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Photochemical degradation of dansyl amino acids on thin-layer chromatography plates
ANALYTICAL
BIOCHEMISTRY
Photochemical
63, 585-591
(1975)
Degradation Thin-Layer
of Dansyl
Chromatography
Amino
Acids
on
Plates
Thin-layer chromatography (tic) quantitative amino acid analysis has been intensely studied by Pataki and co-workers (1,2) who have determined the experimental conditions necessary to obtain suitable precision. D’Souza ec al. (3) have studied the photolysis of dansyl amino acids under various conditions in solution and on paper strips, and Gray (4) Seiler (5), and Zanetta et al. (6) inform on the degradation of dansyl amino acids on tic plates. These previous publications indicate that the determination of flourescent dansyl amino acids on tic plates may be subject to error through photochemica1 degradation. Accordingly we have tried to determine the importance of fluorescence decay under the conditions usually involved in quantitative densitometry. MATERIALS
Dansyl chloride was from Nutritional Biochemical Corporation, all amino acids were from Sigma Chemical Co. The solvents used in the chromatographic separations were Carlo Erba reagent grade; silica gel G was from Merck A.G., Darmstadt. Quantitative determinations were performed with a Uniscan Nester Faust densitometer to which a Beckman 10 in. recorder was adapted. In this instrument the spots are subjected to illumination by a 3660 A mercury lamp mounted on a mobile head which also carries the detector and the fluorescence emitted by the spots during the scan of the tic plate is received by the detector through a yellow filter and a collimator of 2.5 cm length. EXPERIMENTAL
Dansyl derivatives were synthesized by the usual techniques (4,7) ahhough following Spivak et al. (8) dansyl chloride concentration was 20 mM. The experimental conditions identified by Pataki and co-workers (1,2) and Seiler (5,9) as being significant to the reproducibility and precision of the analysis were carefully standardised. The detailed experimental procedure was as follows. The plates were prepared in the Iaboratory with a commercial roller. The gel layer was 0.2 mm thick. After rolling the silica gel on the plate it was allowed to stand until dry 585 Copyright @ 1975 by Academic Press, Inc. Printed All rights of reproduction in any form reserved.
in the United
States.
586
SHORT
COMMUNICATIONS
as judged by eye and then left overnight in a dessicator. The plates were not activated. Amounts varying from 1 to 8 nmoles of amino acid in 5 ,ul acetone: 2 M acetic acid (250:20) were placed on the plates. The solvent systems used for the chromatographic runs are given below: Chloroform: methanol: acetic acid (75 : 20 : 5) for dansyl glutamic acid and dansyl glycine, following Seiler and Wiechmann ( 10). Chloroform : methanol : ammonia (75 : 20 : 5) for dansyl histidine. Toluene : pyridine : acetic acid (150 : 50 : 3.5) for dansyl proline according to Zanetta et al. (6). The chromatographic run took place in the dark. Once the solvent had run IO cm from the seed spot the run was interrupted and the plates were dried in the dark with a warm air blower for exactly 15 min; they were then equilibrated with the laboratory air by standing for exactly 15 min, by this time they had reached room temperature. They were then immediately read on the densitometer. Initial time was taken as the time of the first reading of a spot. RESULTS
Quantitative correlation of dansyl amino acid quantity vs area of the densitogram peak is shown in Table 1 for three amino acids. The calibration was further extended to 15 runs (not included in Table 1); the average correlation coefficient was 0.97 with a standard deviation of 0.03.
TABLE QUANTITATIVE
1
OF DANSYL AMINO ACID AND AREA OF THE DENSITOGRAM PEAK
CORRELATION
BETWEEN
AMOUNT
Peak area dansyl amino acid”
Correlation coefficient”
NC
Dansyl glutamic acid
0.90 0.39
0.94 0.97
5 .5
Dansyl histidine
0.89 0.65
0.96 0.99
6 6
Dansyl proline
1.26 1.02
1.0 1.0
4 5
Dansyl amino acid
n The slope of the regression line varies according to the gain setting of the densitometer. b Correlation coefficient defined as r = x
r3E+/
in nmoles. e Number of experimental points for each line.
n,with y = area and x = amount
587
SHORT COMMUNICATIONS TABLE VALUES
OF THE FLUORESCENCE
Dansyl amino acid
-
Dansyl Dansyl Dansyl Dansyl
k
~~~
glycine proline glycine glycine
(pass-‘)
2 DECAY
k/2.30
0.07 0.05 0.04 0.04
0.04 + 0.01 0.03 k 0.01
CONSTANT
(mini)
Number of complete runs”
* 0.02 k 0.02 -+ 0.02 * 0.01
9 3 lb I<
I’ Each run consisted of six experimental points. *xcTest of reproducibility of results after different air equilibration times in the dark; b15 min; “35 min after b on an equal amount spotted on the same plate (see Fig. 3 and text).
Figure I illustrates the photodegradation effect of successive readings of the same spot. Very significant loss of fluorescence is observed. Figure 2 shows that the disappearance of fluorescence follows apparent first order kinetics when time is expressed as passes of the mercury lamp over the spot. To obtain approximate first order rate constants expressed in min-*, from the experimental data, we measured the time
048
:j\
054
039
048
024
022
008
030
02a
013
056
052
n 033
032
L6 012
0.60
0.63
036
036
015
068
063
041
038
021
3
%Moles
12
10
8
75
6
FIG. 1. Recording of successive readings of a plate spotted with increasing amounts of dansyl glycine. Numbers under the peaks refer to area in cm’.
588
SHORT log A (arbitrary
011
00
COMMUNICATIONS
umts)
5-
cc3 -J-
-oD!
5-
FIG. 2. Disappearance of fluorescence of dansyl glycine as a function of number of readings of the same spot. The straight line corresponds to the equation log A = -0.02 pass-’ + 0.10. Regression coefficient = 0.99.
during which the densitometer head was this allows conversion of degradation minute. The results are shown in Table that the rate of fluorescence decay is glycine and for dansyl proline.
over the spot during the scan; per pass to degradation per 2. It is interesting to observe not very different for dansyl
Ftc. 3. Dansyl glycine fluorescence. Cl) Measurements on the first spot: (0). measurements on the second spot (see text).
SHORT
589
COMMUNICATIONS
TABLE FLUORESCENCE OF DANSYL PROLINE COMPARED TO PLATES KEPT
3 ON PLATES UNCOVERED
EXPOSED TO uv LIGHT IN THE DARK
Time (min)
% Fluorescence remaining on the dark uncovered plates
% Fluorescence remaining on exposed plates
0 25 930
100 62 24
100 4.5 0
To verify the values obtained and to test their repeatability after different air equilibration times the following experiment was performed: two equal quantities of dansyl glycine were spotted on the same plate. The plate was chromatographed and dried according to the procedure described above. The first spot was illuminated for varying intervals of time and the fluorescence was measured after each interval. This first determination took approximately 20 min. Immediately after the first kinetic run was finished the rate of fluorescence decay of the second spot was determined. The experimental points obtained in both experiments are shown in Fig. 3. The values of the rate constants calculated from each set of data are coincident. They are included in Table 2. This experiment was repeated with dansyl proline with essentially identical results. To compare the photodegradation with the nonilluminated fluorescence decay of dansyl amino acids on uncovered silica gel plates (4-6) two plates were spotted with equal amounts of dansyl proline and read at equal times, one was illuminated while the other was kept uncovered in the dark. The results are shown in Table 3. The degradation percentage which we have found is coincident with Zanetta’s result (6). Table 4 shows the result of the following experiment. Two different plates were spotted with equal amounts of dansyl glycine. After the usual chromatographic run to separate the dansyl amino acid from unreacted dansylic acid, both plates were dried and equilibrated according to our standardised conditions. One spot was then subjected to 2 hr illumination under the desitometer head while the other was kept uncovered in the dark. After this treatment both plates were spotted with a TABLE FLUORESCENCE
REGAIN
AND
4
PHOTODEGRADATION
IN DANSYL
GLYCINE
Initial quantity (nmoles)
Treatment
Quantity remaining after second dimension chromatography (nmoles)
4.0 4.0
2 hr uv 2 hr dark, uncovered
0 4
590
SHORT
COMMUNICATIONS
quantity of dansyl glycine equal to the amount initially used. They were then chromatographed in a second direction perpendicular to the first. The plates were then dried, air equilibrated, and read on the densitometer. The plate which had been illuminated showed only the dansyl glycine spot of the standard which had been added before the second chromatography. The plate which had initially been left 2 hr uncovered in the dark showed both the spot of the initial dansyl glycine and that of the second dimension standard. The quantity of dansyl glycine remaining after the dark-uncovered treatment was roughly 100% of the initial amount. The following results follow from our experimental study: Quantitative estimates of dansyl amino acids may be seriously in error unless the exposure time to uv light is very nearly the same for each spot including of course the standards. There is definite experimental evidence of a photochemical degradation of dansyl amino acids on silica gel plates. The fluorescence of a spot, though also time dependent on nonilluminated uncovered plates (Table 3), may be restored by a purely physical treatment [Table 4 and Seiler (5)]. Fluorescence decay under uv light is probably a complex phenomenon comprising at least two distinct processes, actual photochemical decomposition and a reversible purely physical effect. For time intervals as long as half an hour the decay may quite accurately be described by a kinetic equation of the first order in the dansyl amino acid quantity, the actual value of the apparent rate constant depending strongly on the standardization procedure adopted in preparing tic plates for densitometry. The rate of fluorescence decay on dark uncovered plates is smaller than that observed under uv light, but the interference of this effect on fluorescence densitometry is negligible if the readings of unknown and standard spots are recorded in close succession. Exposing a spot for 30 set to the uv light source of a densitometer may reduce the fluorescence to approximately 90% of its initial value; multiple pass readings may be corrected using the kinetic constants corresponding to standardization conditions. It is therefore clear that due attention must be given to photodegradation effects if the dansyl amino acid tic densitometric method is expected to attain an accuracy comparable to other amino acid analytical methods. REFERENCES 1. PATAKI, G., AND WANG, K. T. (1968) J. Chromatog. 37, 499. 2. ZURCHER, H., PATAKI, G.. BORKO, J., AND FREI, W. R. (1969) .I. Chromatog. 3. D’SOUZA, L., BHATT, K., MADAIAH, M., AND DAY, R. A. ( 1970) Arch. Biophys. 141, 690.
43, 457. Biochem.
SHORT COMMUNICATIONS 4.
591
W. R. (1967) in Methods in Enzymology (Hirs, C. H. W.. ed.), Vol. 1 I, p. 139, Academic Press, New York. 5. SEILER, N. (1970) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 18, p. 259, Interscience, New York. 6. ZANETTA, J. P., VINCENDON, G., MANDEL, P., AND COMBOS, G. (1970) J. Chromatog. 51, 441. 7. MORSE, D., AND HORECKER, B. L. (1966) Anal. Biochem. 14, 429. 8. SPIVAK, V. A., SHCHERBUKHIN, V. V., ORLOV, V. M., AND VARSHAVSKY, J. A. M. (197 I) Anal. Biochem. 39, 27 I. 9. SEILER, N., AND WIECHMANN, M. (1966) Z. Anal. Chem. 220, 109. IO. SEILER, N., AND WIECHMANN, J. (1964) Experientia 20, 559. GRAY,
MARIA ISABEL POUCHAN EDUARDO J. PASSERON Research and Development Division Laboratorio ELEA S.A. Saladillo 2468 Buenos Aires, Argentina Received February I I. 1974; accepted July I I, 1974
BIOCHEMISTRY
Photochemical
63, 585-591
(1975)
Degradation Thin-Layer
of Dansyl
Chromatography
Amino
Acids
on
Plates
Thin-layer chromatography (tic) quantitative amino acid analysis has been intensely studied by Pataki and co-workers (1,2) who have determined the experimental conditions necessary to obtain suitable precision. D’Souza ec al. (3) have studied the photolysis of dansyl amino acids under various conditions in solution and on paper strips, and Gray (4) Seiler (5), and Zanetta et al. (6) inform on the degradation of dansyl amino acids on tic plates. These previous publications indicate that the determination of flourescent dansyl amino acids on tic plates may be subject to error through photochemica1 degradation. Accordingly we have tried to determine the importance of fluorescence decay under the conditions usually involved in quantitative densitometry. MATERIALS
Dansyl chloride was from Nutritional Biochemical Corporation, all amino acids were from Sigma Chemical Co. The solvents used in the chromatographic separations were Carlo Erba reagent grade; silica gel G was from Merck A.G., Darmstadt. Quantitative determinations were performed with a Uniscan Nester Faust densitometer to which a Beckman 10 in. recorder was adapted. In this instrument the spots are subjected to illumination by a 3660 A mercury lamp mounted on a mobile head which also carries the detector and the fluorescence emitted by the spots during the scan of the tic plate is received by the detector through a yellow filter and a collimator of 2.5 cm length. EXPERIMENTAL
Dansyl derivatives were synthesized by the usual techniques (4,7) ahhough following Spivak et al. (8) dansyl chloride concentration was 20 mM. The experimental conditions identified by Pataki and co-workers (1,2) and Seiler (5,9) as being significant to the reproducibility and precision of the analysis were carefully standardised. The detailed experimental procedure was as follows. The plates were prepared in the Iaboratory with a commercial roller. The gel layer was 0.2 mm thick. After rolling the silica gel on the plate it was allowed to stand until dry 585 Copyright @ 1975 by Academic Press, Inc. Printed All rights of reproduction in any form reserved.
in the United
States.
586
SHORT
COMMUNICATIONS
as judged by eye and then left overnight in a dessicator. The plates were not activated. Amounts varying from 1 to 8 nmoles of amino acid in 5 ,ul acetone: 2 M acetic acid (250:20) were placed on the plates. The solvent systems used for the chromatographic runs are given below: Chloroform: methanol: acetic acid (75 : 20 : 5) for dansyl glutamic acid and dansyl glycine, following Seiler and Wiechmann ( 10). Chloroform : methanol : ammonia (75 : 20 : 5) for dansyl histidine. Toluene : pyridine : acetic acid (150 : 50 : 3.5) for dansyl proline according to Zanetta et al. (6). The chromatographic run took place in the dark. Once the solvent had run IO cm from the seed spot the run was interrupted and the plates were dried in the dark with a warm air blower for exactly 15 min; they were then equilibrated with the laboratory air by standing for exactly 15 min, by this time they had reached room temperature. They were then immediately read on the densitometer. Initial time was taken as the time of the first reading of a spot. RESULTS
Quantitative correlation of dansyl amino acid quantity vs area of the densitogram peak is shown in Table 1 for three amino acids. The calibration was further extended to 15 runs (not included in Table 1); the average correlation coefficient was 0.97 with a standard deviation of 0.03.
TABLE QUANTITATIVE
1
OF DANSYL AMINO ACID AND AREA OF THE DENSITOGRAM PEAK
CORRELATION
BETWEEN
AMOUNT
Peak area dansyl amino acid”
Correlation coefficient”
NC
Dansyl glutamic acid
0.90 0.39
0.94 0.97
5 .5
Dansyl histidine
0.89 0.65
0.96 0.99
6 6
Dansyl proline
1.26 1.02
1.0 1.0
4 5
Dansyl amino acid
n The slope of the regression line varies according to the gain setting of the densitometer. b Correlation coefficient defined as r = x
r3E+/
in nmoles. e Number of experimental points for each line.
n,with y = area and x = amount
587
SHORT COMMUNICATIONS TABLE VALUES
OF THE FLUORESCENCE
Dansyl amino acid
-
Dansyl Dansyl Dansyl Dansyl
k
~~~
glycine proline glycine glycine
(pass-‘)
2 DECAY
k/2.30
0.07 0.05 0.04 0.04
0.04 + 0.01 0.03 k 0.01
CONSTANT
(mini)
Number of complete runs”
* 0.02 k 0.02 -+ 0.02 * 0.01
9 3 lb I<
I’ Each run consisted of six experimental points. *xcTest of reproducibility of results after different air equilibration times in the dark; b15 min; “35 min after b on an equal amount spotted on the same plate (see Fig. 3 and text).
Figure I illustrates the photodegradation effect of successive readings of the same spot. Very significant loss of fluorescence is observed. Figure 2 shows that the disappearance of fluorescence follows apparent first order kinetics when time is expressed as passes of the mercury lamp over the spot. To obtain approximate first order rate constants expressed in min-*, from the experimental data, we measured the time
048
:j\
054
039
048
024
022
008
030
02a
013
056
052
n 033
032
L6 012
0.60
0.63
036
036
015
068
063
041
038
021
3
%Moles
12
10
8
75
6
FIG. 1. Recording of successive readings of a plate spotted with increasing amounts of dansyl glycine. Numbers under the peaks refer to area in cm’.
588
SHORT log A (arbitrary
011
00
COMMUNICATIONS
umts)
5-
cc3 -J-
-oD!
5-
FIG. 2. Disappearance of fluorescence of dansyl glycine as a function of number of readings of the same spot. The straight line corresponds to the equation log A = -0.02 pass-’ + 0.10. Regression coefficient = 0.99.
during which the densitometer head was this allows conversion of degradation minute. The results are shown in Table that the rate of fluorescence decay is glycine and for dansyl proline.
over the spot during the scan; per pass to degradation per 2. It is interesting to observe not very different for dansyl
Ftc. 3. Dansyl glycine fluorescence. Cl) Measurements on the first spot: (0). measurements on the second spot (see text).
SHORT
589
COMMUNICATIONS
TABLE FLUORESCENCE OF DANSYL PROLINE COMPARED TO PLATES KEPT
3 ON PLATES UNCOVERED
EXPOSED TO uv LIGHT IN THE DARK
Time (min)
% Fluorescence remaining on the dark uncovered plates
% Fluorescence remaining on exposed plates
0 25 930
100 62 24
100 4.5 0
To verify the values obtained and to test their repeatability after different air equilibration times the following experiment was performed: two equal quantities of dansyl glycine were spotted on the same plate. The plate was chromatographed and dried according to the procedure described above. The first spot was illuminated for varying intervals of time and the fluorescence was measured after each interval. This first determination took approximately 20 min. Immediately after the first kinetic run was finished the rate of fluorescence decay of the second spot was determined. The experimental points obtained in both experiments are shown in Fig. 3. The values of the rate constants calculated from each set of data are coincident. They are included in Table 2. This experiment was repeated with dansyl proline with essentially identical results. To compare the photodegradation with the nonilluminated fluorescence decay of dansyl amino acids on uncovered silica gel plates (4-6) two plates were spotted with equal amounts of dansyl proline and read at equal times, one was illuminated while the other was kept uncovered in the dark. The results are shown in Table 3. The degradation percentage which we have found is coincident with Zanetta’s result (6). Table 4 shows the result of the following experiment. Two different plates were spotted with equal amounts of dansyl glycine. After the usual chromatographic run to separate the dansyl amino acid from unreacted dansylic acid, both plates were dried and equilibrated according to our standardised conditions. One spot was then subjected to 2 hr illumination under the desitometer head while the other was kept uncovered in the dark. After this treatment both plates were spotted with a TABLE FLUORESCENCE
REGAIN
AND
4
PHOTODEGRADATION
IN DANSYL
GLYCINE
Initial quantity (nmoles)
Treatment
Quantity remaining after second dimension chromatography (nmoles)
4.0 4.0
2 hr uv 2 hr dark, uncovered
0 4
590
SHORT
COMMUNICATIONS
quantity of dansyl glycine equal to the amount initially used. They were then chromatographed in a second direction perpendicular to the first. The plates were then dried, air equilibrated, and read on the densitometer. The plate which had been illuminated showed only the dansyl glycine spot of the standard which had been added before the second chromatography. The plate which had initially been left 2 hr uncovered in the dark showed both the spot of the initial dansyl glycine and that of the second dimension standard. The quantity of dansyl glycine remaining after the dark-uncovered treatment was roughly 100% of the initial amount. The following results follow from our experimental study: Quantitative estimates of dansyl amino acids may be seriously in error unless the exposure time to uv light is very nearly the same for each spot including of course the standards. There is definite experimental evidence of a photochemical degradation of dansyl amino acids on silica gel plates. The fluorescence of a spot, though also time dependent on nonilluminated uncovered plates (Table 3), may be restored by a purely physical treatment [Table 4 and Seiler (5)]. Fluorescence decay under uv light is probably a complex phenomenon comprising at least two distinct processes, actual photochemical decomposition and a reversible purely physical effect. For time intervals as long as half an hour the decay may quite accurately be described by a kinetic equation of the first order in the dansyl amino acid quantity, the actual value of the apparent rate constant depending strongly on the standardization procedure adopted in preparing tic plates for densitometry. The rate of fluorescence decay on dark uncovered plates is smaller than that observed under uv light, but the interference of this effect on fluorescence densitometry is negligible if the readings of unknown and standard spots are recorded in close succession. Exposing a spot for 30 set to the uv light source of a densitometer may reduce the fluorescence to approximately 90% of its initial value; multiple pass readings may be corrected using the kinetic constants corresponding to standardization conditions. It is therefore clear that due attention must be given to photodegradation effects if the dansyl amino acid tic densitometric method is expected to attain an accuracy comparable to other amino acid analytical methods. REFERENCES 1. PATAKI, G., AND WANG, K. T. (1968) J. Chromatog. 37, 499. 2. ZURCHER, H., PATAKI, G.. BORKO, J., AND FREI, W. R. (1969) .I. Chromatog. 3. D’SOUZA, L., BHATT, K., MADAIAH, M., AND DAY, R. A. ( 1970) Arch. Biophys. 141, 690.
43, 457. Biochem.
SHORT COMMUNICATIONS 4.
591
W. R. (1967) in Methods in Enzymology (Hirs, C. H. W.. ed.), Vol. 1 I, p. 139, Academic Press, New York. 5. SEILER, N. (1970) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 18, p. 259, Interscience, New York. 6. ZANETTA, J. P., VINCENDON, G., MANDEL, P., AND COMBOS, G. (1970) J. Chromatog. 51, 441. 7. MORSE, D., AND HORECKER, B. L. (1966) Anal. Biochem. 14, 429. 8. SPIVAK, V. A., SHCHERBUKHIN, V. V., ORLOV, V. M., AND VARSHAVSKY, J. A. M. (197 I) Anal. Biochem. 39, 27 I. 9. SEILER, N., AND WIECHMANN, M. (1966) Z. Anal. Chem. 220, 109. IO. SEILER, N., AND WIECHMANN, J. (1964) Experientia 20, 559. GRAY,
MARIA ISABEL POUCHAN EDUARDO J. PASSERON Research and Development Division Laboratorio ELEA S.A. Saladillo 2468 Buenos Aires, Argentina Received February I I. 1974; accepted July I I, 1974