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Automated determination of organic sulfur compounds
BIOCHEMICAL
MEDICINE
Automated
33, 211-214
Determination
I. Spectrophotometric
WALTER Department
MARX,
(1985)
of Organic Sulfur Compounds
Measurement of Inorganic Sulfate by Low-Pressure Exchange Chromatography
PRABHAKAR
RAO
qf Pharmacology and Nutrition, 2025 Zonal Avenue,
KAVIPURAPU,
AND
University of Southern Los Angeles, California
Received
April
SAMUEL
P.
California 90033
School
BESSMAN of Medicine,
30, 1984
Attempts are underway in this laboratory to develop an automated method for determination of organic sulfur compounds in biological fluids, using equipment similar to that reported by Bessman et al. for automatic measurement of organic phosphates (1,2). The procedure consists of a chromatographic separation of the sulfur compounds followed by combustion of the separated fractions to inorganic sulfate. The present paper describes the technique involved in the assay of the inorganic sulfate fractions. It is based on a modification of a method developed for the determination of SO, in air (3-5), and it involves an exchange reaction between sulfate and barium chloranilate. METHODS Two Teflon filter circles of 6.5 mm diameter (type LS, pore size 5 nm, Millipore Corp., Bedford, Mass.) were placed at the bottom of a flanged glass column (3 mm inside diameter, 50 mm long) closed at each end with a Teflon disc having a l/32-in. center bore held in place by threaded polypropylene fittings (Altex Scientific Co., Berkeley, Calif.). A suspension of barium chloranilate (J.T. Baker Chemical Co., Phillipsburg, N.J., used without further treatment except for removal of fines) in 70% (v/v) 2-propanol was percolated through the column repeatedly, until the latter was filled to the top. Either unbuffered 70% (v/v) 2propanol, or 0.02 M ammonium acetate in the same solvent, apparent pH 8.68.9, were used as perfusion and sample medium. The solution was passed from a reservoir through the column and then through an uv absorption cell provided with a 313-nm filter (Biochemical UV Monitor, Model 150, Altex Scientific Co., Inc., Berkeley, Calif.), and connected to an integrating recorder (Model 252-A, Linear Instruments Corp., Irvine, Calif.). In order to establish a baseline, it was necessary to perfuse the column and warm up the monitor and recorder for about 18-2 hr. The following adjustments were made: Chart speed, 4 in./hr; integrator, 3000 counts/min: sensitivity range, 211 0006-2944/U
$3.00
Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
212
MARX.
KAVIPURAPU.
AND
BESSMAN
0.05-0.2, depending on sample size; perfusion rate, 0.9 ml/min. When the baseline was stabilized the sulfate samples were injected manually through a septum injector (Chromatronix Laboratory Data Control. Riviera Beach, Fla.) attached at the top of the column using a microsyringe with a needle having a solid point and side port (Unimetrics Corp., Anaheim, Calif.) without interruption of the solvent flow. Integration of the recorded peak areas produced by the chloranilate ion released from the barium chloranilate column resulted in values expressed in arbitrary integration units which corresponded to the amounts of inorganic sulfate applied. in agreement with earlier reports (3-5). RESULTS Reproducibility. When six identical samples containing 7.5 nmole (NH&SO, each were applied consecutively to a barium chloranilate column under conditions described in Fig. 1, peak areas ranging from 1054 to 1199 integration units were obtained with a mean of 1114 integration units and a relative standard deviation of 5.6% (Fig. 1). Sensitivity. Application of 0.05 nmole NaSO, to a barium chloranilate column produced a detectable peak at a sensitivity range of 0.05. However, when sulfate samples below 1.0 nmole were used, a significant rise in the relative standard deviation was observed with decreasing sulfate concentration, indicating a reduction in the precision of the measurements (Table I). For quantitative measurements. therefore, samples containing at least 1 nmole sulfate were used. as far as possible. Calibration curve. Samples of 2.5-20.0 nmole Na,SO, were applied to a barium chloranilate column under conditions shown in Fig. 7. The values for the peak areas recorded, when plotted as a function of the amount of sulfate applied. were found to fall on a straight line (Fig. 2). When the amounts of sulfate were increased further, the slope of the curve was observed to decline gradually. This decline in slope was smaller when a longer column was used.
FIG. I. Reproducibility. Six identical samples of 72 nmole (NH&SO., each dlssol\led in 31% (v/v) 2-propanol containing 0.02 M ammonium acetate. apparent pH X.6, were applied to a barium chloranilate column perfused with the same buffered solvent. Flow rate. 0.9 ml/min. Sensitivity range, 0.2. Numbers under peaks indicate areas expressed in integration units
MEASUREMENT
OF INORGANIC
SULFATE
213
TABLE 1 Influence of Sample Size Na$O, (nmole)
Number of samples
Mean peak area, integr. units
% of peak area (SD)
2.0 1.0 0.5 0.2
6 3 3 3
1935 945 537 150
7.8 6.8 28.5 38.4
Notes. Samples dissolved in 70% (v/v) 2-propanol were applied to a barium chloranilate perfused with the same solvent. Flow rate, 0.5 ml/min. Sensitivity range, 0.05.
column
DISCUSSION
The determination of inorganic sulfate described is based on an exchange reaction between sulfate and barium chloranilate first reported by Bertolacini and Barney (6,7). Although it was indicated that the amount chloranilate ion liberated during this reaction was equivalent to the quantity of sulfate applied (3-5), it was observed in this laboratory that the size and shape of a peak produced by a given amount of sulfate was greatly influenced by experimental conditions. Therefore, it was found to be critical to maintain variables such as flow rate, pH, sample volume, and temperature at a constant level. Experiments are in progress to study in greater detail the effects of these experimental factors as well as of the presence of other ions on the peak areas produced by a given sulfate concentration (8). The procedure represents a convenient method for measuring inorganic sulfate in nanomole quantities; it involves low-pressure exchange chromatography, a technique requiring relatively simple and inexpensive equipment.
nmoles
Ne,SO,
applied
FIG. 2. Calibration curve. Samples of 2.5-20.0 nmole Na$O, were applied to a barium chloranilate column under conditions described in Fig. 1, except for apparent pH. 8.9.
214
MARX.
KAVIPURAPU.
AND BESSMAN
SUMMARY
A method for determination of inorganic sulfate is described which represents the terminal step of an automated assay of organic sulfur compounds in biological fluids. Nanomole quantities of inorganic sulfate were applied to a barium chloranilate column. Corresponding amounts of chloranilate ion, released as a result of an exchange reaction, were then measured by uv absorption at 313 nm. The rcproducibility and sensitivity of the method and a calibration curve are reported. ACKNOWLEDGMENT The authors are much obliged to Dr. Paul J. Geiger for his rntcrest and for- many helpful suggesttons. REFERENCES I. 2. 3. 4. 5.
Bessman. S. P., And. Biochem 59, 574 (1974). Bessman. S. P.. Geiger, P. J., Lu. T. C. and McCabe. E. K. B.. 4&. &&ze/~. 59. 535 t 1974). Laxton. J. W., and Jackson, P. J., J. In.ct. Fltrl 37, 12 (1964). Schafer, H. N. S., Anal. Chem 39, 1719 (1967). Tejada, S. P.. Sigsby, J. E., and Bradow. R. L.. in “Interim Report for Characterizing [Inregulated Emissions, etc.,” EPA 600/2-80-068. p. 446. U.S. Environmental Protection Agency. Research Triangle Park, N.C. 27711. 1980. 6. Bertolacini, R. J.. and Barney. J. E.. 11, Amrl. Chum. 29, 281 (lY571. 7. Bertolacini, R. J., and Barney, J. E.. Ii. Af7d. Chem. 30, 202 (19.58). 8. Marx, W.. and Bessman, S. P.. in preparation.
MEDICINE
Automated
33, 211-214
Determination
I. Spectrophotometric
WALTER Department
MARX,
(1985)
of Organic Sulfur Compounds
Measurement of Inorganic Sulfate by Low-Pressure Exchange Chromatography
PRABHAKAR
RAO
qf Pharmacology and Nutrition, 2025 Zonal Avenue,
KAVIPURAPU,
AND
University of Southern Los Angeles, California
Received
April
SAMUEL
P.
California 90033
School
BESSMAN of Medicine,
30, 1984
Attempts are underway in this laboratory to develop an automated method for determination of organic sulfur compounds in biological fluids, using equipment similar to that reported by Bessman et al. for automatic measurement of organic phosphates (1,2). The procedure consists of a chromatographic separation of the sulfur compounds followed by combustion of the separated fractions to inorganic sulfate. The present paper describes the technique involved in the assay of the inorganic sulfate fractions. It is based on a modification of a method developed for the determination of SO, in air (3-5), and it involves an exchange reaction between sulfate and barium chloranilate. METHODS Two Teflon filter circles of 6.5 mm diameter (type LS, pore size 5 nm, Millipore Corp., Bedford, Mass.) were placed at the bottom of a flanged glass column (3 mm inside diameter, 50 mm long) closed at each end with a Teflon disc having a l/32-in. center bore held in place by threaded polypropylene fittings (Altex Scientific Co., Berkeley, Calif.). A suspension of barium chloranilate (J.T. Baker Chemical Co., Phillipsburg, N.J., used without further treatment except for removal of fines) in 70% (v/v) 2-propanol was percolated through the column repeatedly, until the latter was filled to the top. Either unbuffered 70% (v/v) 2propanol, or 0.02 M ammonium acetate in the same solvent, apparent pH 8.68.9, were used as perfusion and sample medium. The solution was passed from a reservoir through the column and then through an uv absorption cell provided with a 313-nm filter (Biochemical UV Monitor, Model 150, Altex Scientific Co., Inc., Berkeley, Calif.), and connected to an integrating recorder (Model 252-A, Linear Instruments Corp., Irvine, Calif.). In order to establish a baseline, it was necessary to perfuse the column and warm up the monitor and recorder for about 18-2 hr. The following adjustments were made: Chart speed, 4 in./hr; integrator, 3000 counts/min: sensitivity range, 211 0006-2944/U
$3.00
Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
212
MARX.
KAVIPURAPU.
AND
BESSMAN
0.05-0.2, depending on sample size; perfusion rate, 0.9 ml/min. When the baseline was stabilized the sulfate samples were injected manually through a septum injector (Chromatronix Laboratory Data Control. Riviera Beach, Fla.) attached at the top of the column using a microsyringe with a needle having a solid point and side port (Unimetrics Corp., Anaheim, Calif.) without interruption of the solvent flow. Integration of the recorded peak areas produced by the chloranilate ion released from the barium chloranilate column resulted in values expressed in arbitrary integration units which corresponded to the amounts of inorganic sulfate applied. in agreement with earlier reports (3-5). RESULTS Reproducibility. When six identical samples containing 7.5 nmole (NH&SO, each were applied consecutively to a barium chloranilate column under conditions described in Fig. 1, peak areas ranging from 1054 to 1199 integration units were obtained with a mean of 1114 integration units and a relative standard deviation of 5.6% (Fig. 1). Sensitivity. Application of 0.05 nmole NaSO, to a barium chloranilate column produced a detectable peak at a sensitivity range of 0.05. However, when sulfate samples below 1.0 nmole were used, a significant rise in the relative standard deviation was observed with decreasing sulfate concentration, indicating a reduction in the precision of the measurements (Table I). For quantitative measurements. therefore, samples containing at least 1 nmole sulfate were used. as far as possible. Calibration curve. Samples of 2.5-20.0 nmole Na,SO, were applied to a barium chloranilate column under conditions shown in Fig. 7. The values for the peak areas recorded, when plotted as a function of the amount of sulfate applied. were found to fall on a straight line (Fig. 2). When the amounts of sulfate were increased further, the slope of the curve was observed to decline gradually. This decline in slope was smaller when a longer column was used.
FIG. I. Reproducibility. Six identical samples of 72 nmole (NH&SO., each dlssol\led in 31% (v/v) 2-propanol containing 0.02 M ammonium acetate. apparent pH X.6, were applied to a barium chloranilate column perfused with the same buffered solvent. Flow rate. 0.9 ml/min. Sensitivity range, 0.2. Numbers under peaks indicate areas expressed in integration units
MEASUREMENT
OF INORGANIC
SULFATE
213
TABLE 1 Influence of Sample Size Na$O, (nmole)
Number of samples
Mean peak area, integr. units
% of peak area (SD)
2.0 1.0 0.5 0.2
6 3 3 3
1935 945 537 150
7.8 6.8 28.5 38.4
Notes. Samples dissolved in 70% (v/v) 2-propanol were applied to a barium chloranilate perfused with the same solvent. Flow rate, 0.5 ml/min. Sensitivity range, 0.05.
column
DISCUSSION
The determination of inorganic sulfate described is based on an exchange reaction between sulfate and barium chloranilate first reported by Bertolacini and Barney (6,7). Although it was indicated that the amount chloranilate ion liberated during this reaction was equivalent to the quantity of sulfate applied (3-5), it was observed in this laboratory that the size and shape of a peak produced by a given amount of sulfate was greatly influenced by experimental conditions. Therefore, it was found to be critical to maintain variables such as flow rate, pH, sample volume, and temperature at a constant level. Experiments are in progress to study in greater detail the effects of these experimental factors as well as of the presence of other ions on the peak areas produced by a given sulfate concentration (8). The procedure represents a convenient method for measuring inorganic sulfate in nanomole quantities; it involves low-pressure exchange chromatography, a technique requiring relatively simple and inexpensive equipment.
nmoles
Ne,SO,
applied
FIG. 2. Calibration curve. Samples of 2.5-20.0 nmole Na$O, were applied to a barium chloranilate column under conditions described in Fig. 1, except for apparent pH. 8.9.
214
MARX.
KAVIPURAPU.
AND BESSMAN
SUMMARY
A method for determination of inorganic sulfate is described which represents the terminal step of an automated assay of organic sulfur compounds in biological fluids. Nanomole quantities of inorganic sulfate were applied to a barium chloranilate column. Corresponding amounts of chloranilate ion, released as a result of an exchange reaction, were then measured by uv absorption at 313 nm. The rcproducibility and sensitivity of the method and a calibration curve are reported. ACKNOWLEDGMENT The authors are much obliged to Dr. Paul J. Geiger for his rntcrest and for- many helpful suggesttons. REFERENCES I. 2. 3. 4. 5.
Bessman. S. P., And. Biochem 59, 574 (1974). Bessman. S. P.. Geiger, P. J., Lu. T. C. and McCabe. E. K. B.. 4&. &&ze/~. 59. 535 t 1974). Laxton. J. W., and Jackson, P. J., J. In.ct. Fltrl 37, 12 (1964). Schafer, H. N. S., Anal. Chem 39, 1719 (1967). Tejada, S. P.. Sigsby, J. E., and Bradow. R. L.. in “Interim Report for Characterizing [Inregulated Emissions, etc.,” EPA 600/2-80-068. p. 446. U.S. Environmental Protection Agency. Research Triangle Park, N.C. 27711. 1980. 6. Bertolacini, R. J.. and Barney. J. E.. 11, Amrl. Chum. 29, 281 (lY571. 7. Bertolacini, R. J., and Barney, J. E.. Ii. Af7d. Chem. 30, 202 (19.58). 8. Marx, W.. and Bessman, S. P.. in preparation.