I
I
I
20
15
Figure 2. formation
1
5 TIME (minutes) IO
0
No inhibition of polymer
Column, 200 feet, 0.03-inch i.d. coated with 10% Ucon HB 2000. Temperature, 135' C. Sample h e , 1 pl.
RESULTS AND DISCUSSION
Inhibition of D M P polymerization by different concentrations of dimethyl sulfoxide is illustrated in Figure 1. Complete inhibition of polymer formation occurs with a concentration of 1% DMSO for a t least eight hours; further
observation indicated however that a faint yellow color appeared after 20 hours. Where dimethyl sulfoxide was absent, a high percentage of polymer had formed after just two hours, and after eight hours the solution had become dark brown. It has been our experience with the majority of mono- and dicarboxylic acids tested that, esterification reactions which included D M P were loo'% complete within four hours. A concentration of dimethyl sulfoxide as low as 0.25% effectively inhibits significant polymer formation. If the time of reaction for a particular esterification is known, a concentration of dimethyl sulfoxide could be chosen which would completely prevent polymer formation for the duration of the reaction. Figure 2 shows the interference which may be caused during a gas chromatographic analysis by the presence of D M P polymers. Figure 3 represents identical conditions to Figure 2 with the exception that dimethyl sulfoxide had been added to the esterification procedure prior to gas chromatography. Other compounds which were tested as possible inhibitors of the reaction described above and found to be ineffective were: pyridineN-oxide, di-n-hexyl sulfoxide, di-n-hexyl sulfone, and trimethylamine oxide. Tetramethylene sulfoxide, however, inhibited D M P polymerization, but was only 500/, as effective as dimethyl sulfoxide. LITERATURE CITED
(1) Lorette, N. B., Brown, J. H., J . Org. Chem. 24, 261 (1959).
I
20
15
5
IO
TIME (minutes)
Figure 3. Inhibition of polymer forrnation b y dimethyl sulfoxide Chromatographic conditions as in Figure 2
(2) Mason, M. E., Waller, C. R , ANAL. CHEM.36, 583 (1964). ( 3 ) Tove, S B., J . Nutr. 75,361 (1961). ( 4 ) Waller, G. R., Symposium on Gas
Chromatography, 16th Southwest Regional Meeting of A.C.S., December 1-3, 1960, Oklahama City, Okls.
Chemistry Department University of Houston Houston, Texas
P. G. SIMMONDS ALBERTZLATKIS
Use of Sorbitol as Internal Standard in Determination of D-Glucose by Gas Liquid Chromatography SIR: I n adapting the method of Sweeley, Bentley, Makita, and Wells ( 4 ) to the quantitative analysis of D-glucose in commercial corn sugar and corn sirups, several difficulties arose. Sample size could not be controlled well enough to allow direct peak-area measurement. In addition a film, thought to be silicon dioxide, was deposited on the flame detector, greatly reducing its sensitivity. Peak areas were 30% higher in some cases when measurement was made directly after cleaning the detector. An internal standard was then sought. For a material to classify as a n internal standard it should not be present in the sample being analyzed ( 1 ) . I t should be eluted near the sample's components, and the ratio of its peak area to the components' should be near unity. Sorbitol (D-Sorbitol or
D-glucitol) fulfilled these requirements, and its use overcame the difficulties discussed above. A typical chromatogram is seen in Figure 1, which shows sorbitol being eluted between CY-D- and 0-D-glucose. With a total analysis time of '/* hour, up to 16 samples can be analyzed in a normal working day. The new procedure is considerably more rapid than present paper chromatographic techniques, and it offers the obvious advantage over the numerous D . E . (Dextrose Equivalent) methods in directly analyzing a material for its glucose content. EXPERIMENTAL
Reagents. Hexamethyldisilazane was obtained from Peninsular Chemresearch, Gainesville, Fla. ; trimethylchlorosilane from General Electric
Co., Silicone Products Division, Waterford, N. Y . ; a-D-glUCOSe, 0-Dglucose a n d D-sorbitol from Pfanstiehl Laboratories, Inc., Waukegan, Ill.; Enzodex, anhydrous dextrose and corn sirup solids, from Grain Processing. Corp., Muscatine, Iowa. Materials were used as supplied unless indicated otherwise. Apparatus. An derograph A-600-B chromatograph equipped with hydrogen flame ionization detector (Wilkens Instrument and Research, Inc., Waln u t Creek, Calif.) attached to a Leeds and Northrup Speedomax H recorder was used. Columns. During most of this study a 6-ft. X 0.25411. 0.d. coiled, stainless steel column packed with 370 SE-52 on 100- to 120-mesh Chromasorb P was employed. It was obtained from Applied Science Labs, State College, Pa. Operating Conditions. T h e column oven was maintained a t 180' C VOL. 37, NO. 2, FEBRUARY 1965
303
and injector oven a t 230’ C. A flow rate of 25 to 30 ml. per minute of argon or nitrogen gave t h e most efficient performance. T h e following electrometer settings were used: inp u t impedance, l o 9 ohms; output sensitivitv. lx: attenuator. between 8 and 32:’ ’ Samples of 1.0 to 5.0 pl. were injected with a 10.0-~1. Hamilton svringe. Sample size was adjusted to &e &e third to two thirds of the recorder scale. Total retention time was 35 minutes using argon and 29 minutes using nitrogen as carrier gas. Sample Preparation. (A) AKHYDROUS CORX SUGAR. A 1.000-gram sample of anhydrous material was weighed into a 100-ml. volumetric flask. Anhydrous pyridine (stored
I! ll
0-10RWOL
n,
$ -D -GLUCOSE
a
~
D -GLucose
Figure 1. Separation of trimethylsilyl derivatives of Dglucose and D-sorbitol on SE-52 column
a-D-glucose, 25% p-D-glucose, and 50% over KOH pellets) was added, the D-sorbitol; this value multiplied by 100 sample dissolved, and diluted to gave per cent D-glucose : 100 ml. From here the procedure was essentially the same for each type Table 1. Determination of D-Glucose 7% D-glucose = { (ao,/~~(,,.,,,,,)/(A (C) / of material. Two milliliters of the in Anhydrous and Aqueous Samples of A ( S ) s t d ) x 100 solution was pipetted into a 10.0-ml. Corn Sugar volumetric flask with 2.0 ml. of a 10 With aqueous samples the per cent uyo D-Glucose mg./ml. pyridine solution of sorbitol glucose calculated above was divided Nature Gas Paper and 6.0 ml. of pyridine. One milliliter by the sample weight times the per of sample Samplen chrom. chrom.* of the final solution, containing 4 mg./cent solids (expressed as a fraction) a 1-dram vial, and ml., was placed into Anhydrousc 1 96.9 96.0 to give the actual per cent o-glucose. 2 95.1 95.4 the procedure of Sweelev - (,.,L ) was The same applied with corn sirups 3 95.6 953 folloGed. except the final value wa7 divided by 97.5 4 97.5 (B) AQUEOUSCORNSUGAR.The per two or four to correct for the 2 : 1 or 4: 1 5 94.2 94.9 cent. solids iusuallv 20 to 25%) of a ratios of sirup to sorbitol used as dis96.4 6 95.9 sample was determined by measurecussed under Sample Preparation. Aqueous 7 96.4 96.3 ment of refractive index. A 4.0-ml. 8 95.0 94.6 sample was weighed in a 100-ml. voluRESULTS AND DISCUSSION 9 95.5 94.3 metric flask and the sample diluted 10 95.5 95.2 with pyridine. The procedure deThe success of an internal standard a Samples of Grain Processing Corp. scribed in Part A was then followed. is based on the constancy of the ratio Enzodex, anhydrous dextrose, and corn (C) CORNSIRUPS. .&sirup, normally between the component’s peak area and sirup solids. Dextrose is a common a t 80% solids, was diluted with distilled that of the standard at a fixed concenname for D-glucose used by the corn water to 20 to 25% solids and its reindustry. tration. Except with the corn sirups A 4.0-ml. fractive index measured. b Paper chromatographic method the products to be analyzed contained sample of the diluted sirup was weighed adapted from that of Dimler et al. ( 2 ) . 93 to 98% p-glucose, so that a 50-50 and dissolved in pyridine in a 100-ml. Samples normally contained 0.5% mixture of pure D-glucose and sorbitol volumetric flask. With 54 to 72 D.E. water. sirups (25 to 50% D-glucose), 4.0 ml. was picked as the best reference of the pyridine solution, 2.0 ml. of standard. The ratio of this mixture sorbitol, and 4.0 ml. of pyridine were was constant over the range of 2 to 10 Table II. Determination of D-Glucose placed into a 10-ml. volumetric flask. mg./ml. with an average value of 0.886 in Corn Sirups With sirups lower than 54 D.E., 4.0 ml. f 0.OOi. Over a period of 3 months yo D-Glucose of pyridine solution, 1.0 ml. of sorbitol, the ratio varied from 0.852 to 0.893, and 5.0 ml. of pyridine were used. D.E.0 Gas chrom. Paper chrom. although duplicates run the same day One milliliter samples of the final 62 37.2 37.5 never varied more than j~0.01. The mixtures were treated with the silane 62 39 8 39 4 variation did not affect the accuracy of reagents as in Part A. 41.3 62 40 8 Calculations. Peak areas were the method and is thought to be due 31.5 54 31 5 measured using a Gelman planimeter. 52 26 6 26 2 to slight day to day changes in chro42 20.2 19 7 With anhydrous materials the glucose matographic conditions. 16.4 42 16.6 area, iiC0(total a- and 6-D-glucose), The glucose determination of numer9.02 54 9 25 was divided by the sorbitol area, SI, ous corn sugar samples was then sucto give the ratio . 4 ( ~ ) / - 4 ( ~ i ) ~ ~ ~ ~ 1 ~ . a Dextrose equivalent determined by cessfully performed. As seen in Table the method according to Somogyi (3). This ratio was divided by that of a I values were in excellent agreement reference standard, composed of 25% with those obtained by paper chromatography. Precision of duplicate samples was *1.5%. Initial attempts to analyze aqueous Table 111. Recovery of D-Glucose A,dded to Corn Sugars and Corn Sirups solutions of corn sugar were unsuccessD-Glucose ful. Spurious peaks, believed to be a present Total from result of reagent hydrolysis and inD-glucose, sample, D-Glucose recovered, mg./ml. Recovery, complete substitution of the carboFound Sample mg./ml. mg./ml. Added 70 hydrate with trimethylsilyl groups, 2.004 2.000 100.2 Corn siigar 1 3 922 1 918 were obtained. By reducing the total 2.037 2.000 101.8 3 978 1 941 Corn s\igar 2 sugar concentration in l~yridine to 4 2,036 2.000 101.8 3 448 1 412 Corn siriip 1 mg./ml. (2 nig. of glucose and 2 nig. of 2,030 2.000 101.5 Corn sirrip 2 4 305 2 275 sorbitol) the per cent of water in the ~
304
ANALYTICAL CHEMISTRY
system w a s reduced to less than 1%, which eliminated the difficulty. This procedure was used with all materials as discussed under Sample Preparation. Corn sirup samples were analyzed by the same technique. Here the ratio of sirup to sorbitol was increased so that the D-glucose to sorbitol ratio would approximate that of sugar samples. Values obtained for several sirups are shown in Table 11. Precision here was Z ! Z O . ~ ~ ~ . T o determine the accuracy of the method, known amounts of D-glucose were added to two corn sugar and two
corn sirup samples and the mixtures analyzed. The results in Table 111 showed that the recovery of added D-glucose was 1 to 2% high, although values were generally in better agreement with paper chromatographic values as seen in Tables I and I I. ACKNOWLEDGMENT
We would like to thank George G. Hazen and James A. Hause of Merck and Co., and William W.Wells of the University of Pittsburgh for their helpful suggestions during the initial phases of this work.
LITERATURE CITED
( 1 ) Dal Nogare, S., Juvet, R. S., “Gas-
Liquid Chromatography,” Interscience Publishers, Inc., Sew York, 1962, p.
256. ( 2 ) Dimler, R. J., Schaefer, W. C., Wise, C. S., Rist, C. E., ANAL.CHEM.24, 1411 (1952). ( 3 ) Somogyi, lf., J . Bzol. Chem. 160, 61 (1945). ( 4 ) Sweeley, C. C., Bentley, R., Makita, M., Wells, W. W., J . Am. Chem. SOC. 35, 2497 (1963).
R. J. ALEX.4NDER J. T. GARBUTT
Grain Processing Corp. Muscatine, Iowa
Spectrophotometric Determination of Boron Using Barium Chloranilate and Saccharic Acid SIR: Srivastava, Van Buren, and Gesser (4) have described a photometric method for the determination of boron which involves the precipitation of a complex borotartrate, formed upon the addition of barium chloranilate to a solution containing boric and tartaric acids, with subsequent measurement of the released chloranilate ion. We find that certain modifications can significantly improve the sensitivity while retaining the simplicity of the method. EXPERIMENTAL
Reagents. A solution of saccharic acid, buffered to a p H of 8.8, was prepared by dissolving 5.17 grams of the calcium salt of saccharic acid (Nutritional Biochemicals Co.) in a minimum amount of dilute hydrochloric acid. This solution was passed through a column containing a cation exchange resin (Rexyn AG 50H) in the hydrogen form, the effluent being collected in a 250-ml. volumetric flask. T h e column was washed with about 150 ml. of distilled water. To the effluent was added 25.0 ml. of 1 5 M ammonium hydroxide and 60.0 grams of ammonium chloride, a n d diluted t o the mark. The buffered solution was stored in a polyethylene container. A similar solution, buffered to a p H of 9.5, was prepared in similar fashion, except t h a t only 11.55 grams of ammonium chloride were used. Standard boron solutions were prepared from recrystallized and fused reagent grade boric acid. Solutions were prepared containing 21.6 mg. of boron per liter. T h e methyl cellosolve (ethylene glycol monomethyl ether) and barium chloranilate (Fisher Scientific Co.) were reagent grade materials. A Beckman DU spectrophotometer was uied in conjunction with matched 1.0 cm. silica cells for all photometric measurements. A mechanical shaker
was utilized to shake the flasks in which t h e reactions were carried out. Procedure. A series of standard boron solutions was prepared by adding appropriate amounts of t h e stock solution t o 10-ml. volumetric flasks. T h e volume of the standard boron solution was not allowed to exceed 4.0 ml. in a n y case. T o each solution was added 1.0 ml. of either the p H 8.8 or 9.5 saccharic acid solution (depending on the boron concentration) and 5.0 ml. of methyl cellosolve. A blank was prepared in t h e same way. All flasks were filled to the mark with water, and 40 mg. of dry barium chloranilate were added to each. The flasks were then shaken mechanically for about 30 minutes. At the end of the shaking period, the solutions were filtered through a fine paper (Schleicher and Schuell No. 589 Red Ribbon), portions were transferred t o t h e photometric cells, and absorbance measurements were made at 346 mM for t h e p H 9.5 solutions or 355 mp for the p H 8.8 solutions. RESULTS AND DISCUSSION
Initially a number of metal chloranilates, including those of barium, magnesium, calcium, strontium, lead, lanthanum, thorium, cadmium, and mercury were studied. Similarly a variety of organic acids, including citric, malic, galacturonic, mucic, and saccharic, were investigated. This survey indicated that the combination of barium chloranilate with saccharic acid showed the most promise. Gautier and Pignard ( 2 ) have indicated that the barium borotartrate complex is formed a t a p H of 8.8. Various water miscible solvents, including acetone, cellosolve, and methyl cellosolve, were studied a t this pH to investigate their effect on the sensitivity. Methyl cellosolve was selected because
of the good sensitivity and excellent linearity exhibited. A study of the temperature effect showed some variance of sensitivity with temperature. The effect is relatively minor, good sensitivity is obtained a t about 25’ C., and nominal variations from room temperature do not cause significant error. Absorbance measurements are made a t 355 mp as discussed by Srivastava and coworkers (4). Table I shows that at this wavelength Beer’s law is obeyed up to about 10 p.p.m., an improvement over the earlier method in which the upper concentration range is about 3.0 1i.p.m. Even though this saccharic acid procedure extends the concentration
Table 1.
Absorbance Measurements at 355 mp
1.0-cm. path length, pH 8.8 methyl
cellosolve, saccharic acid Boron, p.p.m. Absorbance 2.16 4.33 6.49 8.66
0.401 0.733 1.070 1 ,490
Table II.
Absorbance Measurements at 346 mp 1.0-cm. path length, pH 9.5 methyl
cellosolve, saccharic acid Boron, p.p.m. Absorbance 0.05 0.11 0.22 0.43 0.65 0.87
0.060 0 142 0 258 0 519 0 719 0 XR2
VOL. 37, NO. 2, FEBRUARY 1965
e
305
