Separations of isomeric polyols

ANALYTICAL

BIOCHEMISTRY

26,

(1968)

21$-230

Separations S. A. BARKER,

of

;\I. J. HOW,

lsomeric

Polyols

P. V. PEPLOW,

AND

P. J. SOMERS

Chemistry Department, University of Birmingham, Birmingham

15,

England

Received February 16, 1968 The separat’ion and determination of sugar alcohols is important in plant analysis. They are widely distributed among the different plant families (1). Certain polyols are inhibitors of glycosidases of plant origin (2, 3). Mixtures of sugars and sugar alcohols are formed in the structural analysis of plant and bacterial polysaccharides by periodate oxidation, reduction, and acid hydrolysis (4) (Smith degradation). Partition chromatography on ion-exchange resins can be used to separate sugars and sugar alcohols (5). This paper describes the application of automated chromatography to mixtures of sugars and sugar alcohols using an anion-exchange resin in the molybdate form. METHODS Separations

AND RESULTS

on Dowex

1 (Molybdate)

Preparation of resin. In all preparations of resin and subsequent elutions, boiled and deaerated water was used. AG l-X3 resin (Cl- form, 200-400 mesh, 30 ml) was washed over 3 hr with sodium hydroxide (1 N, 6 liters), and then with water (2 liters). The resin was treated over 2 hr with sodium molybdate (Hopkin and Williams G.P.R., batch 67094, 1 ;1[, 2 liters)-cloudiness in the solution was removed by filtration of the solution at 50”. After washing with water (2 liters), the resin was packed into a jacketed glass column (60 X 2 cm), maintained at 50” by a circulating pump, to give a bed of 530 X 6 mm. Water was passed through the column at a flow rate of approximately 0.25 ml/min for at least 2 hr before use. Analytical systeft&. Sugars were determined by reaction with cysteinesulfuric acid reagent (6). In the measurement of hexoses, the reaction mixture was heated (98”, 3 min), and the absorbance was measured at two wavelengths (404 and 420 mp). 219

220

B.4RKER,

HOW,

PEPLOW,

AND

SOMERS

Sugar alcohols were measured using acetylacetone to determine the formaldehyde released on periodate oxidation (5). The flow diagram of the Technicon AutoAnalyzer system used is shown in Figure 1. Sep~rucions. Aliquots (1.0 ml) of solutions of sugar alcohols were

Heo b

Calorimeters FIG.

1. Automated

Recorder

analysii of sugar alcohols and sugars:

Column eluate Air HI04 (0.015 M) neutralized with Nl% and buffered to pH 7.5 with a phosphate buffer (100 ml/liter) NaABOz (0.25 M) neutralized with HCl to pH 7.0 Acetylacetone (0.02 M) in a solution of ammonium acetate (2 M) and acetic acid (0.05 M) Column eluate Air lrCy&eine HCl (0.07% w/v) in HpS04 (86% v/v) Abeorbances measured in 15 mm flow cells.

Pumping rate, ml min d &hod A 0.23 0.23 0.32 0.16 0.33

Method 0.10

0.16 0.32

B

0.32 0.60

0.10 0.23 0.53

applied to the column and eluted with water. The separation obtained in Figure 2 was of glucose (89 pg), glycolaldehyde (62 pg), glycero1 (104 pg), propane-1,2-diol (42 pg), and ethylene glycol (46 ,,ug) (peak l), D-threitol (68 pg) (peak 2), and erythritol (82 pg) (peak 3) on a resin bed of 530 X 6 mm, using a column flow rate of 0.26 ml/min. Method A (see Fig. 1) of sugar alcohol analysis was used.

POLYOL

SEPARATIONS

Elulion

volume

221

(ml)

FIG.

2. Separation of a complex mixture containing threitol and erythritol: (-) formaldehyde assay (optical density 420 m/L), (- - -) cysteine-HSO, assay (optical density 420 m/l), (. . .) cysteine-H2SOI assay (optical density 404 ma). Peak 1 = glucose, glycolaldehyde, glycerol, propane-l,Z-diol, and ethylene glycol. Peak 2 = n-threitol. Peak 3 = erythritol.

The separations obtained in Figure 3 were of ribitol (180 pg) (peak 1) and arabitol (309 Fg) (peak 2), allitol (143 pg) (peak 3) and mannitol (835 pg) (peak 4), and lactitol (213 pg) (peak 5) and melibiitol (2.42 mg) (peak 6) on a resin bed of 190 X 6 mm, using a column flow

FIQ.

3. Separations of isomeric sugar alcohols: (-) ribitol (peak 1) and arabitol 2) ; (- - -) allitol (peak 3) and mannitol (peak 4) ; (0. .) lactitol (peak 5) and melibiitol (peak 6). Sugar alcohols determined by formaldehyde assay (optical density measured at 420 mp.

(peak

222

BARKER,

HOW,

PEPLOW,

AND

SOMERS

rate of 0.45 ml/min. Method B (see Fig. 1) of sugar alcohol analysis was used. Calibration. The recoveries of the tetritols on eluting from Dowex 1 (molybdate) with water at 50” were determined by applying aliquots (1.0 ml) of solutions of n-threitol (208 pg/ml), and of erythritol (220 pg/ml) to the column. The column was washed with water (50 ml), and the amount of tetritol recovered from the column was determined by assaying the formaldehyde released on periodate oxidation. An aliquot (0.50 ml) of the eluate was treated with periodic acid (0.015 M, pH 7.5, 0.50 ml), and after standing for 20 min at room temperature, unreacted periodate was destroyed by addition of sodium arsenite solution (0.25 M, pH 7.0, 0.50 ml). Acetylacetone reagent (0.02 M, 1.0 ml) was added to the sample solutions and, after mixing, the solutions were heated at 98” for 3 min. After cooling, the absorbances were measured at 415 rnp using a Unicam SP 500 spectrophotometer. Standard solutions of n-threitol (4.2 pg/ml), and of erythritol (4.4 ,pg/ml) were used in the assay. It was calculated that the amounts of threitol and erythritol recovered from the column were 227 and 220 pg, respectively.

FIG. 4. (a) Calibration of Dowex 1 (molybdate) with tetritol. (b) Rate of release of formaldehyde on periodate oxidation of tetritol. (0) n-Threitol. (0) Erythritol.

Aliquots (1.0 ml) of solutions of the tetritols (O-150 pg of each alcohol) were applied to the column, and eluted with water using a flow rate of 0.26 ml/min. The eluate was analyzed for sugar alcohol using Method A. The peaks obtained on the chart recording were integrated by a trapezoid summation. A linear relationship was found between the peak area and amount of added tetritol-the calibrations are shown in Figure 4 (a) .

POLYOL

SEPARATIONS

223

Aliquots (1.0 ml) of solutions containing (i) n-threitol (51 pg/nll), and erythritol (51 pg/ml), (ii) n-threitol (51 &/ml), erythritol (51 pg), ethylene glycol (45 pg/ml), glycerol (57 p&ml), and prwane-l?-diol (77 pg/ml), and (iii) n-threitol (51 pg/ml), crythritol (51 pg/ml), glycolaldehyde (42 pg/ml), and glucose (47 pg/ml) were fractionated by chromatography on Dowex 1 (molybdate) . No interference was found in the determination of the tetritol by compounds which may be formed by periodate oxidation, reduction, and acid hydrolysis of polysaccharides. The areas of the peaks corresponding to threitol and erythritol on the chart recording, using the automated determination of sugar alcohol, were in (i) 2.45 and 4.42 units, (ii) 2.45 and 4.43 units, and (iii) 2.61 and 4.36 units. Periodate oxidation of tetritols. Tetritol solution (31 pg/ml, 20 ml) was treated with periodic acid (0.015 M, pH 7.5, 32 ml) at room temperature. Aliquots (2.6 ml) were removed at intervals (15 min) and added to tubes containing sodium arsenite solution (0.25 M, pH 7.0, 1.6 ml). When all the aliquots had been removed (3 hr), the formaldehyde contents of the periodate-oxidized samples were determined by addition of acetylacetone reagent (0.02 M, 3.2 ml) and heating the solutions at 98” for 3 min. The absorbances were measured at 415 mp after cooling the solutions, and t,he amount of formaldehyde released was calculated. The rates of release of formaldehyde on oxidation of tetritol are shown in Figure 4(b). Selective oxidation of hexitols. Glucitol solution (0.5 M, 10 ml) was treated with sodium periodate (0.05 M, 10 ml). After standing at room temperature for 1 hr, iodate was removed from the solution by addition of barium carbonate. The solution was concentrated to 20 ml in vacua, and sodium borohydride (120 mg) was added over 3 hr. The solution was treated with Dowex 5OW-X8 (H+, 20-50 mesh, 4 gm) and was left to stand for 2 hr at room temperature. The resin was removed by filtration, and the solution was evaporated to dryness in uacuo. Borate was removed from the residue by distillation with methanol (5 x 25 ml). The residue was dissolved in water (25 ml), and an aliquot (0.20 ml) was fract’ionated on Dowex 1 (molybdate) at 50”. Erythritol (69 pg in aliquot applied to column) wa s found in the presence of compounds having elution positions corresponding to glycerol and glucitol. Galactitol solution was oxidized similarly, and the products reduced as above. Analysis of the reduced products by chromatography on Dow-es 1 imolybdate) showed threitol (150 pg in aliquot applied to column) in the presence of compounds having elution positions corresponding to glycerol and galactitol. The presence of tetritol in the products from the partial oxidation and

224

BARKER,

HOW,

PEPLOW,

AND

SOMERS

reduction of glucitol and galactitol was confirmed by gas-phase analysis and mass spectrometry of the trimethylsilyl (TMS) derivatives of the products (7). An aliquot (0.20 ml) of the solution containing the products of the degradation was fractionated on Dowex 1 (molybdate), using a flow rate of approximately 0.23 ml/min. Fractions of column eluate were collected at 3 min intervals, and aliquots (0.40 ml) of those fractions containing the compound believed to be a tetritol were bulked and evaporated to dryness in W.XXO. Aliquots (1.00 ml) of standard solutions of n-threitol (228 pg/ml) , and of erythritol (200 pg/ml) , were separately evaporated to dryness in vacua. The TMS derivatives of the residues were prepared by the addition of pyridine (0.506 ml), hexamethyldisilazane (0.100 ml), and trimethylsilyl chloride (0.050 ml). After shaking well for 15 set, the reaction mixtures were left to stand at room temperature for 2 hr. The solutions were then evaporated to dryness in vacua, and the TMS derivatives were extracted into n-hexane (0.500 ml). An aliquot (5 ,ul) of the solution of TMS derivative in hexane was injected into a Pye 104 gas chromatograph fitted with a column (5 ft X 4 mm) of SE-30 (10% w/w) on Celite (100-120 mesh). The temperature of the column was 153” and, using nitrogen as carrier gas, the flow rate was 40 ml/min. The retention times of the TMS derivatives of the standards of threitol and erythritol were found to be identical (14.6 min) , and the presence of a tetritol in the products from the degradation of the hexitols was confirmed by gas-liquid chromatographic analysis of the TMS ethers. Solutions of the TMS derivatives of the tetritol-(i) for threitol 1.9 pmoles and (ii) for erythritol 1.6 pmoles-in hexane (0.50 ml) was evaporated by removal of hexane in a stream of nitrogen. The mass spectrum of the residue was measured with an AEl MS9 mass spectrometer, using a heated inlet system (180”). The spectra for the TMS ethers of threitol and eryt,hritol are shown in Table 1. Peak abundances are expressed as percentages of the total abundance; peaks having abundances < 1.0% are not reported. Solutions (0.50 ml) of the TMS derivatives of the products, believed to be tetritols, from the degradation of the hexitols were similarly treated, and the mass spect.ra recorded. Production of threitol and erythritol from the oxidation of galactitol and glucitol, respectively, was confirmed. Glucitol solution (0.5 M, 10 ml) was treated with sodium periodate (O.O5M, 10 ml) and, after 1 hr, iodate was removed from the solution by addition of barium carbonate. The solution was deionized by addition of Dowex 5OW-X8 (H’, 20-50 mesh. 4 gm). The resin was removed by

POLYOL

225

SEPARATIONS

filtration, and the solution was concentrated to 25 ml in vacua. An aliquot (0.20 ml) of the solution was fractionated on Dowex 1 (molybdata) (530 X 6 mm). Compounds having elution po’sitions corresponding to glyceraldehyde, erythrose (elution volume 18.4 ml), and glucitol were found. Galactitol solution was similarly oxidized, and analysis of t’he products by chromat’ography on Dowex 1 (molybdak) showed the presence of compounds having elution positions corresponding to glyceraldehyde, galactitol, and a compound believed to be threose (elution volume 14.0 Mass

Spectra

TdBLE 1 of TMS Derivatives Percentage

abundance

khS.3

No.

Threitol

Erythritol

i3 74 75 77 103 116 117 129 133 147 148 189 191 204 205 206 207 217 518 307

31.7 1.9

“0.6 1.9

-

2.

6

2..

‘)

1 .o 8.2

1.7 3.5 1.1 1.3 7,s 1.3 2.2 1.9 6 6.9 1 5

2.

7.4 1.7 2. 4

6.9

1.9 3

6

17 1.4 7.7

1.2 4 1.9 2.9 i ” 1.7 1 0 69 1.7 2 2

2

ml). Fractions of column eluate were collected at 3 min intervals, and aliquots (0.40 ml) of those fractions containing the compound believed to be threose were bulked. The solution was treated wit’h sodium borohydride (40 mg) over 2 hr. Dowex 5OW-X8 (H+, 20-50 mesh, 4 gm) was added to the solution and was left to stand overnight at room temperature. The resin was removed by filtration, and the solution was evaporated to dryness in. varuo. Borat’e was removed by distillation with methanol (4 x 25 ml), and the residue was dissolved in water (0.30 ml). The solution was applied to Dowcx 1 (molybdate) size (530 x 6 mm), and a compound eluted with the same elution volume as threitol.

226

BARKER,

HOW,

Separations

PEPLOW,

on Dowex

AND

SOMERS

1 (Sdfat,e)

Preparation of resin. In all preparations of resin and subsequent elutions, degassed ethanol (86% v/v) was used. AG l-X8 resin (Clform, 200-400 mesh, 30 ml) was washed over 3 hr with sodium hydroxide (1 N, 6 liters), and then with water (2 liters). The resin was treated over 2 hr with sodium sulfate (1 M, 2 liters)-cloudiness in the solution was removed by filtration. After washing with water (2 liters), the resin was packed into a jacketed glass column (72 X 2 cm), maintained at 50” by a circulating pump, to give a resin bed of 580 x 6 mm in ethanol (86% v/v). Ethanol (86% v/v) was passed through the column at a flow rate of approximately 0.3 ml/min for 3 hr before use. Analytical system. The automated determination of sugar alcohols by Method A was used as previously described, except that a lower heating bath temperature (78”) was necessary. Separations. Aliquots (1.0 ml) of solutions of sugar alcohols in ethanol (86% v/v) were applied to the column, and eluted with ethanol (86% v/v). The separation obtained in Figure 5(a) was of propane-1,2-diol (64 ,ag) (peak 1)) ethylene glycol (52 pg) (peak 2), glycerol (88 pg) (peak 3), and n-threitol (48 pg) (peak 4) in ethanol (86% v/v) on a resin bed of 580 X 6 mm using a column flow rate of 0.32 ml/min. The separation obtained in Figure 5(b) was of glycolaldehyde (67 pg) (peak 1)) glycerol (104 pg) (peak 2)) erythritol (135 ,kg) (peak 3)) and xylitol (180 pg) (peak 4) in ethanol (86% v/v) on a resin bed of 580 X 6 mm, using a column flow rate of 0.52 ml/min. No separation of the tetritols was obtained when an aliquot (1.0 ml) of a solution containing n-threitol (52 ,pg/ml) and erythritol (73 pg/ml) in ethanol (86% v/v) was applied to the column, and eluted with ethanol (86% v/v) at a flow rate of 0.40 ml/min. Analysis of Products from Periodate Oxidation

of Several

Carbohydrates

Carbohydrate (4 mg) was treated with periodic acid-(i) methyl (YD-glucopyranoside with 2 X 10m4 mole periodate for 24 hr, (ii) methyl /3-D-maltoside, 1 X 1W mole, 22 hr, (iii) Schardinger /3-dextrin, 1 )( lo+ mole, 67 hr, and (iv) mixture of alcohols of nigeranl tetrasaccharides, 2 X 10-* mole, 14 hr-at room temperature (18”) and the uptake of periodate was followed spectrophotometrically by measuring the absorbance at 222.5 rnp after suitable dilution (8). When no more periodate was consumed, the solution was neutralized by addition of barium ’ O-n-D-&

3)

o-a-D-&

(1 (1

4)

+ --)

4)

O-a-D&C o-a-D-&

(1 (1

+

3)

--)

3)

o-a-D-& o-a-D-&.

(1

-

4)

O-a-D-gk

:

&x-D-&

(1

+

POLYOL

227

SEPARATIONS

carbonate. After concentration of the solution to 20 ml, sodium borohydride (20 mg) was added over 3 hr. The solution was treated with Dowex 5OW-X8 (H+, 20-50 mesh, 4 gm) and was left to stand at room temperature for 1 hr. The resin was removed by filtration and the solution evaporated to dryness in vacua. Borate was removed from the residue by distillation with methanol (4 X 25 ml), and the residue hydrolyzed in sulfuric acid (1 N, 7 ml) at 98” for 3 hr. The hydrolyzate

00

12.8

25.6 Elut~on

Elutlon

384 volume

volume

51.2

640

(ml1

(ml)

FIG. 5. Separations of sugar alcohols: (a) (1) = propane-1,2-diol, (2) = ethylene glycol, (3) = glycerol, (4) = D-threitol; (b) (I) = glycolaldehyde, (2) = glycerol, (3) = erythritol, (4) = xylitol. Sugar alcohols determined by formaldehyde assay (optical density measured at 420 mpL).

was neutralized by addition of barium carbonate, and the solution was passed through a column of Dowex 5OW-X8 (H’, 20-50 mesh) (25 X 0.6 cm). The solution was analyzed by chromatography on Dowex 1 (molybdate) and Dowex 1 (sulfate). The degradations of methyl a-n-glucopyranoside and Schardinger ,8dextrin produced glycerol and erythritol, respectively. Oxidation of methyl

228

BARKER,

HOW,

PEPLOW,

AND

SOMERS

/3-n-maltoside yielded glycerol and erythritol in a mole ratio of 1.0 to 0.93. A mixture of the alcohols of the nigeran tetrasaccharides (O-,-Dglc (1 + 4) o-cl-n-glc (1 + 3) O-a-D-glc (1 + 4) O-a-D-glc and o-a-D-gk (l+ 3) O-cy-n-glc (1 -+ 4) O-a-D-glc (1 + 3) O-a-n-glc) gave on degradation equimolar amounts of erythritol and glucose. Glycerol was detected also. DISCUSSION

The separation and determination of threitol and erythritol is important in structural studies on plant and bacterial polysaccharides using the method of periodate oxidation, reduction, and acid hydrolysis; the production of threitol and erythritol characterizes a 1,4-linked galactoand 1,4-linked gluco-pyranoside unit, respectively. Partition chromatography on Dowex 1 (sulfate) at 50” using ethanol (86% v/v) as eluant failed to separate the tetritols. Moreover, gasliquid chromatography of these isomers as trimethylsilyl ethers (7), using a column of SE-30 (10%) on Celite and nitrogen as carrier gas, did not give a separation. However, different retention times for erythritol and threitol acetates have been reported using butanediol succinate polyester on Chromosorb W (10). Weigel (9) has found that paper electrophoresis in molybdate solution (pH 5.0) effects the separation of the tetritols, but in borate solution (pH 9.2) no separation was obtained. Partition chromatography on Dowex 1 (molybdate) was carried out in a way similar to that described for Dowex 1 (borate), in which borate complexes are applied to the column and eluted with borate solution having varying concentration or pH. It was not possible to elute the complexes of the tetritols, formed in sodium molybdate (0.005 M, pH 5.0)) from Dowex 1 (molybdate) at room temperature using sodium molybdate (1 M, pH 8.5)) or sodium molybdate (1 M) having a pH changing from 8.5 to 12.1. When the tetritols in ethanol (86% v/v) were applied to Dowex 1 (molybdate) (approximately 15 ml) at 50” and eluted with ethanol (86% v/v), they were not eluted in a volume of 220 ml. On passing water through the column at 50”, threitol separated from erythritol. Threitol, which is considered to form the weaker complex with molybdate, was eluted first. The tetritols were not eluted from the column at room temperature using water. This method of separating the tetritols does not require the formation of a complex prior to application to the column, and elutions are carried out with water, which allows an easy recovery of the compounds. Some variation was found in the absolute retention times of threitol and erythritol when different batches of Dowex 1 were used. However,

POLYOL

SEPARATIONS

229

with a given batch of resin, reproducible elution positions were obtained using the regeneration procedure described. One batch of resin was found to give no separation of the tetritols. In the calibration experiment in which different amounts of tetritol were applied to Dowex 1 (molybdate) at 50” and eluted with water, a linear relationship was found for each tetritol between peak area on the chart recording and amount of tetritol added. However, a much greater response to erythritol than threitol was obtained when using the automated determination of sugar alcohol. This is probably caused by the periodate oxidation of erythritol being more favorable for steric reasons than in the oxidation of threitol. Thus, in this case, the nature of the sugar alcohol can be predicted by its rate of oxidation with periodate. Separations of other isomeric alcohols were obtained: lactitol was separated from melibiitol, ribitol from xylitol and arabitol, and allitol from glucitol, galactitol, and mannitol. The separation of allitol and ribitol from other pentitols and hexitols gives a simple method of recovering these rare sugar alcohols from mixtures. The selective oxidation of glucitol and galactitol (11) has been carried out, and by chromatography on Dowex 1 (molybdate) has allowed the isolation of erythrose and threose, respectively. Reduction of the products from the partial oxidation of the hexitols has resulted in the recovery of tetritols (confirmed by gas-phase analysis and mass spectrometry of TMS derivatives) by fractionation on Dowex 1 (molybdate). The reaction path in the oxidation of the hexitols which results in the formation of a tetrose was followed to the extent of 14% and 31% for glucitol and galactitol, respectively. Mixtures of the fragments obtained from t,he periodate oxidation, reduction, and hydrolysis of polysaccharides were analyzed by chromatography on Dower I (molybdate) and Dowex 1 (sulfate). SUMMARY

A method is presented for the automated determination of erythritol and threitol, which may be present in mixtures obtained from t.he partial oxidation of hexitols or oxidation of polysaccharides, by chromatography on Dowex 1 (molybdate). Separations of allitol and ribitol from their isomers are also described. Chromatography on Dowex 1 (sulfate) allowed the separation of propane-1,2-dial, glycolaldehyde, glycerol, erythritol, and xylitol. ACKNOWLEDGMENTS We tha.nk Professor M. Stacey, F.R.S., C.B.E., for his interest, and the Trust for the award of a scholarship (P.V.P.). We also thank Dr. J. R. Majer spectra determinations and R. R. Woodbury for technical assistance.

Fentham for mm

230

BARKER,

HOW,

PEPLOW,

AND

SOMERS

REFERENCES 1. BARKER, S. A., in “Modern Methods of Plant Analysis” (K. Paech and M. V. Tracey, eds.), Vol. II, p. 55. Springer-Verlag, Berlin, 1955. 2. KELEMEN, M. N., AND WHELAN, W. J., Abstracts, 2nd meeting, Fed. European Biochesm. Sot., Vienna, 1965. 3. LEE, E. Y. C., AND WHELAN, W. J., Biochem. J. 95, 27 (1965). 4. AKHER, M., HAMILTON, J. K., MONTOOMERY, R., AND SMITH, F., J. Am. Chem. xoc. 74, 4970 (1952). 5. SAMUELSON, O., AND STROMBERG, H., Carbohydrate Res. 3, 89 (1966). 6. BARKER, S. A., JONES, R. G., LAW, A. R., AND SOMERS, P. J., Carbohydrate Res. (1967). 7. SWEELEY, C. C., BENTLEY, R., MAKITA, M., AND WELLS, W. W., J. Am. Chem. Sot. 85, 2497 (1963). 8. DYER, J. R., Methods Biochem. Anal. 3, 129 (1956). 9. WEIGEL, H., Advan. Carbohydrate Chem. 18,61 (1963). 10. GUNNER, 8. W., JONES, J. K. N., AND PERRY, M. B., Can. J. Chem. 39, 1892 (1961). 11. SCHWARZ, J. C. P., J. Chem. Sot. 1957, 276.