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Separation of choline from some bases on dowex columns
197
SHORT COMMUNICATIONS
for fluctuations of room temperature and flow rate. The controller probe is mounted in the middle well, in the water moving most rapidly. This assures the most rapid response of the probe. To minimize heat loss due to evaporation, covers (Parafilm, American Can Co., with holes to accommodate reaction vessels) were placed on the water surface of each well. With baths with either 10 or 20 wells, temperature control in all wells of *O.O5’C was obtained. This degree of temperature control was found in all parts of the wells except for an area immediately in front of the outflow slot. The temperature variation was measured with thermistors (time constant 5 set) . Due to heat exchange with the room, the temperature gradient is slightly nonlinear. Baths of this type have been used with well sizes ranging from 12.5 X 12.5 x 10 cm deep to 4.5 cm wide X 11 cm long X 12 cm deep. With a bath with 20 wells the largest temperature range used was 25” and the smallest was 10”. The lowest temperature used was 10’ and the highest 45”. 1. OPPENHEIMW, 2. MILLER, R.
C. N., AND
REFERENCES DROST-HANSEN, W. J., J.
J., AND DAVEY, C. B., Sod Sci. 101,418
Bacterial. (1966).
80, 21 (1960)
il. R. OVERMAN RAYMOND J. MILLER Agronomy Department University of Illinois Urbana, Illinois 61803 Received January 6, 1967
from
Separation of Choline Some Bases on Dowex Columns
Since choline is important in metabolic processes, it is desirable to be able to isolate and quantitate this substance. Some quantitative assay methods are relatively insensitive and others may lack specificity. A commonly used assay method is the periodide test (1)) which is very sensitive for detection of relatively small amounts of choline but which also gives positive reactions with many bases. We have utilized a column chromatographic method for the separation of some of the interfering compounds. This method is fast and reproducible and gives almost concc plete recovery of the substances. The general approach was to chromatu&
198
SHORT
COMMUNICATIONS
graph different bases on the Dowex 50-X8 column of a Beckman. Spinco amino acid analyzer and to collect the fractions with a fraction collector. The bases were then quantitated by the periodide method. Eqerimental Procedure. The solid compounds were taken to constant weight in a vacuum desiccator. A stock solution of each compound, with concentrations ranging from 0.165 to 7.14 ~moleJm1, was prepared in the same buffer that was used as eluant in the Dowex columns. This buffer was the pH 5.28 citrate buffer that Spackman, Moore, and Stein (2) used to elute basic amino acids from Dowex columns, but with three important exceptions. Caprylic acid and Brij-35 were deleted, because these substances interfered with the periodide test. Phenol is not used in our amino acid analyses, and was also omitted from the buffer. The Dowex 50-X8 column, bead form, 6.5 X 0.9 cm, was the same column that is usually used to separate the basic amino acids in the Beckman Spinco amino acid analyzer. The column was equilibrated with the buffer, some of which was taken from the column outflow for use as reagent blanks in the periodide test. In order to determine the position of elution, a 1 ml aliquot of a stock solution was placed on the column. The flow rate was 68 to 70 ml/hr and the eluant was collected in 1 ml aliquots in a fraction collector (Gilson Medical Electronics, Middleton, Wisconsin). These aliquots were analyzed by the periodide method of Hayashi et al. (1)) but with a slight modification. The addition of 0.2 ml of acetate buffer was omitted from the procedure. Potassium iodide (0.1 ml of 0.1 M) was added to each 1 ml eluant fraction and to 1 ml of eluant buffer for a reagent blank. Then extraction into 4 ml of ethylene dichloride reagent (0.5% I, in ethylene dichloride) was done. Absorption was measured at 385 rnp with a Gilford spectrophotometer 300 (Gilford Instrument Co., Oberlin, Ohio). Recovery of each of the bases from the column was determined in separate experiments. The absorption of 1 ml of stock base was measured by the periodide method. One milliliter of stock solution was applied to the column and the absorption of each fraction was measured. Since preliminary studies indicated linear standard curves, the ratio of the sum of the absorbancies of the fractions to the absorbance of the stock solution was taken as per cent recovery. Molar extinction coefficients were calculated. in order to compare the. relative absorbancies of. the periodide reaction’ptioducts.. ~Ekjwimiintal Results and Discussion. The compounds tested, lthe ‘cbncentrations ‘of stock solutions, the calculated-molar. extinction coe%icien&a; and the average per cent recoveries are shown in Table 1. Figure 3. shows! the posit%ons of elution of ten bases tested; eight’are completely.separable from choline. The trailing edge of betaine overlapped the leading ‘edge of choline. More complete separation of these two compounds might be
SHORT
199
COMMUNICATIONS
Betaine Triet&jami:l+, I 0
15
30 Eluant
15
ii 0
(ml)
FIQ. 1. Eluant pattern of different bases from Dowex 50-X8 column. The quantities of material applied to the column are given in Table 1, to demonstrate the number of milliliters of eluant in which the compounds were detectable with the periodide test.
effected by collecting appropriate aliquots in the fraction collector. The only base which significantly overlapped choline was tetramethylammonium. However, a longer column than 6.5 cm, and alteration of the pH of the buffer, may effect complete separation. The data for the amino acids lysine, histidine, and arginine are not included because these amino TABLE 1 Concentrations,
Calculated
Molar Extinction
Compound
Stock concentrstioll, ~mole/ml
0.358 0.165 0.252 0.324 0.348 0.342 0.523 0.489 0.466 1.502 7.143
Choline chloride Thiamine hydrochloride o,L-Carnitine chloride Tetramethylammonium bromide Betaine hydrochloride Diphenhydramine hydrochloride Trimethylamine hydrochloride n,cTryptophan Ephedrine sulfate Tryptamine hydrochloride Triethylamine hydrochloride 0 385 q,
Gilford spectrophotometer
Coefficients and Per Cent Recoveries M&I extinction co&icient~
4,050 14,545 6,865 4,877 4,023 3,392 3,231 2,822 2,210 1,643 377
Averaee
7%
WCOPWy
99.9 92.0 95.4 99.0 95.2 80.1 93.8 103.0 87.3 73.3 98.0
300.
acids are not periodide positive. The usual elution sequence on these columns is lysine, histidine, ammonia, and arginine. Choline elutes between histidine and ammonia. The usefulness of this system is that it takes less than one hour for elution of all the bases tested, and in most instances the separation from choline is complete. ACKNOWLEDGMENTS This investigation was supported in part by U. S. Public Health Service Grants He-08446 and 5TlGM-125 from the National Institutes of Health. Mrs. Susan Kobeck was very helpful in obtaining some of the experimental results.
200
SHORT
COMMUNICATION
REFERENCES 1.
HAYASHI,
2. SPACKMAN,
M., UNEMOTO, T., D. H.,
STEIN,
AND
MIYAKI, K., Chem. Pharrn. Bull. 10,633 (1902) S., Anal. Chem. 30, 1185 (1958).
W. H., AND MOORE,
K. GOURLEY~ CHARLES D. H.L&
WILLIAM
SEYMOUR
BAKERMAN
Departments of Biochemistw and of Pathology and Oncology University of Kanwe Medical Center Kansas City, Kansas 6f31W Received January 9,1967 1 Taken in part from M.S. the& of W. G. st University ’ Medical student summer Fellow.
of Kaums, 1967.
SHORT COMMUNICATIONS
for fluctuations of room temperature and flow rate. The controller probe is mounted in the middle well, in the water moving most rapidly. This assures the most rapid response of the probe. To minimize heat loss due to evaporation, covers (Parafilm, American Can Co., with holes to accommodate reaction vessels) were placed on the water surface of each well. With baths with either 10 or 20 wells, temperature control in all wells of *O.O5’C was obtained. This degree of temperature control was found in all parts of the wells except for an area immediately in front of the outflow slot. The temperature variation was measured with thermistors (time constant 5 set) . Due to heat exchange with the room, the temperature gradient is slightly nonlinear. Baths of this type have been used with well sizes ranging from 12.5 X 12.5 x 10 cm deep to 4.5 cm wide X 11 cm long X 12 cm deep. With a bath with 20 wells the largest temperature range used was 25” and the smallest was 10”. The lowest temperature used was 10’ and the highest 45”. 1. OPPENHEIMW, 2. MILLER, R.
C. N., AND
REFERENCES DROST-HANSEN, W. J., J.
J., AND DAVEY, C. B., Sod Sci. 101,418
Bacterial. (1966).
80, 21 (1960)
il. R. OVERMAN RAYMOND J. MILLER Agronomy Department University of Illinois Urbana, Illinois 61803 Received January 6, 1967
from
Separation of Choline Some Bases on Dowex Columns
Since choline is important in metabolic processes, it is desirable to be able to isolate and quantitate this substance. Some quantitative assay methods are relatively insensitive and others may lack specificity. A commonly used assay method is the periodide test (1)) which is very sensitive for detection of relatively small amounts of choline but which also gives positive reactions with many bases. We have utilized a column chromatographic method for the separation of some of the interfering compounds. This method is fast and reproducible and gives almost concc plete recovery of the substances. The general approach was to chromatu&
198
SHORT
COMMUNICATIONS
graph different bases on the Dowex 50-X8 column of a Beckman. Spinco amino acid analyzer and to collect the fractions with a fraction collector. The bases were then quantitated by the periodide method. Eqerimental Procedure. The solid compounds were taken to constant weight in a vacuum desiccator. A stock solution of each compound, with concentrations ranging from 0.165 to 7.14 ~moleJm1, was prepared in the same buffer that was used as eluant in the Dowex columns. This buffer was the pH 5.28 citrate buffer that Spackman, Moore, and Stein (2) used to elute basic amino acids from Dowex columns, but with three important exceptions. Caprylic acid and Brij-35 were deleted, because these substances interfered with the periodide test. Phenol is not used in our amino acid analyses, and was also omitted from the buffer. The Dowex 50-X8 column, bead form, 6.5 X 0.9 cm, was the same column that is usually used to separate the basic amino acids in the Beckman Spinco amino acid analyzer. The column was equilibrated with the buffer, some of which was taken from the column outflow for use as reagent blanks in the periodide test. In order to determine the position of elution, a 1 ml aliquot of a stock solution was placed on the column. The flow rate was 68 to 70 ml/hr and the eluant was collected in 1 ml aliquots in a fraction collector (Gilson Medical Electronics, Middleton, Wisconsin). These aliquots were analyzed by the periodide method of Hayashi et al. (1)) but with a slight modification. The addition of 0.2 ml of acetate buffer was omitted from the procedure. Potassium iodide (0.1 ml of 0.1 M) was added to each 1 ml eluant fraction and to 1 ml of eluant buffer for a reagent blank. Then extraction into 4 ml of ethylene dichloride reagent (0.5% I, in ethylene dichloride) was done. Absorption was measured at 385 rnp with a Gilford spectrophotometer 300 (Gilford Instrument Co., Oberlin, Ohio). Recovery of each of the bases from the column was determined in separate experiments. The absorption of 1 ml of stock base was measured by the periodide method. One milliliter of stock solution was applied to the column and the absorption of each fraction was measured. Since preliminary studies indicated linear standard curves, the ratio of the sum of the absorbancies of the fractions to the absorbance of the stock solution was taken as per cent recovery. Molar extinction coefficients were calculated. in order to compare the. relative absorbancies of. the periodide reaction’ptioducts.. ~Ekjwimiintal Results and Discussion. The compounds tested, lthe ‘cbncentrations ‘of stock solutions, the calculated-molar. extinction coe%icien&a; and the average per cent recoveries are shown in Table 1. Figure 3. shows! the posit%ons of elution of ten bases tested; eight’are completely.separable from choline. The trailing edge of betaine overlapped the leading ‘edge of choline. More complete separation of these two compounds might be
SHORT
199
COMMUNICATIONS
Betaine Triet&jami:l+, I 0
15
30 Eluant
15
ii 0
(ml)
FIQ. 1. Eluant pattern of different bases from Dowex 50-X8 column. The quantities of material applied to the column are given in Table 1, to demonstrate the number of milliliters of eluant in which the compounds were detectable with the periodide test.
effected by collecting appropriate aliquots in the fraction collector. The only base which significantly overlapped choline was tetramethylammonium. However, a longer column than 6.5 cm, and alteration of the pH of the buffer, may effect complete separation. The data for the amino acids lysine, histidine, and arginine are not included because these amino TABLE 1 Concentrations,
Calculated
Molar Extinction
Compound
Stock concentrstioll, ~mole/ml
0.358 0.165 0.252 0.324 0.348 0.342 0.523 0.489 0.466 1.502 7.143
Choline chloride Thiamine hydrochloride o,L-Carnitine chloride Tetramethylammonium bromide Betaine hydrochloride Diphenhydramine hydrochloride Trimethylamine hydrochloride n,cTryptophan Ephedrine sulfate Tryptamine hydrochloride Triethylamine hydrochloride 0 385 q,
Gilford spectrophotometer
Coefficients and Per Cent Recoveries M&I extinction co&icient~
4,050 14,545 6,865 4,877 4,023 3,392 3,231 2,822 2,210 1,643 377
Averaee
7%
WCOPWy
99.9 92.0 95.4 99.0 95.2 80.1 93.8 103.0 87.3 73.3 98.0
300.
acids are not periodide positive. The usual elution sequence on these columns is lysine, histidine, ammonia, and arginine. Choline elutes between histidine and ammonia. The usefulness of this system is that it takes less than one hour for elution of all the bases tested, and in most instances the separation from choline is complete. ACKNOWLEDGMENTS This investigation was supported in part by U. S. Public Health Service Grants He-08446 and 5TlGM-125 from the National Institutes of Health. Mrs. Susan Kobeck was very helpful in obtaining some of the experimental results.
200
SHORT
COMMUNICATION
REFERENCES 1.
HAYASHI,
2. SPACKMAN,
M., UNEMOTO, T., D. H.,
STEIN,
AND
MIYAKI, K., Chem. Pharrn. Bull. 10,633 (1902) S., Anal. Chem. 30, 1185 (1958).
W. H., AND MOORE,
K. GOURLEY~ CHARLES D. H.L&
WILLIAM
SEYMOUR
BAKERMAN
Departments of Biochemistw and of Pathology and Oncology University of Kanwe Medical Center Kansas City, Kansas 6f31W Received January 9,1967 1 Taken in part from M.S. the& of W. G. st University ’ Medical student summer Fellow.
of Kaums, 1967.