Quantitative Aspects of Spectrodensitometry of Thin Layer Chromatograms Joseph C. Touchstone, Sidney S. Levin, and Taras Murawec The Steroid Laboratory, Department of Obstetrics and Gynecology and Harrison Department of Surgical Research, School of Medicine, Uniaersity of Pennsylvania, Philadelphia, Pa. I9104
A survey of operating parameters for optimal quantitative spectrodensitometry of thin layer chromatograms (TLC) is described. Transmittance is more sensitive than reflectance for absorbance, fluorescence quenching, and fluorometric modes of quantitation of TLC. Linear calibration curves were obtained at the nanogram level using a double beam densitometer with absorbance measurements of azo derivatives of estrogens and with natural fluorometric determinations of Rhodamine-B, 2,7-dichlorofluorescein, and quinine sulfate. Linearity is dependent upon proper choice of scanning wavelengths. Absorbance and fluorescence characteristics for quinine sulfate on silica gel layers are different from those found in sulfuric acid or alcohol. Some of the parameters of preparation of the TLC are discussed. As OBSERVED in this and other laboratories ( I ) , quantitative thin layer chromatography (TLC) has been limited by tedious elution techniques, and by the faster but less accurate visualization methods. With the recent advent of reliable spectrodensitometers, in situ measurements of TLC have become possible. However, there is some confusion as to the parameters of direct densitometry due to lack of information based on theoretical, as well as practical applications. This paper describes the in situ quantitative spectrodensitometry of materials separated by TLC and includes a comparison of transmittance and reflectance methods in terms of sensitivity. The results indicate that with proper usage, quantitation at the nanogram level can be both reproducible and reliable. EXPERIMENTAL
Apparatus. The double beam spectrodensitometer was a Schoeffel Model 3000 instrument provided with photomultiplier tubes for both transmission and reflectance. A mercury-xenon lamp was used. The instrument was provided with interference wedge monochromators (400-700 nm) between the TLC plate and the photomultiplier. The fluorescence accessory had a Corning 3-74 filter passing only the light above 400 nm. This, along with the adjustable interference wedge monochromator assured that the measured signal was the emission phenomenon and not transmitted from other spectral components contained in the source. The densitometer included an analog computer which renders and converts photomultiplier signals to provide the following parameters: (1) a linear double beam density readout up to O.D. 3 according to the equation Absorbance or O.D. Difference
= =
Reference Signal or Sample Signal log Reference Signal log Sample Signal (1) log
(2) a linear single beam density readout where the reference signal is replaced by electrical reference signal (1) E. Sawicki, E. W. Stanley, and H. Johnson, Microchem. J., 8, 257 (1964).
858
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
Absorbance or O.D. Difference
=
log
Electrical Reference (2) Sample Signal
(3) linear signal readout for the recording of deviation from a 100% condition as encountered in transmission and reflection measurements Output = T % or Output
=
R %
(3)
(4) linear emission readout with adjustable background subtraction reading; only the signal produced emission (5) linear ratio readout output =
Sample Signal Reference Signal
(4)
A range control for 100-fold attenuation in six steps is provided. Materials and Methods. The thin layer plates, silica gel G and GF, 250-p thick were obtained from Photo Scientific Research, Chester, Pa. These were scored into IO-mm lanes with a Schoeffel Scoring Machine. A 10-pl Hamilton syringe was used for spotting. Chromatograms were developed in 7 X 22 X 22 cm glass tanks containing 100 ml of solvent as indicated below. All plates were developed until the solvent reached 1 cmfrom the top; Rhodamine B and 2,7-dichlorofluorescein, 30 % propionic acid in isopropanol; quinine sulfate, 2 developments in the same direction with 20% methanol in benzene; azotized Estrogens (synthesized as described previously) (21, 20 % benzene in butylacetate; corticosterone (B), 17-hydroxydesoxycorticosterone (S), progesterone (P) 17-hydroxyprogesterone (17-P) and desoxycorticosterone (DOC), 20 % acetone-isopropyl ether (3). Since double beam TLC scanners have only been introduced recently and measurements of this highly scattering media require different treatment than liquids in double beam spectrophotometry, the following discussion appears apropos. Spectral characteristics of the illuminating source and the response curve of the photomultipliers (Figure 1) show the mercury bands of the xenon-mercury lamp at 312/313 and 365/366. The band at 365/366 nm, is considerably more intense than other wavelengths. The xenon lamp has a curve of intensity rising gradually from 200 to a hump at approximately 500 nm and then leveling off. The photomultipliers used in this work had a maximum response to light of 380 nm. Before this wavelength there was a shoulder, then a continual decline in response. Above this wavelength the response gradually declined and became almost zero at 700 nm. Figure 2 illustrates the importance, in quantitative work, of operating at the peak of absorption of excitation bands. In the case of 2,7-dichlorofluorescein scanning away from the maximum of excitation (315 nm) resulted in nonlinear curves. Scanning at 315 nm gave a linear calibration curve. Peak areas calculated from the height times the width at half height were corrected for the appropriate instrument attenuation. All values are reported using the same gain. ~
~~
~
(2) J. C. Touchstone, A. K. Balin, and P. Knapstein, Steroids, 13, 199 (1969). (3) S. S. Levin, J. C. Touchstone, and T. Murawec, J. Chromatogr., 42, 129 (1969).
-
Activation
Emission
Wavelength in nm.
Figure 1. Activation and emission spectra for quinine sulfate on silica gel G as obtained on the Schoeffel Spectrodensitometer Emission obtained by activation 312/13 nm. Activation spectrum obtained at emission 450 nm. The fine line shows the intensitylines of the light source. XeHg The range control was found to be linear, so correction was valid. Base-line change was corrected, during each wavelength change. All peak area calculations reported in this paper represent the average of 3-5 determinations. All quantitative results are based on determinations taken at the peak of absorption spectra (X max). Scoring to give IO-mm parallel lanes limits the sample travel within lanes since there is no migration across the scored lines. The entire width is thus scanned by the 10-mm beam of the densitometer. Samples were applied on alternate lanes. The empty lanes served as reference for the double beam instrument. When the zone between the scored lines is not covered by the spot, the energy passing through or reflected from the plate must be averaged by the photomultiplier. Since the outer edge of the spot will show less absorption than the inner portion, this could be a source of error which will increase as the ratio of spot width to lane width decreases. Sample Application. Application is a factor in linearity of the calibration curves. Streaked samples provide more uniform dispersion across the band. Highly concentrated areas of the applied spot should be avoided since the solvent tends to flow around rather than through a spot, resulting in uneven sample diffusion. Spot travel with an Rf of at least 0.25 should be a prerequisite for quantitation. High Rf (over 0.75) values may result in too dispersed spots that could be contaminated by the solvent front. Solvent systems used in this work provided separations in which the spots were kept within the middle half of the plate. This is in agreement with Thomas et d., who reported that areas were more constant within the R, range 0.3-0.8 ( 4 ) . (4) A. E. Thomas, J. E. Scharoun, and H. Ralston, J . Amer. Oil Cltem. Soc., 42, 790 (1965).
8-
76hi- 5-
E
s 0
e
4-
P
- 5
4
2-
//
0
I-
"
5
10
20
15
25
50 nq.
Amount of 27-dichlorofluorercsin in spot
Figure 2. Change of linearity due to change in exciting wavelength 0
310nm, A 300nm,
W
320 nm, 0 290nm
RESULTS
The reproducibility of spotting and scanning was determined by scanning (after development) a single zone of azotized estriol (0.5 pg) 10 times. The standard deviation was 0.3 for the recorded peak having a height of 141 mm. A plate of silica gel G was streaked in 8 lanes with the same amount of the estriol derivative and each lane scanned twice after development. The standard deviation was 0.5 %. Table I gives a comparison of results obtained with a given ANALYTICAL CHEMISTRY, VOL. 43, NO. 7 , JUNE 1971
859
Transmitted Light Silica Gel F 254
Table I. Spotted us. Streaked Sample Application [peak area (mm2)] Spotted" Streakeda Pg EI EP E3 El E1 E3 0.5 630 600 625 630 650 600 0.4 555 540 575 546 558 630 420 0.3 396 470 400 426 400 295 0.2 3 50 322 322 342 270 Azotized estrone (El), estradiol-17p (E2)and estriol (E3)were separated on scored silica gel G plates and scanned after development. @
Table 11. Transmittance us. Reflectance Determination of Azotized Estrogens Double Beam Mode, 410 nm [peak area (mm2)] Steroid , Estrone Estradiol-17P Estriol
Single Beam Reflected Light Silica Gel F 2 5 4
d Single Beam
Pg
Ta
Rb
T
1260 490 1260 0.4 980 1078 496 0.3 760 350 882 0.2 532 588 238 0.1 216 90 252 e T = transmittance. R = reflectance. Average T/R & std dev = 2.47 =k 0.30. 0.5
R
T
R
490 434 322 217 128
1080 990 780
441 420 258 200 112
550
232
Dual Beam
e6
beam operation can be seen in Figure 3. Factors affecting the base line of the densitometer include light scattering, variation in matrix thickness, and solvent impurities. The last results from contaminants either eluted from the absorbent plate with the solvent front or present in the solvent. The base line is more stable with double beam scanning in both transmission and reflectance modes of operation (Figure 3). Transmittance us. Reflectance in Quantitation of Absorbance in the Visible Range. Estrone, estradiol-17/3, and estradiol were coupled with Fast Violet Salt B(6-benzoylamino-4-methoxy-5-toluidine,diazonium salt, Borden Chemical Co., Philadelphia, Pa.) and separated by TLC. The yellow colored azo derivatives separated well (Figure 4) on silica gel layers and can be quantitated in amounts as low as 20 ng. A comparison was made between reflectance and transmittance in double beam scanning of these chromatograms. Table I1 shows that the transmission mode is
Figure 3. Comparison of single beam us. double beam scanning of five steroids (left to right) B, S, 17-P, P , and DOC separated by TLC This illustrates the base-line distorting effect of solvent front on the base line of single beam scans amount of colored azotized estrone, estradiol-1 7/3, and estriol when spotted or streaked on individual thin layer plates. In contrast to unscored plates, there appears t o be little difference between the results when obtained on scored plates if the R f is sufficient for diffusion of the spot across the lane. Dual Beam GS. Single Beam Operation. Dual beam instrumentation compensated for much of the background produced by the variables of TLC and is prerequisite for the quantitation of compounds at low concentration. The comparison of the effect of a TLC plate by both single and dual
Chart speed = 4 "/m in. 2 x plate speed
d
P
Figure 4. Densitometry of azotized estrone, estradio1-17@,and estriol. Each peak represents 0.5 bg steroid (410 nm)
e
x
9
5
Q
P
Effect of R f on spot characteristics a n d peak area 860
ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
Steroid
-
P
F1 436 0.4 1078 0.8 1596 1.2 Fl/Abs i std dev 1 . 8 f 0.26 1.9 i 0.45 2.1 f 0.25 1 . 5 f 0.20 1 . 6 r t 0.21 4 Activating light was 250 nm. (This wavelength, although not maximal for absorbance or excitation, gave maximal quenching.) Abs = absorbance on Silica Gel G (nonfluorescent plates). c F1 = fluorescence quenching in Silica Gel GF25a plates. l.(g
Absb 253 372 663
Table 111. Absorbance us. Fluorescence Quenching. Quantitation of Five Steroids (Reflectance Only). Both Photomultiplier Slits 1.0 mm' B S DOC 17P Flc Abs F1 Abs F1 Abs F1 364 504 347 280 240 334 404 1078 558 988 426 990 792 396 882 1344 684 1424 668 1190 1108
Abs 312 646 890
Table IV. Transmission us. Reflectance Fluorescence Quenching" (peak areas mm2),Both Photomultiplier Slits 1.0 mm Steroid B S DOC 17P P l.(g Tb Re T R T R T R T R 1105 750 1118 754 592 597 916 847 495 910 2.0 422 357 678 374 645 592 260 536 315 1.o 5 20 3 26 539 350 539 495 232 490 234 448 224 0.8 266 416 270 151 442 456 396 175 400 180 0.6 142 156 145 218 91 252 80 167 70 0.4 202 T/R f std dev 2.2 + 0.52 1.9 0.29 2.3 Z!C 0 . 7 3 1 . 4 f 0.16 1 . 6 f 0.09 (This wavelength, although not maximal for absorbance or excitation, gave maximal quenching.) a Activating light was 250 nm. T = transmittance. cR = reflectance.
more sensitive (2.47 f 0.30) in determinations of absorbance of the colored zones. We have previously described the use of this method for quantitation of estriol in biological samples (5). Detection of Compounds Which Absorb Ultraviolet Light. Absorption of ultraviolet light can be determined on plates with or without fluorescent media. However, this method is limited to those compounds which are strongly absorbent in the ultraviolet region. For fluorescence quenching, the compounds must absorb near the wavelength necessary to activate the phosphor. Migrants that absorb ultraviolet light in the range of the activating light of the phosphor will prevent fluorescence or phosphorescence of the plate region into which they migrated to a degree indicative of the quantity present. Table I11 shows the difference between absorbance on plain plates and plates containing phosphor for 5 steroids separated and scanned by reflectance. The steroids were streaked on silica gel and silica gel GFz5., plates. Fluorescence quenching on silica gel GF254 plates appeared to be more sensitive than absorbance on plain silica gel. Table IV gives a comparison of the transmission us. reflectance studies of the same 5 steroids in fluorescence quenching. The mean ratio between transmittance and reflectance in this mode of determination was 1.88. These determinations were done at gain equivalent to full scale recorder deflection of 0.2 optical density unit. Therefore, the minimum amount of steroid as determined by fluorescence quenching would be 0.1 pg in a 2 X 10 mm streak. The limit for other compounds will be dependent on the extinction coefficient. Linear calibration curves for steroids have been obtained using this method of determination (6). ( 5 ) J. C . Touchstone,A. K.
Balin, and T. Murawec, J . Chromatogr. Sci., 8, 81 (1970). (6) P. Knapstein, J. C . Touchstone, P. Menzel, and G. W. Oertel, J. Chromafogr.,44, 190 (1969).
Table V. Effect of Medium on Spectral Characteristics of Quinine Sulfate Optical Maxima of Spectra (nm) Medium Absorption Excitation Emission 0.1N HzSOa ( 7 ) 250, 315, 350 340, 390 458, 466 Glycerol ( 7 ) 315, 363 340, 390 450 Alcohol 280, 333 337 370 Silica gel 315, 365 450
Determination of Fluorescence. The nature of the solution medium can affect the absorption maximum of dissolved compounds in solution with a Cary Model 14 spectrophotometer and in the TLC with the spectrodensitometer. As seen in Table V, quinine sulfate in silica gel thin layers showed a shift of the maximum of the absorption spectrum. The effect of the medium on absorbance and fluorescence of quinine has been studied by other authors (7). In double beam operation the wedge monochromators for both sample and reference beams are adjusted for the maximum emission passage of the energy. However, in fluorescence, energy is absent in the reference lane which results in an insufficiently illuminated reference photomultiplier that cannot be balanced against the sample photomultiplier. The reason being that the absorbance of activating energy must be treated in a logarithmic fashion while the emission at a different wavelength must be a simple linear algebraic expression. Absorption is subtractive while fluorescence is additive to the photomultiplier response. This is too complicated for the computer when the double beam mode is used, because the reference lane will show no emission. Therefore, the single beam operation was favored. The activation and emission spectra of Rhodamine B (Figure 5 ) , 2,7-dichlorofluorescein (Figure 6), and quinine sulfate (Figure 1) by both transmittance and reflectance (7) R. F. Chew, Anal. Biochem., 19, 374 (1967). ANALYTICAL CHEMISTRY, VOL. 43, NO. 7, JUNE 1971
861
E miss i o n
Activation
Activation
650
7501
550
650-
Emission
A
550-
450
450-
250 (L‘ 150
‘ ‘”1 f 3
4
400:
a
9
.z 0
d
A
550-
300-
J‘\
Reflectance
200-
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450
\
I OJ 1
I 250150-
\
1
1
I
,
1
1
1
1
1
l
l
i
460 400 500 520 540 Wavelength in nm
\
Figure 6. Activation (at emission 510 nm) and emission (at activation 310 nm) spectra for 2,7-dichlorofluorescein by reflectance and transmission determination
50I
.
I
’
I
540 560 580 600 W a v e l e n g t h i n ‘nm
260 280 300 320 340
Figure 5. Activation (at emission 570 nm) and emission (at activation 300 nm) spectra for Rhodamine B by reflectance and transmission determination by single beam operation are shown. A survey was made of the effect of wavelength on the emission in terms of intensity bands known to be present in the light sources. As seen in Figures 1,5, and 6, the activation spectra may show the effect of the high energy bands of the light source; however, peak excitation may not necessarily occur in these regions. The densitometer used in the present work will detect 0.5 and 1.O nanogram spots of Rhodamine B at 50 of maximum sensitivity of the instrument. Below this level the detection is influenced by the nature of the layer. Figure 7 shows the calibration curve for Rhodamine B determined by both transmission and reflectance. Transmittance gave higher response than reflectance measurements, i.e., it was 1.7 0.67 times more sensitive than reflectance. In contrast to results reported by others (8), these curves are linear. Janchen (9) has discussed the effect of the developing solvent on linearity of calibration curves in terms of resultant Rf differences. Figure 8 shows the calibration curve for quinine sulfate obtained by transmission. The curve obtained resulted from activation at either 312/13 or 365/66 nm. The response curves from measurement of emission by transmission at 450 nm with activation at both 312/13 and 365/66 nm were the same. Responses at 650 nm are not linear over the whole range and represent only second order trans-
*
mission (IO) which can easily be suppressed by a secondary filter. Transmission was more than twice as effective as reflectance for scanning the fluorescence. In the transmission and reflectance mode of detection, monochromatic radiation can be focused on the TL; therefore, the emitting area can be considered to function as a secondary light source. Fluorescent emission will radiate into all directions, hence placement of the emission analyzer in reference to distance from this secondary source of energy is as important as its distance from the source. Its dimensions determine the size of cone of intercepted emission energy and thereby influence the photomultiplier signal. It is quite obvious that in the transmission mode the intercepting aperture can be placed closer to the plate than in an angular restrictive management such as in the reflection. DISCUSSION
The experiments described in this paper point out some of the requirements of instrumentation necessary for the most sensitive and satisfactory operation. To date, little has been published relative to the proper mode of design and preparation of the optical equipment. The modes of operation, single beam or double beam, reflectance or transmission, will be determined not only by the nature of the compound under study, but also by the wavelengths required for scanning. An evaluation of the possibilities for quantitation of materials separated by TLC showed fluorescence measurements to be the most sensitive. Sawicki et al. ( I ) , have reported an extensive study of the spectrofluorometric characteristics of a large number of aromatic compounds. Hamman and Martin (11) have investigated the direct spectrometric quantitation of steroids
(8) E. J. Shellard, in “Quantitation Paper and Thin Layer Chromatography,” by E. J. Shellard, Ed., Academic Press, New
York, 1967, p 63. (9) D. E. Janchen, in “Quantitative Paper and Thin Layer Chromatography,” E. J. Shellard, Ed., Academic Press, New York, 1968, p 71. 862
ANALYTICAL CHEMISTRY, VOL. 43,
NO. 7, JUNE 1971
(10) Sidney Udenfriend, “Fluorescence Assay in Biology and
Medicine,” Academic Press, New York, 1962, p 111. (11) B. L. Hamman and M. M. Martin, Anal. Biochern., 20, 423 (1967).
4009
E x p a n s i o n 2x
Wavelength In nm Activation ( A ) Emission (E) 650 450
I
3500Q
650 450
3000-
E F h
2500-
t
P
$ 2000-
e
1500-
1000-
500-
1
IO
30
50
1
1
70
1
1
1
1
90 IO
1
1
1
1
1
1
1
1
1
130 150 170 190 ng
Amount of Ouininr Sulfote in Spot
6
~b
1’5
20
215
ng
Amount of Rhodamine B in spot
Figure 7. Calibration curve for serial amounts of Rhodamine B separated by TLC by both transmittance and reflectance separated on TLC. Sprayed chromatograms were scanned and accuracy and reproducibility were described. A later report (12) described problems in accuracy and reproducibility, but these may be related to difficulty in reproducibility of spraying a reagent on TLC. Goldman and Goodall (13) have investigated the theoretical possibilities of in situ quantitative analysis of the thin layer chromatograms by densitometry. They have utilized a simplified Kubelka-Munk equation (14) to express the absorption coefficient in terms of two variables, the background absorbance and the curvature of response. The experimental studies showed the validity of the KubelkaMunk theory. On theoretical grounds they indicated that quantitation by transmission with the thin layer plate was superior to reflectance. However, Stahl and his coworkers (15) state that “spectrophotometric reflectance measurements on thin layer chromatograms are much better to assess.’’ These workers also used a modified Kubelka-Munk equation to correct for the uncontrolled variables. Both of the above groups used single beam instruments and had to provide in their calculations a compensatory correction in order to provide linear calibration curves. (12) B. L. Hamman and M. M. Martin, J . Lab. Clin. Med., 71, 1028 (1968). (13) J. Goldman and R. R. Goodall, J. Chromatogr., 32, 24 (1968). (14) P. Kubelka and F. Z . Munk, Tech. Physik., 12, 593 (1931); P. Kubelka, J. Opt. SOC.Amer.., 38,448 (1948). (15) E. Stahl and H. Josh, in C . Zeiss, Publication S-50-690-E, 1968.
Figure 8. Calibration curve for quinine sulfate at emission 450 and 650 nm Results, herein, using a split beam instrument, indicate that transmission measurements can be more suitable than reflectance measurements in fluorescence and fluorescence quenching methods, as well as in measurements of absorbance of colored compounds. The use of double beam instrumentation overcomes some of the faults due to nonuniformity of layer thickness, background scatter, and sample application. The use of scored lanes on the thin layer matrix offers some advantage in that the size of the spot is controlled in one dimension in all sample applications, particularly if streaking is used. In situ quantitation offers many advantages over the older elution methods, particularly at nanogram to microgram concentrations. It should be pointed out in this context that all experiments reported here have been with samples 2 pg or less. For fluorescence determinations, nanogram amounts were used. Previous investigators worked with concentrations as high as 50 pg or more. It is difficult to obtain linear calibration curves with spots containing high concentrations of sample. ACKNOWLEDGMENT
We wish to express appreciation to Dr. Otto Rosenthal for constructive criticism of the manuscript. RECEIVEDfor review July 8, 1970. Accepted February 25, 1971. Presented in part at the Eastern Analytical Symposium, New York, N.Y., November 19, 1970. This work was supported in part by USPHS Grants HD-1199, Am-K14,013 and AM-04484. National Science Foundation Grant GB-8366 and a Grant from the Ford Foundation also supplied support.
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