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A simple two-column system for the rapid analysis of plasma amino acids
SHORT
A Simple
159
COMMUNICATIONS
Two-Column System for the Rapid Analysis of Plasma Amino Acids’
The procedure of Spa&man et al. (1) for the automatic analysis of amino acids represents one of the most important contributions to analytical chemistry. In recent years several commerical concerns have modified the original apparatus and their equipment is in general use throughout the country. The original Moore-Stein machine required two chromatographic columns for a complete analysis, one for the acidic and neutral amino acids and the other for the more basic components. The instrument was altered by Technicon (Technicon Instruments Inc., Chauncey, N. Y.) to permit the use of a single column, and then in an attempt to decrease the time required for the assay of a sample the equipment was recently redesigned to incorporate five individual columns which are used in sequence. By forcing buffer through each of the new type of columns at high pressures, five samples could be analyzed in 24 hr. The present report describes a method of analyzing four complete samples a day with the old machine at an additional cost of only about 15% of the original purchase price. The apparatus has two adsorption columns and differs from the original and from the multiple-column equipment of Technicon in one very important respect: two different samples can be analyzed in parallel simultaneously, an expedient which enables the operator to compare a normal sample with an abnormal one on the same chart under conditions which are nearly identical. The instrument employed was purchased from Technicon and the basic components still resemble the original AutoAnalyzer as described by Skeggs (2). In addition to the incorporation of most of the parts of the AutoAnalyzer, the original amino acid analyzer had a 130-cm chromatographic column filled with an adsorbent (Chromobeads) , a high-pressure controlled-flow pump which forced buffers through the column, and another pump which circulated hot water through the water jacket to control the column temperature. With the two-column system the volume of the buffers in the automatic pH gradient chamber (Autograd) was doubled without changing the pH gradient of the original equipment. For compactness the columns were mounted on the same frame and buffers were forced at 1.0 ml/min ‘Supported
in part
by
a grant
from
the
National
Institutes
of Health,
H 020152.
160
SHORT
COMMUNICATIONS
through each by two identical constant-volume pumps operating under twice the original rate of flow. To ensure simultaneous delivery of identical buffers to both columns the flow from the Autograd was divided into two streams with a Y-tube. Two independent gages were used to register the pressure of the respective columns. Foam rubber insulated water jackets were connected in series and then the columns mere maintained at identical temperatures by the thermoregulated pump furnished with the original equipment. The proportioning pump in the new two-column system was equipped with eight plastic tubes (solvaflex) instead of the original four. Two heating coils were installed in the reaction chamber, and two sets of glass tubing were required to carry the final colored solution to each of two calorimeters which were connected to the Bristol recorder independently. A third calorimeter is connected in series with the second to detect and measure proline if it is present in the sample since it is fitted with a 440-rnp filter instead of the 570-rnp filter of Nos. 1 and 2 calorimeters (Fig. 1). Although proline is not estimated on the effluent from the column which goes to
FIG. 1. 1, Autograd; 2 and 2A, adsorption columns; 3 and 3A, pressure gages device to remove air reading from O-600 or &lo00 lb/sq in.; 4 and 4A, debubbling introduced in connecting the Autograd, bottles containing 0.2 N NaOH or pH 2.875 buffer. The controlled volume pumps receive fluid from the T (4A): 5 and Pa., O-1009 lb/sq in. capacity; 5A, Minipumps, Milton Roy Co., Philadelphia, 7, heating bath; 7A, cooling coils; 8, 6, proportioning pump; 6A, mixing coils; 8A, and 8B, calorimeters 1, 2, and 3, respectively; 9, Bristol a-channel recorder; 11, llA, and llB, combination transformer 10, Formica-topped folding table; and voltage-stabilizer; 12, 12-gal Pyrex bottle for ninhydrin; 13, nitrogen flow indicator; 14, combination pump and water heater for heating the columns.
SHORT
FIG. 2. Double peritoneal injection apparently derived protein-free filtrate 45°C for the first 1.0 ml/min.
COMNUNICATIONS
161
chromatogram of plasma of a turt.le before and after intraof 10 mmoles ornithine per/kg. The new components were from the catabolism of ornithinc. Two ml of a l/IO alcoholic was added to each column. The column temperature was 90 min and 60” for the remainder of the run. Flow rate was
the single calorimeter (No. I), this is not usually a serious handicap since proline is one of the minor constituents in body fluids. No other modifications of the principal components of the system were required. A complete double run requires 10.5 hr of operation followed by a IO-min wash with 0.2 N NaOH and about a 30-min wash with pH 2.875 buffer to regenerate and clean the columns. The higher flow rate reduces not only operating time but cleaning time as well. We were agreeably surprised to find that increasing the flow rate did not reduce the resolving power of the system. An important incidental convenience was an increase in simplicity and a decrease in both the quantity of paper required and the length of each record. Six feet of paper is much easier to handle than twelve and the use of the doublecolumn apparatus resulted in the use of one-fourth as much paper per sample as was required by the original apparatus. Reducing the ninhydrin requirements to half was a considerable saving in both the reagents and the time required for their preparation. It should be emphasized that, for those operators requiring only a single column for occasional analyses, the time needed for a complete chromatogram may be reduced to one-half by doubling the pump pressures without any other changes in the existing system.
162
SHORT COMMUNICATIONS REFERENCES
1. 2.
D. H., STEIN, W. H., AND MOORE, S. Anal. Chem. 30, 1190 (1958). L. T., JR., Am. J. Clin. Path. 28, 311 (1957).
SPACKMAN, &EGGS,
ROLAND A. COULSON~ THOMAS HERNANDEZ VIRGIL BYERS Departments of Biochemistry and Pharmacology Louisiana State University of Medicine New Orleans, Louisiana Received September 16, 1964 ‘With
the technical
Spectral
Shifts
assistance of Jacob Watson.
in the
Reaction
with
of N-Ethylmaleimide
Proteins
N-Ethylmaleimide (NEM) is widely used as a sulfhydryl group reagent. Its spectrum has a maximum at 306 mp which disappears on reaction with sulfhydryl groups (l), and the absorbance decrease forms the basis of a quantitative method for the determination of these groups in proteins (2-4). During an investigation into the rate of reaction of NEM with /3-lactoglobulin (BLG) in urea solutions,1 it was observed that, when the urea concentration was greater than about 5 M, the decrease in absorbance corresponded to the presence of two sulfhydryl groups per protein molecule, which is in agreement with the known cysteine content of this protein (5). However, when the urea concentration was about 65 M, the decrease in absorbance was significantly greater than expected, and was strongly dependent on the urea concentration. This may be seen from the representative results shown in Table la.* There have been a number of reports that NEM is not specific for sulfhydryl groups when a large excess of the reagent is used (6-8), but a nonspecific reaction cannot be responsible for the anomalous decrease ‘Leslie, J., and Gorin, G., unpublished results. “The BLG used was obtained from Pentex, Inc., Kankakee, homogeneous by immunoelectrophoresis.
Illinois.
It was
A Simple
159
COMMUNICATIONS
Two-Column System for the Rapid Analysis of Plasma Amino Acids’
The procedure of Spa&man et al. (1) for the automatic analysis of amino acids represents one of the most important contributions to analytical chemistry. In recent years several commerical concerns have modified the original apparatus and their equipment is in general use throughout the country. The original Moore-Stein machine required two chromatographic columns for a complete analysis, one for the acidic and neutral amino acids and the other for the more basic components. The instrument was altered by Technicon (Technicon Instruments Inc., Chauncey, N. Y.) to permit the use of a single column, and then in an attempt to decrease the time required for the assay of a sample the equipment was recently redesigned to incorporate five individual columns which are used in sequence. By forcing buffer through each of the new type of columns at high pressures, five samples could be analyzed in 24 hr. The present report describes a method of analyzing four complete samples a day with the old machine at an additional cost of only about 15% of the original purchase price. The apparatus has two adsorption columns and differs from the original and from the multiple-column equipment of Technicon in one very important respect: two different samples can be analyzed in parallel simultaneously, an expedient which enables the operator to compare a normal sample with an abnormal one on the same chart under conditions which are nearly identical. The instrument employed was purchased from Technicon and the basic components still resemble the original AutoAnalyzer as described by Skeggs (2). In addition to the incorporation of most of the parts of the AutoAnalyzer, the original amino acid analyzer had a 130-cm chromatographic column filled with an adsorbent (Chromobeads) , a high-pressure controlled-flow pump which forced buffers through the column, and another pump which circulated hot water through the water jacket to control the column temperature. With the two-column system the volume of the buffers in the automatic pH gradient chamber (Autograd) was doubled without changing the pH gradient of the original equipment. For compactness the columns were mounted on the same frame and buffers were forced at 1.0 ml/min ‘Supported
in part
by
a grant
from
the
National
Institutes
of Health,
H 020152.
160
SHORT
COMMUNICATIONS
through each by two identical constant-volume pumps operating under twice the original rate of flow. To ensure simultaneous delivery of identical buffers to both columns the flow from the Autograd was divided into two streams with a Y-tube. Two independent gages were used to register the pressure of the respective columns. Foam rubber insulated water jackets were connected in series and then the columns mere maintained at identical temperatures by the thermoregulated pump furnished with the original equipment. The proportioning pump in the new two-column system was equipped with eight plastic tubes (solvaflex) instead of the original four. Two heating coils were installed in the reaction chamber, and two sets of glass tubing were required to carry the final colored solution to each of two calorimeters which were connected to the Bristol recorder independently. A third calorimeter is connected in series with the second to detect and measure proline if it is present in the sample since it is fitted with a 440-rnp filter instead of the 570-rnp filter of Nos. 1 and 2 calorimeters (Fig. 1). Although proline is not estimated on the effluent from the column which goes to
FIG. 1. 1, Autograd; 2 and 2A, adsorption columns; 3 and 3A, pressure gages device to remove air reading from O-600 or &lo00 lb/sq in.; 4 and 4A, debubbling introduced in connecting the Autograd, bottles containing 0.2 N NaOH or pH 2.875 buffer. The controlled volume pumps receive fluid from the T (4A): 5 and Pa., O-1009 lb/sq in. capacity; 5A, Minipumps, Milton Roy Co., Philadelphia, 7, heating bath; 7A, cooling coils; 8, 6, proportioning pump; 6A, mixing coils; 8A, and 8B, calorimeters 1, 2, and 3, respectively; 9, Bristol a-channel recorder; 11, llA, and llB, combination transformer 10, Formica-topped folding table; and voltage-stabilizer; 12, 12-gal Pyrex bottle for ninhydrin; 13, nitrogen flow indicator; 14, combination pump and water heater for heating the columns.
SHORT
FIG. 2. Double peritoneal injection apparently derived protein-free filtrate 45°C for the first 1.0 ml/min.
COMNUNICATIONS
161
chromatogram of plasma of a turt.le before and after intraof 10 mmoles ornithine per/kg. The new components were from the catabolism of ornithinc. Two ml of a l/IO alcoholic was added to each column. The column temperature was 90 min and 60” for the remainder of the run. Flow rate was
the single calorimeter (No. I), this is not usually a serious handicap since proline is one of the minor constituents in body fluids. No other modifications of the principal components of the system were required. A complete double run requires 10.5 hr of operation followed by a IO-min wash with 0.2 N NaOH and about a 30-min wash with pH 2.875 buffer to regenerate and clean the columns. The higher flow rate reduces not only operating time but cleaning time as well. We were agreeably surprised to find that increasing the flow rate did not reduce the resolving power of the system. An important incidental convenience was an increase in simplicity and a decrease in both the quantity of paper required and the length of each record. Six feet of paper is much easier to handle than twelve and the use of the doublecolumn apparatus resulted in the use of one-fourth as much paper per sample as was required by the original apparatus. Reducing the ninhydrin requirements to half was a considerable saving in both the reagents and the time required for their preparation. It should be emphasized that, for those operators requiring only a single column for occasional analyses, the time needed for a complete chromatogram may be reduced to one-half by doubling the pump pressures without any other changes in the existing system.
162
SHORT COMMUNICATIONS REFERENCES
1. 2.
D. H., STEIN, W. H., AND MOORE, S. Anal. Chem. 30, 1190 (1958). L. T., JR., Am. J. Clin. Path. 28, 311 (1957).
SPACKMAN, &EGGS,
ROLAND A. COULSON~ THOMAS HERNANDEZ VIRGIL BYERS Departments of Biochemistry and Pharmacology Louisiana State University of Medicine New Orleans, Louisiana Received September 16, 1964 ‘With
the technical
Spectral
Shifts
assistance of Jacob Watson.
in the
Reaction
with
of N-Ethylmaleimide
Proteins
N-Ethylmaleimide (NEM) is widely used as a sulfhydryl group reagent. Its spectrum has a maximum at 306 mp which disappears on reaction with sulfhydryl groups (l), and the absorbance decrease forms the basis of a quantitative method for the determination of these groups in proteins (2-4). During an investigation into the rate of reaction of NEM with /3-lactoglobulin (BLG) in urea solutions,1 it was observed that, when the urea concentration was greater than about 5 M, the decrease in absorbance corresponded to the presence of two sulfhydryl groups per protein molecule, which is in agreement with the known cysteine content of this protein (5). However, when the urea concentration was about 65 M, the decrease in absorbance was significantly greater than expected, and was strongly dependent on the urea concentration. This may be seen from the representative results shown in Table la.* There have been a number of reports that NEM is not specific for sulfhydryl groups when a large excess of the reagent is used (6-8), but a nonspecific reaction cannot be responsible for the anomalous decrease ‘Leslie, J., and Gorin, G., unpublished results. “The BLG used was obtained from Pentex, Inc., Kankakee, homogeneous by immunoelectrophoresis.
Illinois.
It was