Quantitative Determination of Water in a Hydrophilic Macromolecular Sample (Dextran) by Gas Chromatography Wallace M. Pasika and Arthur C. West I11
Chemistry Department, Texas A&M University, College Station, Texas 77843 METHODSFOR THE QUANTITATIVE assay of water in carbohydrates by gas chromatography have been reported ( I , 2). The technique is primarily based on the dissolution of the sample and the injection into a gas chromatograph. This technique unfortunately cannot be used for “large” carbohydrate molecules such as polysaccharides or the higher carbohydrate oligomers. With the latter materials, the injection syringe and column become gummed and column bleed persists for a considerable period thereafter because of the polysaccharide decomposition. Herein we describe a quantitative gas chromatographic method for water that was developed for a polysaccharide which alleviates the above mentioned problems. In principle, the method can be utilized for any kind of sample for which a solvent and precipitant exist. Both, however, must be fairly good solvents for water. EXPERIMENTAL
Apparatus. A Hamilton 701N microliter syringe with a Chaney adapter was used throughout this study. Four injections were made with each solution. Because the peaks were very narrow, the concentration of HzO was assumed to be proportional to the peak height. These were measured in millimeters ( ~ k 0 . 5mm) from the base line and averaged. The column was a six-foot length of ‘ / I in. aluminum tubing filled with PoraPak QS (mesh 80-100) through which helium flowed at a rate of 120 ml/min. Temperature parameters of the Varian Aerograph 1860 during the study were oven, 225 “C; detector, 250 “C; and injector, 225 “C. The thermal conductivity cell power level was set at 150 mA and the attenuation at 1X. Reagents. Regent grade N,N-dimethyl formamide (DMF) dried over Linde 4A Molecular Sieves, was further distilled with the aid of a spinning band column under reduced pressure. The middle cut collected was stored over Linde 4A molecular sieves until needed. Spectral grade dimethylsulfoxide (DMSO) was dried over anhydrous magnesium sulfate and distilled under reduced pressure. A middle cut was collected over Linde 4A Molecular Sieves and stored until required. Deionized distilled water was used throughout. The dextran was supplied by Dr. A. Jeanes of the Northern Utilization Laboratories in Peoria. The sample was a “linear” dextran designated “Partially Degraded Dextran from Mesenteroides NRRL B-2-b B-512, Clinical Size, Fraction PP58-B,” whose physical form was dense and grainy because of alcohol dehydration. Procedure. In an appropriate size volumetric flask, the “wet” sample is dissolved in a small volume of dry solvent. The precipitant is added to this solution as the contents of the flask are agitated vigorously. Upon complete precipitation of the sample, the flask is filled to the fiducial mark with the precipitant. Centrifugation of the stoppered flask gives a solids free supernatant. After standing an appropriate period, the supernatant is assayed with the aid of a gas chromatograph. A study of the possible effect of different variables on the analysis are listed below under various headings. (1) Nut. Bur. Stand. (U.S.), Misc. Pub/., 255, Research Highlights, December 1963, p 96. (2) D. W. Vomhof and J. H. Thomas, ibid., Tech. Note 507, Organic Chemistry Section, July 1969, p 111.
Table I. Effect of DMF:DMSO Ratio on Water Peak Height (H20 concn 10 pl/ml soh) Vol Vol Solution ml ml Av sample DMF DMSO Peak height (mm & s)
z
1’ 2’ 3’ 4/ 5’
0
z
100 60 40 10 0
40 60 90 100
109, 109, 110, 110 90,91, 94, 91 90, 89, 90,95 91, 89, 88, 89 89, 90, 89, 88
110 i 0.81 9 2 & 1.7 91 i 2.7 89 f 1.3 89 3~ 0.81
Table 11. Solution (with DMSO) and Precipitation (with DMF) Characteristics of Dextran Solu-
tion DMSO, Solubility Precipitation sample ml DMF characteristics characteristics 1 1.O a Readily soluble Complete s o h no ppte 2 0.5 Readily soluble Ppte settles with solution, turbidity 3 0.2 With slight Ppte settles, clear superwarming natant 4 0.1 a With slight Ppte settles, clear superwarming natant a to 5 ml of solution. 5
Table 111. HzO Analysis of Dextran Supernatants (after standing 6 hours) Solu-
tion sample 1 2 3 4
Av
Type Direct injection Solvation replacement Solvation replacement Solvation replacement
Peak height
(mm f s)
150, 149, 160, 149 82, 82, 82, 82 80, 86, 80, 84 82, 84, 83
152 i 5 . 4 82 i. 0.0 83 =k 3.2 83 i 1 . 0
Effect of Ultimate DMF-DMSO Ratio on Water Peak. To check whether the ratio of DMF:DMSO in the final solution (supernatant) has any effect on the water peak chromatograms, the chromatograms of synthetic supernatants of varying D M F :DMSO but constant water concentration were obtained. The supernatant solutions were prepared by diluting 2.5 ml of distilled water to 5 ml with D M F in a volumetric flask from which 0.1 ml was pipetted into 5-ml volumetric flasks and diluted to 5 ml with various volume ratios of D M F : DMSO. This gave solutions with concentrations of 10 pl HzO/ml solution. The results are presented in Table I. Effect of DMF:DMSO Ratio on Precipitation of Dextran and Amount of Water Detected. The ease of dissolution and precipitation of the dextran is outlined in Table 11. In all cases 0.1 gram of dextran (initial water approximately 13%) were weighed into each of the volumetric flasks. The samples once in solution (in varying amounts of DMSO) were allowed to stand for 2 hours and then diluted (with accompanying precipitation) to 5 ml with DMF. The stoppered mixtures were centrifuged after standing 6 hours. All supernatants were clear. Small aliquots of these solutions were added to ethanol to check whether all the dextran had been precipitated in each sample preparation. Injections of 1 p1 of the supernatants gave the results found in Table 111. After each in-
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Table IV. H20 Analysis of Dextran Supernatants (Standing 24 hours and 36 hours) Peak height average (mm f s) Solution sample Type 24 hr 36 hr 1 Direct injection ... 153 i 20 2 Solvation replacement 145 =t7.4 145 i 14 3 Solvation replacement 145 i: 11 8 145 f 2.5 4 Solvation replacement 144 i 1 . 3 142 i 17 Table V. Comparison of Various Water Assays on “Wet” B-512 Dextran Technique Vac-drying Abderhalden Direct injection Solvation replacement Vac drying-solvation replacement
Hz0,
Z
12.99 16.91 16.87 12.99
+ 3.91
16.90
jection of sample No. 1, the injection needle had to be cleaned. After 24 hours and 36 hours, the same supernatants were chromatographed with the results presented in Table IV. Effect of DMS0:Dextran Ratio on H 2 0 Peak Height. “Wet” dextran (0.1000 gram) was weighed into a 10-ml volumetric flask and dissolved in 1.0 ml of DMSO. After 2 hours, D M F was added to precipitate the dextran and bring the volume to 10 ml. This preparation scheme gave a system that had a DMF:DMSO ratio identical to that of sample 2 of Table I1 but with a higher DMS0:dextran ratio. The solution was centrifuged and allowed to stand for 42 hours after which the supernatant was chromatographed. Water Assay of “Wet” B-512 Dextran by Direct Injection of Its Solution into the Gas Chromatograph. “Wet” B-512 dextran (0.1001 gram) was dissolved in 1 ml of DMSO in a 5-ml volumetric flask and made up to volume with DMF. After standing 24 hours, it was injected into the gas chromatograph. An HzO standard solution of 25 p1 of HzO and 0.5 ml of DMSO and D M F to 5 ml of solution was used as the standard. The peak height of a blank of DMSO and D M F in the ratio used in the sample preparations was subtracted from all sample peaks heights. (This analysis was very arduous because of gumming, etc.) Water Assay of “Wet” B-512 Dextran by Our Solvation Replacement Method. The specifics of the analysis have already been described (sample 2, Table 11). The water standard was that used in the preceding section. Water Assay by a Combination of Drying in an Abderhalden Apparatus and the Solution Replacement Method. “Wet” B-512 dextran (0.1008 gram) was subjected to high vacuum in Aberhalden Drying Apparatus at room temperature for 12 hours after which the sample still under vacuum had its temperature raised to 65 “C by refluxing methanol. The heated evacuation was carried out for another 12 hours. The sample was then cooled in a desiccator over CaCh and subsequently weighed. The per cent moisture was determined to be 12.99 (Table V). Some of this “dried” sample was then subjected to our solvation replacement technique. The amount of water contained in the “dried sample” was 3.91 making a total of 16.90% HyOfor the “wet” B-512 dextran.
z
z
RESULTS AND DISCUSSION The apparent differences in the averages of Table I arise from the fact that the DMSO employed had a small GC peak very near where the water appeared. Despite a very determined effort to rid our DMSO of extraneous peaks, we were 276
unsuccessful. It may well be that we did in fact start out with pure DMSO but upon contact with the hot injection port, etc., some of the DMSO underwent a thermal reaction giving the impression that impurities were present in the original DMSO. (DMSO upon distillation is known to give mercaptans, etc.) This being the case, the greater the amount of DMSO injected, the greater would be the variation (increase) in average peak height of water. Table I indicates this trend and also indicates no serious error present in our analysis if the amount of DMSO is kept less than some 15 volume (any other system may have another limit). The DMS0:dextran ratio does not affect the peak height of the HzO peak. O n the basis of the result obtained with sample 2 of Table I1 and apropos of the dilution factor, the peak height expected for the above supernatant was 66 mm. The peak height obtained experimentally was 67 mm. From Table I1 it is obvious that if DMSO is present in greater than 15 volume %, precipitation of the dextran does not occur. (This critical parameter would have to be determined individually for any other system assayed.) Analysis by the direct injection gave an average peak height of 147 mm i 5.6 for five injections of 1 pl of solution. The average peak height for five injections of 1 p1 of the standard water solution was 201 mm i 15. Appropriate calculations indicated that the per cent HzO in our “wet” dextran was 16.91 %. The data in Table 111indicate that on initial precipitation a fair amount of water is “sorbed” in the gel-like dextran precipitate. This sorbed water, however, can be made available for analysis by simply letting the precipitate-supernatant system stand. Table IV indicates that >90% of this sorbed water will diffuse into the supernatant within 24 hours because of replacement solvation of the macromolecule-DMSO or D M F for H 2 0 . It is interesting to hypothesize on the difference (some 8 mm) between the peak heights (Table IV) for the direct injection and solvation replacement technique. It may be that this difference represents water bound to the macromolecule at room temperature as opposed to water that loosely solvates the macromolecule (the bulk of the difference (69 mm) in peak heights of Table 111). CONCLUSION Assaying by vacuum drying does not give satisfactory water analysis. Others are of the same opinion (3, 4). Direct injection gives total analysis but with concomitant difficulties. Our “solvation replacement” technique gives as good a n analysis as the direct injection technique without the inherent problems. ACKNOWLEDGMENT We thank Dr. A. Jeanes for the dextran sample and discussions. RECEIVED for review August 6, 1970. Accepted October 27, 1970. We appreciate the support given us by the Agricultural Research Service, U. S. Department of Agriculture through Grant No. 12-14-100-9159(71) administered by the Northern Utilization Research and Development Division, Peoria, Ill. (3) N. N. Hellman, Cereal Chem., 28 (l), 71 (1951). (4) M. Friml and R. Lejkova, Listy Cukrol;., 84, 230-5 (1968); Chem. Abstr., 70, (14) 59116R.
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