Anal. Chem. 1981, 53, 1357-1361
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Thin-Layer Chromatography with in Situ Multichannel Image Detection of Fluorescent Compounds M. L. Gianelli, J. B. Callis,” N. H. Andersen, and G. D. Christian Deparfment
of Chemistry B G 10, University of
Washington, Sea ftle, Vashington 98 195
A multichannel detector for In situ analysls of Huorescent materials on thin-layer plates is evaluated. Optical system 1 Is designed to obtain a fluorescence spectrum from each position along the elution axis of a one-dlmenslonal plate without need for mechanical scanning. By use of tetraphenylporphine (H,TPP) and octaethyiporphlne ( H,OEP) as test materials, it is shown how overlapping spots can be resolved into their components. Optical system 2 Is designed to obtain a quantitative two-dimensional fluorescence image of a single, two-dlmensionai chromatogram or of multiple, one-dimensional chromatograms. A dynamlc range 01 fluorescence intensity of over 500:l can be measured on a single plate, and the detection limits extend as low as 3 pg for H2TPP. Imaging detectors allow rapid, In situ qualitative and quantitative information on fluorescent materlais to be obained and might allow the analyst to take full advantage of the parallel processing capabliltles of thin-layer plates for routine assays.
Recent advances in separation power coupled with methodology for elimination of active sites have so greatly improved the capacilities of thin-layer chromatography that the term “high-performance thin-layer chromatography” (HPTLC) does not appear a t all unwarranted (1). At the same time, advances in technology have made quantitative analysis of the plates possible in situ by scanning densitometry (2). Fluorescence densitometry in particular has proven to be of significant value in many applications due to its sensitivity and selectivity (3-5). In clinical and environmental analysis, the use of HPTLC is especially appealing compared to gas-liquid chromatography (GLC) or high-performance liquid chromatography (HPLC) because it is quite feasible to develop many plates/samples simultaneously. Unfortunately, there still remains the difficulty of quantitatively analyzing the plates. The present generation of mechanical scanners simply do not provide the high throughput desired; moreover, any attempt to speed up mechanical scanning is likely to suffer a signal-to-noise degradation inversely proportional to the decreased time spent in the scanning process. One possible solution to this problem is to use an imaging detector to quantitatively measure light reflected or emitted from the plate. The capabilities of imaging detectors are by now well appreciated in the scientific community and their usefulness in chemistry has been amply demonstrated (6). We have been especially impressed with these devices as fluorescence detectors (7-11). In this paper, we report on the use of a digital imaging photometer based upon a S I T vidicon as a two-dimensional fluorescence detector. We have used this device in two ways: (a) in conjunction with a monochromator to analyze both the wavelength of emission and retention time of each spot on a one-dimensional chromatogram and (b) in conjunction with a simple optical filter to analyze either two-dimensional chromatograms or multiple one-dimensional chromatograms
simultaneously. In both approaches no mechanical scanning is required. The results contained herein demonstrate the feasibility of this approach.
EXPERIMENTAL SECTION Apparatus. Two different optical configurations were used and are presented schematically in Figure 1. Both systems used a 150-W Xenon arc lamp as excitation source. Light from the arc was collected by a quartz f l l . 0 lens focused to infinity to produce nearly uniform illumination of the entire plate. A band of excitation wavelengths was selected by means of various Coming glass filters; infrared light was eliminated by means of a Schott KG-3 glass filter. In the first configuration(Figure 1)a Jarrell-Ash 82-415 quarter-meter Ebert monochromator equipped with a 295 l/mm grating blazed at 400 nm was used to analyze the emitted light. The exit slit and holder were removed from the housing and the entire exit plane image was focused onto the SIT vidicon by means of an f/2.5 lens with 2:l demagnification. In the second configuration, Figure 1 minus the monochromator and fixed lens, various Corning and Schott glass filters were used to isolate the fluorescence emission from the thin-layer plates and the resultant fluorescence image was focused directly onto the photo surface of the vidicon with an f/2.5 lens with approximately 2:l demagnification. The digital SIT vidicon and computer system has been previously described (7). Reagents. For spotting on the thin-layer plates, chromatographically pure free base octaethyl-, etio- and tetraphenylporphines were dissolved in spectroquality dichloromethane from Matheson, Colem and Bell. Thin-Layer Chromatography. Porphyrin mixtures were chromatographed on Whatman KClereversed-phase microslides. The two-porphyrin mixture was developed in methanol-acetone (80:20); the three-porphyrin mixture was developed in the same manner, dried, and then redeveloped in 100% acetonitrile. All development solvents were spectroquality reagents from Matheson, Coleman and Bell. Data Acquisition and Analysis. Fluorescence images were collected with the digital photometer operated at a resolution of 64 X 64 elements. A total of 512 successive images were summed together for improvement of the signal-to-noiseratio. An automatic shutter was then closed and 512 successive “ d a r k images were subtracted from memory to eliminate dark noise arising from systematic variation of leakage current from element to element. Data were then analyzed with a PDP 11/04 computer using a series of programs described in the Ph.D. thesis of Johnson (12). Data were displayed on a video monitor or Tektronix 4006 terminal. For rank analysis and hard copy graphics display, the data were transmitted via phone line to the University Computer Center where it was analyzed with programs developed by Warner et al. (13).
THEORY Optical System 1. The optical system of Figure 1 was designed to produce an emission spectrum from every point along the retention axis of a one-dimensional chromatogram without any mechanical scanning. The manner in which this was accomplished is given in Figure 2. Suppose that we have a two-component mixture of fluorophores a and b whose fluorescence spectra (Figure 2A) and relative retention profiles (Figure 2B) are highly overlapping. A mixture of these components when chromatographed will produce a thin-layer plate
0003-2700/81/0353-1357$01.25/00 1981 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
function of position along the retention axis indexed by i and a function of wavelength of emission indexed by j . In the case where the number of photons absorbed is small and concentrations of interacting species are low, interaction between components is negligible, and energy transfer rates are low, one may write
I
MONOCHROMATOR
T uu
SIT CAMERA
rL
VIDEO MONITOR
Flgure 1. Optical system 1 for producing emission spectra from materials on thin-layer plates. A two-dimensionalfluorescence Image of a thin-layer plate is obtained by removal of the monochromator and focal lens and replacing with a filter (optical system 2). A
B I
D
C
I
M = [mi,]= kaktiky? = k a k t k @ y k k=l
thin-layer plates without mechanical scanning: (2A) emission spectra of components a and b; (28) image of thin-layer plate spotted with a and b; (2C) image of video monitor of 2B; (2D) isometric projection of 2 c . which, when viewed under a uniform excitation light, would have the appearance of Figure 2B, a single unresolved spot. Suppose that a lens of appropriate focal ratio is used to project a fluorescent image of the thin-layer plate onto the entrance slit of an analyzing monochromator such that the long axis of the slit is aligned parallel with the thin-layer development axis and such that the center of the spots is centered on the slit. Suppose further that the exit slit assembly has been removed from the monochromator to allow the two-dimensional detector to view the resultant image. From our previous work (7) it is known that the monochromator faithfully preserves the information concerning the distribution of fluorescence along the long axis of the slit. Thus, the y axis of the exit plane image represents the retention axis of the thin-layer plate, while the x axis represents the wavelength of the fluorescence. The expected image for the compounds of Figure 2A is shown in Figure 2C as it might appear on a television monitor display. Once the image is captured quantitatively, various data analysis and display modes may be invoked; for example, Figure 2D shows how the data might appear when formated as an isometric projection with the vertical axis representing fluorescence intensity and all hidden lines removed for clarity. The data from the above experiment are most conveniently represented as a matrix M of intensity values which are a
(1)
In this expression we have assumed that r fluorescent species have been chromatographed (indexed by k ) and that each has a unique fluorescence spectrum characterized by a vector y k = [yj]and a retention time characterized by a vector tk [t,]which expresses the relative concentration of the particular fluorophore along the retention axis. The scalar, c y k , contains the concentration dependence of the fluorescence intensity and is proportional to the fluorescence quantum efficiency as well. The symbol @ denotes the outer product operation. The bilinear form of eq 1 suggests that certain powerful theorems of linear algebra may be used to analyze the data. Indeed there is an obvious similarity between eq 1of this paper and eq 4 of Warner et al. (13) wherein data from a video fluorometer in the form of an emission excitation matrix is discussed. In analogy with that work, we can assert that: (a) the rank of M is a lower bound to the number of independently emitting species on the plate, and (b) the eigenvectors of the real square symmetric matrices MTM and MMT are linearly related to the fluorescence and retention vectors. Thus one can obtain a rather exhaustive qualitative analysis of the materials if they are unknowns. In the case of known components, the techniques of least squares (13, 14) and rank annihilation may be used for quantitative analysis (15, 16). In practice, the use of a multichannel imaging device produces distortions of the data in a characteristic manner which requires correction. This point has been carefully considered by our astronomer colleagues (17). It is easily shown that eq 1 must be modified as follows: Mobsd
Flgure 2. Concept for producing emission spectra from materials on
k=l
=
Ccukt,kY,k%] + P , O , + 4,
k=l
(2)
where uII is the relative sensitivity of each pixel, p V , the background fluorescence of the plate as imaged onto the detector, and d,, the dark signal from each pixel. Corection for the dark signal is easily obtained by closing a shutter in front of the camera and collecting a “dark frame” D E [d,] averaged over the same time as the light frames. A “blank frame” B is also obtained from the plate prior to spotting and developing, and stored in the computer for later substraction. Obviously (3) €3 = [PI]% + dl11 Correction for the variation in pixel to pixel relative sensitivity can be obtained by means of a “flat field” correction F obtained by shining a white light source onto the plate and collecting the radiation from it. In this case (4) F = [ P u , ~ l+~ yd,,I where u, is the relative intensity collected along the long axis of the slit. uI is the relative spectral output of the lamp and a constant of proportionality. With the above data in hand, one obtains a corrected matrix MCo”as follows r
Mcorr= (Mobsd- B)/(F
- D) = Cako,q, k=l
(5)
where y k = c y k / & 0,= t r / u ,and p , = x k / U ] , Obviously eq 5 and 1 are identical for the purposes of rank analysis, and our previous work may be taken over unmodified. Optical System 2. The optical system of Figure 1 was modified to obtain a two-dimensional fluorescent image of the
ANALYTICAL CHEMISTRY. VOL. 53, NO, 9. AUGUST 1981
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For quantitative purposes, we assumed that concentration waa proportional to the total fluorescence from the spot minus the total background signal estimated for the spot.
Fbure 3. Image 01 a m8n-aycr chromalagram 01 a mirr-re 01 n,OEP ana h2TPP proarc& 01 oplcal syslem 1. raw aala as aospayea on a video mon lor
plate by removal of the monochromator and isolating the fluorescence by introducing colored glass filters in front of the detector. In this case. the ability to obtain wavelength information is forfeited in favor of simultaneously analyzing several one-dimensional chromatograms or one two-dimensional chromatogram. Our previous commenb regarding flat field, dark field. and blank field corrections apply to optical system 2,and a strategy similar tn that of the previous sectinn must be used to obtain a quantitative analysis. Unfortunately, due to irreproducihility in positioning the plate for background dark and flat field frames, there remained a small hackground signal which without further CD rection frustrated analysis at the subnanogram level. We found it sufficient tn use a simple interactive level thresholding algorithm in which the operator determined the spot size and the computer obtained the hnckground signal per pixel from an average of all of the pixels immediately adjacent to the spot.
RESULTS AND DISCUSSION Optical System 1. We evaluated optical system 1for ita ability to provide a qualitative analysis of a thin-layer plate using rank analysis reduction of the data. Accordingly, a mixture of free base tetraphenylporphine (H,TPP) and free base odaethylporphine (H,OEP) was chromatographed so that i t exhibited only a single unresolved spot similar to Figure 2B. Figure 3 shows a video monitor display of the output image from the monochromator. Only the upper half of the retention axis was imaged onto the entrance slit of the analyzing monochromator. The single spot has now been resolved into two distinct species with the added dimension of wavelength of emission. The substance with the largest R, value is H,OEP which is confirmed by the emission peaks at 624 and 686 nm. The substance with the lesser R, value is H,TPP which is confirmed by the emission peaks at 652 nm and the shoulder at 718 nm. Figure 4 shows contour map representations of the raw data, the blank, the flat field, the data minus the blank, and the fully corrected data according to eq 5. Figure 5 shows linear plots of the total emission spectra and total elution data, together with selected rows and columns providing spectra and retention profiles, respectively, of each compound. Examination of Figures 3-5 shows that the optical system of Figure 1does indeed provide us with data in the manner shown in Figure 2. One can quite clearly observe that two components are present. Reference to Figure 4B shows that the blank fluorescence was not at all negligible for porphyrins at the concentrations used while reference to Figure 4C shows that the plate is not irradiated uniformly nor is the Dixel-nixel sensitivity uniform. Table I gives the resulta of a rank analysis of the data of Figure 4A,E. It can be seen that the first two eigenvalues of
Flgura 4. log wntou maps 01 data acquired by optical system 1: (A) data 01 Figure 3 (B) blank l a plate: (C) Ilat flew (D) raw data wllh blank SuDhaCtBd; (E) data corrected lor blank and flat field. The ratio 01 the most intense pixel 01 A to the intense pixe 01 B is 7.26.
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Table 1. Eigenvalues" two components 6U x 60 two components 40 x 4 0 two components 4 0 x 40 flat field corrected a
lirst
second
1 . 9 9 x 10" 4 . 3 8 K 10" 1.13 X 10"
2.99 x 101" 6 . 1 3 x 10" 7.50 x i o v
Noise a t the edges was deleted by cropping to a 40
X
lhird 1.65 x
inx
1 . 0 4 X 10' 3.17 x 10'
2/3
181 589 237
40 matrix.
Figue 6. Dywmic range study. Various concentratims of H,TF'P were spotted on a thin-layer plate. and their fluorescent image was obtained with optical system 2. Isometric piojections of fully corrected data. lly -Ls
Ilr
C M L S
Figure 5. Linear plots of both emission and retention data of Figure 4D. (A) scan 01 total emission spectra, sum of all the rows; (B)scan of emission of H2TPP and H,OEP by selecting two individual rows of pixels: (C) total retention data. sum of all the columns; (D) chromatographic profiles of H;TPP and H,OEP by scanning two individual columns by pixels. of the retentinn-time matrix are rather larger than the third (and succeeding) eigenvalues demonstrating that rank analysis may be a useful tool in determining the number of compounds spotted on the plate. A comparison of the ratio of eigenvalues with and without flat field corection shows that this potential sou~ce of error is negligible compared to others. Also, we found that data from the edges of the thin-layer plate contributed only noise, and accordingly such data were eliminated from the rank analysis. Clearly, the multichannel capability of optical system 1 offers an advantage over conventional thin-layer plate scanners. AU mechanical scanning of the plate has been eliminated while taking the spectrum of every spot along the retenion axis. One may also choose to focus in on only one or two unresolved spots by changing the focal point of the lens hetween the plate and the monochromator and readjusting the plate accordingly. Disadvantages of our system are (a) that correction must be made for pixel-pixel variation in sensitivity and (b) that only a limited central region of each spot is analyzed. The pixel-pixel variation may be partially overcome by suitable flat field correction. Disadvantage (b) may be overcome, at least partially, by means of a cylindrical lens system which images more of each spot onto the entrance slit of the analyzing monochromator by compressing the focus of the spot in the X direction while maintaining the spatial resolution in the retention direction. A possible compromise optical system would use a stepper motor to drive the thin-layer plate past the entrance slit of the analyzing system. The optics would be devised so that the retention axis of the plate would be normal to the long axis of the slit. This configuration would be identical with the conventional scanners except that all spectral information would be acquired a t onre using a multichannel detector. We
are currently building such a system to test this approach. Our feasibility study shows that rank annihilation is likely to he of great value in analyzing retention-emission data. In its present form, the algorithm does have the disadvantage that it cannot successfully separate two substances which have identical or similar emission characteristim, even though they might have rather different retention times. For example, a three-component mixture of H2TPP, H2Etio (free base etioporphine), and H20EP porphyrins yields a retention--time matrix which has only two principal components, based on eigen analysis. This happens hecause the spectra of H2Etio and H,OEP are nearly identical. However, if the algorithm were to be modified to recognize the fact that each component elutes as a single spot of predictable half-width, the separation into three components is quite straight forward (18). Optical System 2. We evaluated system 2 for its ability to provide a quantitative analysis of a thin-layer plate in two dimensions. We were particularly interested in the capability to operate over a wide dynamic range with very small amounts of materials. As a test of this, we placed nine spots of H2TPP a t amounts varying from 3 pg to 1.5 ng. Figure 6 gives the fully corrected data as isometric projections. It can be safely concluded that quantitation is quite feasible in the picogram region and that a t least a 2.5 orders of magnitude range of H2TPP can be quantitated on one plate. The only disappointment is that the calibration curves are not quite linear. While the correlation coefficient was very high (0.996), the slope of a log-log plot was only 0.779. For a truely linear relationship, the value of the slope should have been equal to 1.0. This nonlinearity could be due to aggregation of the compound on the plate, allowing self-quenching to occur and thus lowering the fluorescence at higher concentrations. Uchiyama and Uchiyama (19)have shown that spraying the plate with a suitable nonviscow solvent can often prevent self-quenching due to aggregation. The linearity of the multichannel detector has been carefully checked (7) and this has been eliminated as a source of error. To e n s u e that development of the plate does not somehow prevent quantitation, we have placed three spots of H2TPP (3 pg, 9 pg, 1.5 pg) on a aemud plate and developed it.. An isometric projection of the image is shown in Figure 7. While
ANALYTICAL CHEMISTRY, VOL. 53. NO. 9.
1' , '
,/
Thin-layer chromatogram of H,TPP at three different concentrations: isometric projection.
Flgure 7.
it is difficult to observe the 3-pg spot by eye, summation of the pixels at the R,value expected for H2TPPand correction for the background gave a signal considerably above the noise level for the plate. The linear correlation coefficient in this case was greater than 0.999. In this case, the limit of visual detection probably lies somewhere between 3 and 9 pg. Increased amounts of fluorescent compounds may also be measured until saturation of the camera has occurred. When this happens, suitable neutral density filters may be placed in front of the camera to cut down the intensity of light reaching it. Further studies (20) have been carried out with prostaglandins derivatized by the method of Dunges (21). Under chromatography conditions optimized for separation of prostaglandin derivatives and their metabolites, we again obtained excellent calibration profiles extending from 100 pg to 20 ng and reconfirmed the necessity for background and flat field corrections. Optical system 2 is perhaps the most useful apparatus for routine determinations of fluorescent compounds on thin-layer plates. We have demonstrated that in situ quantitation of fluorescent materials a t the picogram level is feasible. Moreover, a dynamic range of 5001 may be covered on the same plate. We believe that a number of improvements can be made to further decrease the detection limits and increase the precision of the analysis. At present, the major problem is the high blank values of the plate. Some of this blank obviously arises from undesired fluorescence of impurities in the plate; hopefully, this will be remedied by manufacturers in the future. Another contributor to the blank fluorescence of the plate arises from excitation light leaking through the analyzing filter. Specially constructed interference filters on fluorescence-free glass could eliminate much of this problem. Finally, the blank problem was greatly increased by our inability to reposition the plate after chromatography. With the present blank fluorescence, we must operate the camera a t sensitivities 2 orders of magnitude less than its ultimate sensitivity. Indeed, if blank fluorescence could be elimated entirely, we calculate that as few as lo5 molecules per spot could be detected. We shall leave for future studies the important questions of resolving and quantitating overlapping spots using optical
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1981
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system 2. Here, we merely point out that four variables provide separation of components on a two-dimensional chromatogram: wavelength of emission, wavelength of excitation, retention value along the first elution axis, and retention value along the second axis. Such data may be conveniently thought of as a tensor of rank 4. Strategies for evaluation of such data are currently under development in our laboratories. Finally, we should mention the applicability of multichannel detectors to other problems in spatially resolved analytical chemistry. Obviously one can use multichannel detectors for UV-Vis, IR, and Raman imaging of thin-layer plates as well. One exciting development in our laboratory involves use of an improved multichannel detector for two-dimensional scintillation autography. Also one can use our system for other problems as well, such as the spatial mapping of catecholamines in brain slices and the enumeration of proteins with two-dimensional gel electrophoresis.
ACKNOWLEDGMENT We are indebted to Biswanath De for his experimental assistance and to Niels Andersen and Ernest Davidson for useful discussion. We would also like to thank Leon Hershberger and Chu-Ngi Ho for their technical assistance and helpful advice. The chromatographically pure free base octaethyl-, etio-, tetraphenylporphines were the kind gift of Martin Gouterman. LITERATURE CITED (1) Zlatkis. A,: Kaiser, R. E. "High Perlorrnance Thin Layer Chromatography", Eisevier Scientific: Amsterdam. 1977. (2) Touchstone. J. C.: Sherma. J. '"Densilometry in Thin Layer Chromatography": Wiley: New York. 1979. (3) Haefelfinger. P. J. Chromfqr. 1979. 162, 215-222. (4) Gupta, R. N.: Eng, F.: Lewis. 0. AMI. Chem. 1978, 50. 197-199. (5) Doss. M. In ''Progress in Thinlayer Chromatqraphy and Related Methods": Niederwieser. A,. Pataki, G.. Ed.: Ann Arbor Science: Ann Arbor. MI. 1972. VoI. 111. pp 145-176. (6) Talmi. Y.. Ed. ACS Symp. Ssr. 1979. No. 102. (7) Johnson. 0. W.; Gladden, J. A,: Callis, J. B.: Chrlstlan. G. 0. Rev. Scl. Insfmm. 1979, 50, 118-126. (8) Warner, 1. M.: Fogany, M. P. Sheliy. 0. C. Anal. Chlm. Ada 1979. 109. 361-372. (9) Talmi. Y.: Baker. 0. C.: Jadamec. J. R.: Saner. W. A. Anal. Chem. 1978. 50. 936A-952A. (10) VoDinh. T.: Johnson. 0. J.: WinefMner. J. 0. Specfmhfm. A m . Pari A 1977. 33A, 341-345. (11) CURiS, T. G.; Sei-, W. R. J. Chromfqr. 1977. 134. 513-518. (12) Johnson, 0. W. Ph.0. Dissertation. Universw of Washington. Seanle. WA. 1978. (13) Warner. 1. M.: Callis, J. 8.: DavMsan. E. R.: Christian. G. 0. Clln. Chem. 1978, 22, 1483-1492. (14) Warner. I. M.: Davidsan. E. R.; Christian. G. 0. Anal. Chem. 1977. 49. 2155-2159. (15) Ho. C. N.: Chrislian. G. 0.: Davidson. E. R. Anal. Chem. 1978. 50, 1108-1113. (16) Ho. C. N.: Christian, G. D.: Davidsan. E. R. Anal. Chem. 1980, 52. 1071-1079. (17) McCord. T. 8.; Frankston. M. J. Appl. Opt. 1975. 14. 1437-1446. (IS) Appeiolf. C.: Davidson. E. R.. unpublished resuns. (19) Uchiyama. S.: Uchiyama. M. J. Chromatogr. 1978, 153. 135-142. (20) Andersen. N. H. submmed for publication in Proceedings of the U.S.-Italian Joint Symposium an Prostaglandins and Cardiovascular Disease. HwLon. TX. NOY 1979. (21) Dunges. W. Anal. Chem. 1973, 45963.
RECEIVED for review October 6,1980. Accepted April 24,1981. This research was supported in part by National Institutes of Health Grant GM-22311.
