- Email: [email protected]
Identification and estimation of indole analogs by gas-liquid chromatography
ANALYTICAL
lndole
13, 116-120
BIOCHEMISTRY
Identification Analogs by
(1965)
and Estimation of Gas-Liquid Chromatography
R. D. DEMOSS From
the
Department Illinois, Received
AND
VIVIAN
of
Microbiology, Urbana, Illinois April
GAGE Uniuersity
of
22, 1965
Gas-liquid chromatography (GLC) has not been employed extensively for the analysis of indole analogs. Lloyd et al. (2) examined the behavior of certain indole alkaloids on a GLC column, but the alkaloids are generally of more complex structure than those indole analogs which would be useful in studies of tryptophan biochemistry. Both Janak and Hrivnac (1) and Mossini and Vitali (3) employed somewhat higher temperatures, 2OO-225”C, and carrier gas-flow rates, 70 ml/min, the combination of which would not separate satisfactorily the simpler indole analogs we wanted to study. As will be described elsewhere, some of the indole analogs employed are acceptable as substrates for the tryptophan synthetase of Escherichia coli and the product is the corresponding tryptophan analog. In most paper, column, and thin-layer chromatographic procedures tested, the tryptophan analogs and the indole analogs often behaved indistinguishably from the parent compound, and appreciably greater analysis time was required. GLC treatment of the indole analogs, as described below, provided sufficient separation to permit identification and quantitative estimations of the indole analogs. Consequently, tryptophan analogs to be analyzed may be converted to indole analogs by enzymic degradation with tryptophanase from E. coli, and subjected to GLC. MATERIALS
AND
METHODS
Indole analogs were obtained commercially (Aldrich Chemical Co., Regis Chemical Co.). Aqueous solutions were prepared by prolonged (when necessary) shaking of a suspension of the analog at 37°C. Usually, overnight shaking was sufficient to dissolve the suspended material. To avoid, insofar as possible, artifacts due to microbial contamination, sterile flasks were employed for the preparation of solutions of analogues. GLC was performed in an Aerograph model 665 instrument fitted with 116
G-L
CHROMATOGRAPHY
OF
117
INDOLES
a hydrogen-flame ionization detector. A column, 5 ft X 1,s in., of 5% silicone oil DC-550 on Chromosorb W, hexamethyldisilazane treated, SO/l00 mesh (Wilkens Instrument and Research, Inc.) was maintained at a constant temperature as indicated, or, for identification purposes, was programed for a linear increase in temperature. The injector, with Pyrex liner, and detector temperatures were maintained at 215” and 156”C, respectively. The nitrogen carrier gas-flow rate was 15 ml/min, and the hydrogen flow rate was approximately 15 ml/min. RESULTS
AND
DISCUSSION
Table 1 lists the retention times, relative to indole, of the indole analogs studied. Two columns, packed by different operators, were employed. Relative retention times were nearly identical in the two columns. A clear identification of the analog is possible under the conditions employed. The identification may be made more emphatic by preliminary extraction procedures to concentrate the indole solution. The toluene extract of an aqueous solution may be reduced to a small volume, or dried at low temperature (about 6O”C), the residue dissolved in water, and the aqueous solution or the concentrated toluene solution subjected to GLC. The extraction procedure is not useful for quantitative estimation, owing to the appreciable volatility of most of the indole analogs. The toluene extract is not employed at lower temperatures owing to interference, by tailing of the toluene, in identification of the early emergent peaks. RELATIVE
RETENTION
TABLE TIMES
1 FOR INDOLE Column
Analog
Indole 5-Fluoroindole 1,2-Dimethylindole 2,7-Dimethylindole 2,5-Dimethylindole 5-Chloroindole 5-Methoxyindole 5-Bromoindole 5-Methoxyindole 5-Methylindole 5-Aminoindole 5-Nitroindole 5-Cyanoindole 0 Eit.her b Indole
aqueous retention
(A) or toluene (T) time was 5.5 min.
ANALOGS
SOlIl.~
te%pnCp’*
Relative retention time,” min
A A A A A A A A A T T T T
160 162 163 163 163 162 164 163 200 200 200 200 200
1.00 1.17 1.73 2.02 2.36 3.24 3.54 5.24 0.86 0.56 1.47 2.41 2.87
solutiona
were
employed.
118
DEMOSS
AND
GAGE
A programed column temperature increase permits a more convenient identification routine. Figure 1 shows a typical analysis of a mixture of
200 160 OC
160 140
0 TIME,
MINUTES
Fro. 1. Temperature programed gas-liquid chromatogram of indole analogs. A mixture of analogs was prepared in aqueous solution. The sample volume was 1.0 ~1. Initial column temperature was 140°C. Immediately upon injection of the sample, the column temperature was increased linearly at the rate of 2O/min as shown. Peak identities, with retention times (min) are: (1) indole, 6.1 i (2) 5-fluoroindole, 7.0; (3) 1,2-dimethylindole, 9.0; (4) 2,5-dimethylindole, 11.3; (5) bchloroindole, 13.9; (6) 5-bromoindole, 18.1.
analogs: they are well separated and form reasonably sharp peaks. The procedure is highly reproducible. Figure 2 relates peak height to amount of indole in l.O-~1 samples. Similar relationships were obtained with 5-fluoroindole or 5-chloroindole. The GLC method provides a useful relationship for quantitative estimation of indole and indole analogs, providing uniform sample volume is employed and providing column temperatures are chosen such that the peak to be estimated emerges with less than 6-min retention time. The peak height measurement method is less reliable if the retention time is greater than about 6 min. It is necessary to inject a constant sample volume to obtain reproducible results. The use of varying sample volume of a standard indole solution led to variable peak heights. The relationship of retention time as a function of column temperature for the halogenated indole analogs is illustrated in Fig. 3. The method described here provides a rapid and accurate estimation for indole and some of its analogs. The method has been employed in
G-L
CHROMATOGRAPHY
I 0
.oe
OF
I
I
.04 /.~g
.06 INDOLE/pI
119
INDOLES
I
I
I
.06
.I0
.I2
FIG. 2. Peak height as a function of concentration. Appropriate dilutions of indole in water were prepared such that a constant sample volume, 1.0 81, could be injected. Column temperature was 16OC.
this laboratory for following enzymic reactions involving either indole utilization or indole production. Samples of the entire reaction mixture may be injected directly, without treatment, with no apparent change in column characteristics after 1000 estimations, though it is, of course, advisable to replace the injector septum and the Pyrex injector liner periodically. SUMMARY
The qualitative and quantitative estimation of indole and a series of simple indole analogs is described. The method may be employed for the direct estimation of indole analogs in enzyme reaction mixtures.
120
DEMOSS AND GAGE
60
IO
0 120
140 160 TEMPERATURE,
180 OC
200
FIQ. 3. Retention time as a function of column temperature. Aqueous l.O-pl samples of a solution containing three analogs for temperature range 120”-16O”C, or four analogs for temperature range 170”-200°C were injected. Retention time was calculated as interval between time of injection and time at which analog in question began to enter the detector and is the mean value for at least six trials at each temperature. ACKNOWLEDGMENT This investigation was supported in part by Public Health from the National Institutes of Health.
Service grant E-2971
REFERENCES 1. LLOYD, H. A., FALES, H. M., HIQHET, P. F., VANDEN HEUVEN, WILDMAN, W. C., J. Am. Chem. Sot. 82, 3791 (1960). 2. JANAK, J., AND HRIVNAC, M., Coil. Czech. Chem. Commun. 25, 3. MOSSINI, F., AND VITALI, T., Ric. Sci. Rend. [Al 1, 244 (1961) Abstr. 57, 66175: (1962).
W. J. A., AND 1557 (1960). ; cited in Chew.
lndole
13, 116-120
BIOCHEMISTRY
Identification Analogs by
(1965)
and Estimation of Gas-Liquid Chromatography
R. D. DEMOSS From
the
Department Illinois, Received
AND
VIVIAN
of
Microbiology, Urbana, Illinois April
GAGE Uniuersity
of
22, 1965
Gas-liquid chromatography (GLC) has not been employed extensively for the analysis of indole analogs. Lloyd et al. (2) examined the behavior of certain indole alkaloids on a GLC column, but the alkaloids are generally of more complex structure than those indole analogs which would be useful in studies of tryptophan biochemistry. Both Janak and Hrivnac (1) and Mossini and Vitali (3) employed somewhat higher temperatures, 2OO-225”C, and carrier gas-flow rates, 70 ml/min, the combination of which would not separate satisfactorily the simpler indole analogs we wanted to study. As will be described elsewhere, some of the indole analogs employed are acceptable as substrates for the tryptophan synthetase of Escherichia coli and the product is the corresponding tryptophan analog. In most paper, column, and thin-layer chromatographic procedures tested, the tryptophan analogs and the indole analogs often behaved indistinguishably from the parent compound, and appreciably greater analysis time was required. GLC treatment of the indole analogs, as described below, provided sufficient separation to permit identification and quantitative estimations of the indole analogs. Consequently, tryptophan analogs to be analyzed may be converted to indole analogs by enzymic degradation with tryptophanase from E. coli, and subjected to GLC. MATERIALS
AND
METHODS
Indole analogs were obtained commercially (Aldrich Chemical Co., Regis Chemical Co.). Aqueous solutions were prepared by prolonged (when necessary) shaking of a suspension of the analog at 37°C. Usually, overnight shaking was sufficient to dissolve the suspended material. To avoid, insofar as possible, artifacts due to microbial contamination, sterile flasks were employed for the preparation of solutions of analogues. GLC was performed in an Aerograph model 665 instrument fitted with 116
G-L
CHROMATOGRAPHY
OF
117
INDOLES
a hydrogen-flame ionization detector. A column, 5 ft X 1,s in., of 5% silicone oil DC-550 on Chromosorb W, hexamethyldisilazane treated, SO/l00 mesh (Wilkens Instrument and Research, Inc.) was maintained at a constant temperature as indicated, or, for identification purposes, was programed for a linear increase in temperature. The injector, with Pyrex liner, and detector temperatures were maintained at 215” and 156”C, respectively. The nitrogen carrier gas-flow rate was 15 ml/min, and the hydrogen flow rate was approximately 15 ml/min. RESULTS
AND
DISCUSSION
Table 1 lists the retention times, relative to indole, of the indole analogs studied. Two columns, packed by different operators, were employed. Relative retention times were nearly identical in the two columns. A clear identification of the analog is possible under the conditions employed. The identification may be made more emphatic by preliminary extraction procedures to concentrate the indole solution. The toluene extract of an aqueous solution may be reduced to a small volume, or dried at low temperature (about 6O”C), the residue dissolved in water, and the aqueous solution or the concentrated toluene solution subjected to GLC. The extraction procedure is not useful for quantitative estimation, owing to the appreciable volatility of most of the indole analogs. The toluene extract is not employed at lower temperatures owing to interference, by tailing of the toluene, in identification of the early emergent peaks. RELATIVE
RETENTION
TABLE TIMES
1 FOR INDOLE Column
Analog
Indole 5-Fluoroindole 1,2-Dimethylindole 2,7-Dimethylindole 2,5-Dimethylindole 5-Chloroindole 5-Methoxyindole 5-Bromoindole 5-Methoxyindole 5-Methylindole 5-Aminoindole 5-Nitroindole 5-Cyanoindole 0 Eit.her b Indole
aqueous retention
(A) or toluene (T) time was 5.5 min.
ANALOGS
SOlIl.~
te%pnCp’*
Relative retention time,” min
A A A A A A A A A T T T T
160 162 163 163 163 162 164 163 200 200 200 200 200
1.00 1.17 1.73 2.02 2.36 3.24 3.54 5.24 0.86 0.56 1.47 2.41 2.87
solutiona
were
employed.
118
DEMOSS
AND
GAGE
A programed column temperature increase permits a more convenient identification routine. Figure 1 shows a typical analysis of a mixture of
200 160 OC
160 140
0 TIME,
MINUTES
Fro. 1. Temperature programed gas-liquid chromatogram of indole analogs. A mixture of analogs was prepared in aqueous solution. The sample volume was 1.0 ~1. Initial column temperature was 140°C. Immediately upon injection of the sample, the column temperature was increased linearly at the rate of 2O/min as shown. Peak identities, with retention times (min) are: (1) indole, 6.1 i (2) 5-fluoroindole, 7.0; (3) 1,2-dimethylindole, 9.0; (4) 2,5-dimethylindole, 11.3; (5) bchloroindole, 13.9; (6) 5-bromoindole, 18.1.
analogs: they are well separated and form reasonably sharp peaks. The procedure is highly reproducible. Figure 2 relates peak height to amount of indole in l.O-~1 samples. Similar relationships were obtained with 5-fluoroindole or 5-chloroindole. The GLC method provides a useful relationship for quantitative estimation of indole and indole analogs, providing uniform sample volume is employed and providing column temperatures are chosen such that the peak to be estimated emerges with less than 6-min retention time. The peak height measurement method is less reliable if the retention time is greater than about 6 min. It is necessary to inject a constant sample volume to obtain reproducible results. The use of varying sample volume of a standard indole solution led to variable peak heights. The relationship of retention time as a function of column temperature for the halogenated indole analogs is illustrated in Fig. 3. The method described here provides a rapid and accurate estimation for indole and some of its analogs. The method has been employed in
G-L
CHROMATOGRAPHY
I 0
.oe
OF
I
I
.04 /.~g
.06 INDOLE/pI
119
INDOLES
I
I
I
.06
.I0
.I2
FIG. 2. Peak height as a function of concentration. Appropriate dilutions of indole in water were prepared such that a constant sample volume, 1.0 81, could be injected. Column temperature was 16OC.
this laboratory for following enzymic reactions involving either indole utilization or indole production. Samples of the entire reaction mixture may be injected directly, without treatment, with no apparent change in column characteristics after 1000 estimations, though it is, of course, advisable to replace the injector septum and the Pyrex injector liner periodically. SUMMARY
The qualitative and quantitative estimation of indole and a series of simple indole analogs is described. The method may be employed for the direct estimation of indole analogs in enzyme reaction mixtures.
120
DEMOSS AND GAGE
60
IO
0 120
140 160 TEMPERATURE,
180 OC
200
FIQ. 3. Retention time as a function of column temperature. Aqueous l.O-pl samples of a solution containing three analogs for temperature range 120”-16O”C, or four analogs for temperature range 170”-200°C were injected. Retention time was calculated as interval between time of injection and time at which analog in question began to enter the detector and is the mean value for at least six trials at each temperature. ACKNOWLEDGMENT This investigation was supported in part by Public Health from the National Institutes of Health.
Service grant E-2971
REFERENCES 1. LLOYD, H. A., FALES, H. M., HIQHET, P. F., VANDEN HEUVEN, WILDMAN, W. C., J. Am. Chem. Sot. 82, 3791 (1960). 2. JANAK, J., AND HRIVNAC, M., Coil. Czech. Chem. Commun. 25, 3. MOSSINI, F., AND VITALI, T., Ric. Sci. Rend. [Al 1, 244 (1961) Abstr. 57, 66175: (1962).
W. J. A., AND 1557 (1960). ; cited in Chew.