Separation of diacetyl, acetoin, and 2,3-butylene glycol by salting-out chromatography

ANALYTICAL

BIOCHEMISTRY

Separation

22,

154-160 (1968)

of Diacetyl,

Glycol

Acetoin,

by Salting-Out

R. A. SPECKMAN Department

of Food

Science

and

and

2,3-Butylene

Chromatography AND

E. B. COLLINS

Technology,

Davis, California

University

of California,

96616

ReceivedFebruary 10, 1967 Lack of a method for quantitative separation of mixtures of compounds in microgram quantities has hampered studies of the mechanisms by which diacetyl (2,3-butanedione) , acetoin (3-hydroxy-2-butanone) , and 2,&butylene glycol (2,3-butanediol) are formed from pyruvate, and the metabolic interrelationships among these compounds. Diacetyl and acetoin have commonly been determined as the sum of both, by the calorimetric procedure of Westerfeld (1) or by the steam-distillation procedure of van Niel et aE. (2) as modified by Michaelian and Hammer (3) or Prill and Hammer (4). The latter procedure can be used to determine diacetyl and acetoin differentially, but large quantities of the compounds are required and the method has inherent disadvantages, including possibilities of thermal decomposition, chemical rearrangement, and oxidation of the compounds being separated. Gas chromatography can be used for determining diacetyl and acetoin, but this method requires clarification of biological test systems and is not readily adaptable to quantitative studies. Acetoin, diacetyl, and 2,3-butylene glycol are neutral, water-soluble, structurally related, organic nonelectrolytes, representing three levels of oxidation of the same four-carbon skeleton. This paper presents a procedure for separating them quantitatively by salting-out chromatography, which is the separation of water-soluble nonelectrolytes by elution with an aqueous salt solution through a column of ion-exchange resin. Preliminary results with this method substantiate its usefulness (5-7). The procedure is based on the report of Sargent and Rieman (8)) who found that separation of organic nonelectrolytes was greatly improved when a salt solution was used instead of water as eluant on columns of ion-exchange resin. They reported separating several series of closely related compounds with the new procedure. Additionally, the present paper includes modifications of the method reported by Happold and Spencer for determining 2,3-butylene glycol (9). 154

SEPARATION

OF

MATERIALS

DIACETYL

AND

ACETOIN

155

AND METHODS

Columns. Analytical-grade Dowex l-X8 anion-exchange resin in the chloride form, 200-400 mesh, washed alternately with 1 N NaOH and 1 N HCI, was used for experiments involving columns of two sizes with different physical and chemical parameters. Column I had a diameter of 1.9 cm and was packed to a bed height of 27 cm with resin slurried in 3.0 M (NH,) $0,. The resin in this column was converted to the sulfate form by washing with 3.0M (NH,)$O, until tests for chloride ions in the effluent were negative. Column II had a diameter of 3.5 cm and was packed to a bed height of 54 cm with resin slurried in saturated Na,SO,. This column was washed with saturated Na,S04 until tests for chloride ions in the efluent were negative. Xeparation, Elution, and Collection. All separations were made at room temperature; 1 ml samples of solutions containing 25 to 100 pg of each of the compounds to be separated were placed on top of the resin beds. Diacetyl, acetoin, and 2,3-butylene glycol were separated and eluted by washing columns I and II, respectively, with 0.5M (NH&SO, at a flow rate of 0.5 ml/min and 0.5 M Na,SO1 at a flow rate of 1.5 ml/min. Effluents were collected and distributed into t.est tubes by a fraction collector. Fraction sizes were determined by a drop counter, synchronized with the collector. Conversion of 2,S-Butylene Glycol to Acetoin. Column II, with 0.5 M Na,SOa as eluant, proved advantageous in the separation and determination of 2,3-butylene glycol. After separation, the glycol was converted to acetoin by the following procedure, based on the oxidation of 2,3-butylene glycol with bromine water, first reported by Happold and Spencer (9) : (1) To 5 ml of the glycol fraction plus 4 ml of water in a red, light-resistant., 15 mm X 15-cm test tube capped with a 12 mm rubber septum, 4 ml of bromine water was added with a syringe. (2) The mixture was heated for 3 min in the dark in a bath of boiling water. (3) Next, the mixture was transferred to a water bath at room temperature and held for 15 min in the dark. (4) Finally, a small amount of Na,SO, (on a spatula point) was added to neutralize excess bromine water. Determination of Diacetyl or Acetoin. With diacetyl, acetoin, and 2,3hutylene glycol separated, and the glycol oxidized to acetoin, the Westerfeld method (1) was used for their determination. Chemicals. Diacetyl and acetoin were obtained from Eastman Organic Chemicals; 2,3-butylene glycol and esterified tu-acetolactate were obtained from K & K Laboratories, Jamaica, New York. An infrared spectrum determined for the compound received from K & K Laboratories indicated that it was the acetoxy ethyl ester of a-acetolactate.

156

SPECKMAN

AND

COLLINS

a-Acetolactate was prepared from the ester by saponifying with two equivalents of cold 0.025 N NaOH, added slowly to the system held at 5°C and continuously purged with a stream of nitrogen. The creatine and recrystallized su-naphthol, used as reagents for the Westerfeld test, were respectively obtained from Calbiochem and Fisher Chemical Co. RESULTS

AND

DISCUSSION

Nature of the Eluad. The following eluant salts were compared on columns with the dimensions of column I except that the resin was left in the chloride form for studying the chlorides: (NH,),SO,, NH,Cl,

WAVELENGTH

(mu 1

FIG. 1. Absorption spectra of eluant salt solutions after oxidation with bromine water and addition of Westerfeld reagents; 5 ml of a 0.5 M eluant-salt solution was oxidized with bromine water (see text), Westerfeld reagents were added, and the absorption spectrum was determined with a Beckman model DB spectrophotometer: (A) N&O,; (B) (NH,)zSOa.

NaCl, and Na.,S04. For each test, 1 ml of an aqueous solution containing 100 pg each of diacetyl, acetoin, and 2,3-butylene glycol was placed on the column. Each of the four salts, at a concentration of 0.5 M and a flow rate of 0.5 ml/min, with a fraction size of 1.6 ml (32 drops), separated diacetyl and acetoin, but only (NH,),SOi completely separated acetoin and 2,3butylene glycol. The compounds emerged from the column in decreasing order of polarity, irrespective of the salt used: 2,3-butylene glycol, acetoin, and diacetyl. Recoveries were quantitative with each of the salts, showing that adsorption of the compounds to the column was not a

SEPARATION

OF' DIACETYL

AND

ACETOIIG

157

problem. Recoveries of the three compounds in over 100 determinations ranged from 95 to 104%. The compounds appeared earlier in the effluent volume with the ammonium salts, and in more compact bands. Resolution was somewhat better with the sodium salts and with the sulfates. An increase in concentration of each of the eluant salts resulted in an increase in elution volun~e for each component of the mixture, but resolution of the compounds was not improved. No further attempts were made to get better resolution of acetoin and 2,3-butylene glycol on column I with the three salts not giving excellent resolution.

FIG. 2. Elution pattern of a mixture of 2,3-butylene glycol, acetoin, and diacetyl chromatographed on Dowex l-X8 anion-exchange resin in the sulfate form. A 1 ml aqueous solution containing 100 pg each of 2,3-butylene glycol (A), acetoin (B), and diacetyl (C) was added to a 3.5 x 54 cm column. Elution was with 0.5M NazSOl at a Aow rate of 1.5 ml/min. Fractions of 6 ml were collected, and those containing glycol were oxidized to acetoin (see text). Subsequently, all fractions were analyzed by the Westerfeld procedure (1).

Analysis of Fractions Containing Diacetyl or Acetoin. The Westerfeld method (1) was used. With 0.5 M Na,SOa as eluant, no difficulties were encountered. With 0.5 M (NH,),SO, as eluant, no difficulties were encountered if the aliquots used in the Westerfeld test were no larger than 3 ml. Larger aliquots resulted in a white precipitate. Analysis of Fractions Containing 2,J-Butylene Glycol. Difficulties were encountered in the oxidation and determination of 2,3-butylene glycol separated on column I by washing with (NH,),SO,. Upon addition of the Westerfeld reagents, following oxidation of the glycol to acetoin

158

SPECKMAN

AND

COLLINS

with bromine water (9), a masking emerald-green color was encountered. The green color appeared upon addition of the reagents and likely resulted from their reacting with products from a reaction of (NH,).SOa with the bromine water. Absorption spectra in Figure 1 indicate such a reaction. One method found for avoiding the green color was to ensure complete oxidation of the (NH,)&$O, by increasing the heating period required for oxidizing the glycol. We found a 45 min heating period satisfactory. Another method was to use 0.5 M Na,SO, as eluant in place of the (NH,),SO,. With this substitution, a 3 min heating period was found to be satisfactory for oxidizing the glycol, but column I did not give

80A

20-

EFFLUENT

VOLUME

(ml)

3. Elution pattern of a mixture of acetoin and diacetyl chromatographed on Dowex l-X8 anion-exchange resin in the sulfate form. A 1 ml aqueous solution conta.ining 199 pg each of acetoin (A) and diacetyl (B) was added to a 1.9 x 27 cm column. Fractions of 1.5 ml were collected during elution with 0.5 M (NH,)ZSO, at a flow rate of 0.5 ml/min. Fractions were analyzed by the Westerfeld procedure (1). FIG.

clear-cut separation of acetoin and 2,&butylene glycol with 0.5M Na,SO, as eluant. The possibility was investigated of using a larger column with 0.5M NazSOa as eluant. Column II, described in the section on methods, was prepared and tested. This larger column accomplished excellent separation of all three compounds, with the flow rate increased to 1.5 ml/min and the fraction size to 6.2 ml (128 drops). Figure 2 shows the separation of diacetyl, acetoin, and 2,3-butylene glycol. Figure 3 shows the separation of diacetyl and acetoin with column I. Where 2,3-butylene glycol is

SEPARATION

OF

DIACETYL

AKD

15Y

ACETOIN

not involved or its determination is not required, column I with 0.5M (NH,),SO, as eluant is satisfactory. Interfering Substances. Application of a column chromatographic procedure to complex biological test systems requires investigation of the possibility that other substances might interfere with the results. Pyruvatc, citrate, and sugars arc ruled out by the fact that they do not react with the reagents of the Westerfeld test to give the typical red color. Acetaldehyde gives an atypical color, but 24 hr is required (lo), and we found acetaldehyde to appear in an effluent volume region different from that of diacetyl, acetoin, or 2,3-butylene glycol. Thiamine is not normalIy present in the large amounts reported to produce a redcolored complex with reagents of the Westerfeld test (10). A compound of considerable concern was a-acetolactate. This acid is stable above neutrality (11). Thus, for testing 8a-acetolactate, converted to the free-acid form as indicated in the section on methods, the eluant salt solutions were adjusted to pH 7.0, to avoid the possibility of oncolumn decarboxylation. a-Acetolactate did not move in the same effluent volume regions as diacet,yl, acetoin, or 2,3-butylene glycol. Columns I and II are being applied in studies dealing with diacetyl biosynthesis and the metabolic interrelationships among diacetyl, acetoin, and 2,3-butylene glycol (6, 7). Considerable data accumulated to date show that the addition directly to the columns of complex biologica materials, e.g., cell-free extracts, does not interfere with their efficiency in quantitatively separating diacetyl, acetoin, and 2,3-butylene glycol. SUMMARY

Salting-out chromatography was found suitable for separating microgram quantities of diacetyl, acetoin, and 2,3-butylene glycol at room temperature. One milliliter of a solution containing 25 to 100 pg of each of the compounds to be separated is applied to the top of a column of Dowex l-X8 anion-exchange resin in the sulfate form. The compounds are separated and eluted from a 1.9 X 27 cm column with 0.5 M (NH,),SO, at a flow rate of 0.5 mI/min, or from a 3.5 x 54 cm column with 0.5 M NatSO at a flow rate of 1.5 ml/min. The compounds emerge from the columns quantitatively in decreasing order of polarity: 2,3butylene glycol, acetoin, and diacetyl. The separated 2,3-butylene glycol is oxidized to acetoin with bromine water. With the compounds separated and the glycol converted to acetoin, the Westerfeld method can be used to complete the quantit,ative determinations. ACKNOWLEDGMENT This National

investigation Institutes

was supported in part by of Health, U. S. Public Health

research Service.

grant

EF-138

from

the

160

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AND

COLLINS

REFERENCES 1. WESTERFELD, 2.

3. 4. 5. 6. 7. 8.

9. 10.

11.

W. W., J. Bid. Chem. 161, 495 (1945). VAN NIEL, C. B., KLUWER, A. J., AND DERX, H. G., Biochem. Z. 210, 234 (1929). MICHAELIAN, M. B., AND HAMMER, B. W., Iowa Agr. Expt. Sta. Res. Bull. No. 179, 201 (1935). PRILL, E. A., AND HAMMER, B. W., Iowa State Coil. J. Sci. 12, 385 (1938). SPECKMAN, R. A., AND COLLINS, E. B., J. Dairy Sei. 48, 766 (1965). SPECIZMAN, R. A., AND COLLINS, E. B., Bacterial. Proc., 1966, 6. SPECKMAN, R. A., AND COLLINS, E. B., J. Dairy sci. 49, ‘701 (1966). SARGENT, R., AND RIEMAN, W., J. Org. Chem. 21, 594 (1956). HAPPOLD, F. C., AND SPENCER, C. P., Biochim. Biophyls. Acta 8, 18 (1952). YATCXI-MANZO, E., RODDY, F., YOUNT, R. G., AND METZLER, D. E., J. Biol. Chem. 234, 733 (1959). KOBAYASHI, Y., AND KALNITSKY, G., J. Biol. Chem. 211, 473 (1954).