed at the silver surface (SERS, surface-enhanced Raman scattering) (4,5) give new potential for analytical applications of Raman spectrometry (6). Thus spraying paper chromatograms with silver colloidal solution produces a strong intensity for resonance Raman scatterers from separated dyes (7,8).This surfaceenhanced resonance Raman scattering (SERRS)spectrometry has permitted in situ colored substances down to 2 ng/cm2 to be detected and identified from chromatogram spots (7). However, investigations with colorless sustances (nonresonance Raman scatterers) in silver colloidal solution have also shown strong enhancement of vibration intensity far from resonance conditions (4,5). Thus purine derivatives from nucleic acids can be detected a t concentrations as low as 10" M and identified by the fingerprint region of their spectra (below 2000 cm-I) (9,10). Furthermore application of minute volumes of silver colloidal solutions with guanine derivatives on HPTLC silica gel 60 support enables SERS spectral recordings to be carried out at subnanogram levels (11). In this paper a method has been developed to record in situ HPTLC chromatogram spots of alkylated nucleic purine derivatives. The presence and the variable extent of such modified nucleic bases in biological extracts have been correlated to tumorigenicity (12, 13).
EXPERIMENTAL SECTION Guanine (Gua), adenine (Ade),and purine were obtained from Serva, Heidelberg (FRG). 1-Methylguanine (1-MeGua), 3methylguanine (3-MeGua), 7-methylguanine (7-MeGua), and 9-methylguanine (SMeGua) were purchased from Fluka,Neu-Ulm (FRG). 1-Methyladenine(1-MeAde) and 1-methybypoxanthine (1-MeHyp) were purchased from Sigma (St. Louis, MO). OeMethylguanine (OB-MeGua)was prepared in our laboratory (10). All other chemical reagents were of analytical quality and were
purchased from E. Merck (Darmstadt, FRG). HPTLC plates of silica gel 60 (10 cm X 10 cm) without fluorescent indicator are produced by E. Merck. The silver colloids were prepared by use of Creighton's procedure (5) by the reduction of AgN03 with M N&Hk In a typical experiment, one volume part of AgN03 is added dropwise to three volume parts of 2 X M NaBH,, cooled in an ice bath, and mixed vigorously. The yellow brownish silver colloidal solution was stored at 5 "C for weeks without any change in color. For HPTLC chromatography, single-compoundstock solutions of nucleic purine derivates were prepared to yield a concentration of about M in ethanol/water (1/1)or methanol (guanine 5 X lo4 M). Application of volumes of solution down to 100 nL has been undertaken with a 1-pL Hamilton syringe in conjunction with a micrometer. The nucleic purine derivative separation was accomplished by using a mixture of chloroform,methanol, and ammonia (60:201) (14). Development time for ascending chromatograms takes about 15 min in a covered glass tank. After drying, HPTLC plates are sprayed to wetness with silver colloidal solution by a spray atomizer. Colored spots arising on the HPTLC plate are used to locate the separated nucleic purine derivatives. The spot color varies from pale yellow through orange to violet depending on the nature and concentration of compounds. As already observed in solution (9-11) aggregated silver colloids induced by adsorption of purine derivatives are responsible for the colored spots. HPTLC plates are analyzed at room temperature by a computer-controlled double beam spectrometer: Spex double monochromator 14018 (0.85 m, f/7.8), Datamate DM 1, cold photomultiplier (RCA 31034 A) operated in the photon counting mode. Monochromator slits were selected so as to provide better than 8 cm-* band-pass. The excitation wavelength was the 514.5-nm line of an argon ion laser (Spectra Physics, Model 2020-03) with 10 mW of power. HPTLC-SERS spectra were measured in a typical 90"scatteringarrangement. Spot diameters are about 2-4 mm and the laser beam focused on HPTLC plates is approximately 1.5 mm in length and 0.1 mm in width. Spectra were obtained with a scanning speed (accumulation time) of 2 cm-'/1.5 s for ratios of substance quantity/spot from 10 to 60 ng. RESULTS AND DISCUSSION Previous observations have led to the conclusion that normal Raman spectrometry is not a very promising technique for in situ analysis of TLC spots (3). Indeed very high con0 1987 American Chemical Society
526
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
736
\
I 1328
W SHIFT (crn
-'
)
Figure 2. SERS of HPTLC spot in ring bathing mode frequency region (33 nglspot): (a) 7-MeGua; (b) 3-MeGua; (c) Oe-MeGua; two scans. I50
550
950
1350
1754
RAMAN SHIFT (cm - 1 )
Figure 1, SERS of HPTLC spot: (a) 30 ng of 1-MeAde; (b) 55 ng of Ade; (c)base line in the absence of compounds; three scans.
centrations of compounds (microgramlevels) and rather high laser power (0.2 W) were required. However the high-quality spectra in Figure 1, parts a and b, where only about 30 ng (1-MeAde)was necessary, show that the contribution of SERS from silver colloids now creates a powerful method of investigation. Figure IC also illustrates the negligible SERS background effect of HPTLC silica gel plate. Comparison of these HPTLC/SERS spectra with normal Raman spectra of molecules in aqueous solution shows significant differences: (a) The relative intensities are changed due to a vibrationdependent surface-enhancement scattering mechanism. Several authors have thus proposed that local electromagnetic field and possible charge transfer at the silver colloid surface affect the polarizability and scattering intensity of adsorbed molecules (4). (b) Band shifts can occur (e.g., shift of ring breathing mode of adenine from 724 to 736 cm-'1. (c) Band broadenings are observed in the HPTLC/SERS spectra. For the identification of HPTLC spots, scanning in breathing vibration regions 600 and 700 cm-', for guanine and adenine derivatives, respectively, gives rapid information on methylation sites of nucleic purine derivatives, Figures 1and 2. RF values and characteristic breathing mode vibrations of the compounds investigated by HPTLC/SERS are reported in Table I: In the case of unresolved spots, complementary frequencies are reported which discriminate further nucleic bases in the solvent system used (14). Limits of detection have been estimated to be less than 5 ng/spot when excited with a 10-mW 514.5-nm laser, i.e., under no resonance Raman conditions for nucleic purine derivatives (15). However SERS detection appears to depend on different factors which must be considered before any usual application. Thus close approach to or direct contact with silver colloids of investigated molecules are prerequisite for Raman scattering enhancement. Spectroelectrochemical study of the adsorption behavior of nucleic acids units at the silver electrode has shown the preponderance of interfacial conditions (6). Thus the varying
Table I. RFValues and HPTLC/SERS Frequencies of Nucleic Purine Derivatives" compound
RF values
HPTLC/SERS freqs, cm-l
Gua 1-MeGua 3-MeGua 7-MeGua 9-MeGua
0.09 0.26
653 655, 1700
0.21 0.34
646
08-MeGua
0.56
Ade
0.38 0.09 0.42 0.40
1-MeAde 1-MeHyp purine
0.26
650, 710 656 620 736 726 720, 1704 812
" HPTLC silica gel 60 plate with mixture of chloroform-methanol-ammonia (60201). All reported Raman frequencies in cm-' have a precision within 1 4 cm-'. electrical charge density and hydrophilic character of the silver surface may influence SERS detection. Furthermore the size and dimension of silver colloids are also important (4). Control and optimization of these parameters would enlarge the feasibility of HPTLC/SERS detection to a maximum number of chemical compounds.
ACKNOWLEDGMENT The authors are indebted to N. von Riesenbeck for her skillful technical assistance. Registry No. Gua, 73-40-5; Ade, 73-24-5;1-MeGua,938-85-2; 3-MeGua, 2958-98-7; 7-MeGua, 578-76-7;g-MeGua, 5502-78-3; 1-MeAde,5142-22-3;1-MeHyp,1125-39-9;OB-MeGua, 20535-83-5; purine, 120-73-0;silver, 7440-22-4. LITERATURE CITED (1) Hezel, U. 8. Journal of Chromfography Library; Zlatkls. A,, Kaiser, R. E., Eds.; Elsevler: Amsterdam, 1077; Vol. I X ; Chapter 8. (2) Zuber, G. E.; Warren, R. J.; Begosh, P. P.; O'Donnell, E. L. Anal. Chem. 1004. 58, 2035-2939. (3) Smcfrosc. . . Gomez-Taylor, M. M.: Kuehl, D.; Grlfflths. P. R. ADD/. .. . 1978, 30,-447-452. (4) Chang, R. K.; Furtak, T. E. Surface Enhanced Raman Scafferlng; Plenum: New York, 1082. (5) Creighton, J. A,; Blatchford, C. G.; Albrecht, M. G. J . Chem. SOC., Faraday Trans. 2 107% 75, 700-798.
Anal. Chem. 1007, 5 9 , 527-530 (6) Koglin, E.: SBquaris, J. M. Topics in Current Chemistry; Boschke, F. L.. Ed.: Springer-Verlag: Berlin, 1986; Vol. 134, Chapter 1. (7) Tran, C. D. J. Chromafogr. 1084, 292. 432-438. (8) Tran, C. D. Anal. Chem. 1984, 5 8 , 824-826. (9) Koglin, E.; SGquaris, J.-M.; Fritz, J. C.; Valenta, P. J . Mol. Sfrucf. 1084, 114, 219-223. (10) SBquaris, J.-M.; Fritz, J.; Lewinsky, H.; Koglin, E. J . Colloid Interface Sci. 1085, 105, 417-425. (11) S6quaris, J.-M.: Koglin, E. Fresenius' Z . Anal. Chem. 1085, 321, 758-759. (12) Craddock, V. M. Nature (London) 1970, 2 2 8 , 1264-1268. (13) Lawley, P. D. ChemiceiCarcinogensandDNA; Grover, P. L., Ed.; CRC Press: Boca Raton, FL, 1979: Vol. I, Chapter 1. (14) ISSaq, H. J.; Barr, E. W.; Zielinski, W. L. J . Chromatogr. 1977, 131, 265-273.
527
(15) Fodor, S. P. A.; Rava, R. P.; Hays, T. R.; Spiro, T. G. J. Am. Chem. SOC. 1985, 707, 1520-1529.
Jean-Marie L. S6quaris* Eckhard Koglin Institute of Applied Physical Chemistry Nuclear Research Centre (KFA) D-5170 Juelich Federal Republic of Germany RECEIVED for review May 23,1986. Accepted October 1,1986.
Evolving Factor Analysis for the Resolution of Overlapping Chromatographic Peaks Sir: The introduction of commercial diode array detectors in liquid chromatographyhas opened a new dimension in data analysis to the analytical chemist. Classical single response detectors such as the single wavelength UV-vis detectors, the refractive index detectors, or the conductivity detectors yield a one-dimensional data vector, the one response vs. the retention time. The array detector, however, provides a twodimensional data matrix, the absorbance against retention time and wavelength. This second dimension enables the application of the powerful techniques of factor analysis ( I ) . The ultimate goal of the deconvolution of any chromatogram is the decomposition of the data matrix Y into the product of a concentration matrix C and an absorbance matrix A
Y=CA
(1)
The rows of Y (dimension S X W) are formed by the S absorption spectra measured with progressing elution and its columns are formed by the absorption profiles measured at W different wavelengths. The concentration profiles of the N components form the columns of C (S X N) and their absorption spectra constitute the rows of A ( N X W) (1, 2 ) . Equation 1is the matrix formulation of Beer's law. It has to be noted at this point that eq 1 is not restricted to UV-vis absorption detection. GC/MS or GC/IR spectra can be described similarly if the IR spectra are calculated in absorption instead of transmission units. The singular value decomposition of Y yields the product of three matrices, U (S X N),S ( N x N),and V ( N x W) (2, 3)
Y = usv
(2)
U is formed by the significant eigenvectors of YYt and V by those of YtY. The columns of U as well as the rows of V are orthogonal, UW = VVt = I (identity matrix). S is a diagonal matrix, its elements are the positive square roots of the significant eigenvalues of YYt or YtY in descending order. This decomposition is completely abstract, the matrices U and V have no chemical or physical meaning (abstract factor analysis). If one is not willing to achieve a complete resolution by an optimal choice of all chromatographic parameters (e.g., a time-consuming optimization of a ternery solvent gradient), there are two fundamentally different software approaches to tackle the chemically meaningful decomposition according to eq 1. The deconvolution can be achieved either by a nonlinear least-squares fit to a given function or chemical model ( 4 , 5 )or by the application of a model-free curve res-
olution technique (6-20). Least-squaresfits of chromatograms are hampered by the fact that there is no general analytical function to describe the concentration profiles of different components in a real chromatogram. Simple functions like the Gaussian or the lognormal curve are only appropriate in special cases. Other analytical functions have been tested (4) and fits with general functions are very cumbersome and the computing times are considerable (5). Computer programs for least-squares fits have reached a high level of sophistication in the field of kinetics or equilibrium investigations where eq 1 is also used to describe the measurement (21-24). In these cases however an explicit or implicit function to describe C is derivable from a suitable chemical model, where C is determined either by a set of differential equations (22) or by the use of the law of mass action (21,23,24). The lack of a proper function to describe elution profiles in chromatography makes a model-free deconvolution of chromatograms highly desirable (25). Most of the model-free deconvolution programs are based on the pioneering work of Lawton and Sylvestre (6). Their self-modelingcurve resolution is based on the evident fact of the nonnegativity of the absorption spectra, yielding a range of possible spectra which is usually narrow enough to be chemically useful. The method has found its main application in chromatography (7,8). The big disadvantage is its intrinsic limitation to two-component systems. Nevertheless extensions to more complex mixtures have been described (9-13), but problems have to be overcome in these cases as graphical display of the space spanned by U or V is not trivial. With a switch to polar coordinates ( 1 0 , I I ) as well as by projections on hyperplanes defined by just two of the eigenvectors (12, 13),valuable information is lost. In addition the results obtained for such multicomponentsystems are usually much less well-definedbroad ranges, as long as the acceptable range of solutions is defined only by the nonnegativity of the physical parameters of concentration and absorptivity (13). Further restrictions such as maximum dissimilarity of all spectra (12) or minimum envelope of the concentration profiles (11)have been suggested to obtain unique solutions, but obviously these assumptions are not in general backed by physical reality. Recently, promising experiments have been undertaken independently in different research groups to deconvolute the data by variants of iterative target transform factor analysis (ITTFA) (14-16). These methods have no general restrictions about the maximal number of components. ITTFA can be divided into two parts, in the first part, initial estimates for the concentration profiles C of all components have to be made, and these are then refined in the second part by an
0003-2700/87/0359-0527$01.50/00 1987 American Chemical Society
