stantially less reactive than all the other chelates. It was conclusively established by isolation and characterization of the final products and by a spectrophotometric examination of the organic phase a t various stages of the reaction that the Fe(I1) and Co(I1) chelates did not undergo any oxidation reactions. The current-time curve obtained with the free ligand, under the same experimental conditions as the metal chelates is included in Figure 1 for purposes of comparison. The bromination of the free ligand takes place more slowly than the Fe(II), Cu(II), Mn(II), and Ni(1I) chelates but the Co(I1) chelate reacts somewhat more slowly than the free ligand. Hence, with the exception of the Co(1I) cheIate, the effect of formation of a metal chelate ring is to increase the rate of electrophilic substitution in the 7-position in the heterocyclic ligand. Several variables, such as dissolved oxygen and glass surface area which have been reported to influence the rates of bromination in low polarity solvents, had a negligible effect on the metal chelate brominations. The possibility of radical reactions arising from photoinitiation or other mechanisms was eliminated. The behavior
of the Co(I1) chelate is clearly anomalous but in the absence of any further experimental evidence any explanation that might be advanced for its anomalous behavior would only be speculative. Under the experimental conditions that were employed in this study, the free ligand, 5-chloro-8-quinolinol, is not extracted into the aqueous phase. The monobromination of this compound in the organic phase follows second-order kinetics, first-order with respect to bromine and first-order with respect t o the ligand. The second-order rate constant in the water saturated chloroform solution is of the order of IOa l./mole . sec, which is about l o 3 times smaller than the rate constant for the monobromination in water (2). With the exception of the anomalous Co(I1) chelate, the coordinated ligand was found to react about 1.5 times faster than the neutral form of the free ligand. RECEIVED for review April 5, 1971. Accepted July 1, 1971. The authors are grateful t o the National Science Foundation for financial assistance.
Characterization and Quantitative Analysis of D-Glucose for Use in Clinical Analysis Bruce Coxon and Robert Schaffer Institute for Materials Research, National Bureau of Standards, Washington, D.C. 20234 The results from analysis of a-D-glucose and p-Dglucose by a variety of spectrometric, chromatographic, and polarimetric techniques are presented and discussed. The apparent values of purity obtained for a-D-glucose by differential scanning calorimetry are discussed in relation to the content of p anomer as determined by gas chromatography and proton magnetic resonance spectrometry. It i s shown that, during melting, both a-D-gIUCOSe and p-D-glUCOSe anomerize rapidly to give an equilibrated mixture of these anomers.
FORABOUT 60 YEARS, the National Bureau of Standards has made available a Standard Reference Material Dextrose, for the purpose of standardization of chemical methods of analysis. In recent years, developments in the standardization of clinical analysis techniques have resulted in a national requirement for more highly characterized, certified samples Of D-glucose, suitable for use in both chemical and biochemical methods of analysis, and particularly in those in which enzymes are employed. Thus, clinical chemists require a D-glUCOSe standard reference in connection with analyses of blood and urine in the detection and diagnosis of diabetes and other diseases. One of the more important parameters of a clinical standard reference material is its quantitative punty. We have successfully applied differential scanning calorimetry (DSC) to determination of the punty of a number of crystalline, clinical standard reference materials, including cholesterol, 4-hydroxy-3-methoxy-~~-mandelic acid (VMAj, D-mannitol, and urea (1-3). For determination of the purity of D-glucose, (1) R.Schaffer, Ed., Nai. Bur. Stand. (U.S.), Tech. Note, 451(1968). (2) Ibid., 507 (1969). (3) Ibid., 547 (1970).
the alternative method of phase solubility analysis ( 4 , 5 ) is unsuitable, because of the likelihood of anomerization during the long periods required for equilibration of the excess of solute with the solvent. Since a-D-glUCOSe is both cheaper and more widely available than @-D-ghCOSe,a-D-ghIcose (Dextrosej was selected for consideration as a clinical standard reference material. It has been known for many years ( 6 ) , that crystalline a-D-glucose, unless very specially purified, contains a small proportion of the p-D anomer. For the purpose of clinical analysis, the presence of some p anomer is unimportant, since the standard material is used in aqueous solution, in which anomerization occurs rapidly in any case. Nevertheless, proper characten’zation and appreciation of the purity of the ff-D-glUCOSe depends on knowledge of the content of @ anomer. Thus, we attempted the application of the DSC method t o a-D-glUCOSe, where we anticipated that the impurity total determined might represent the sum of the contents of 0 anomer, hydrates of a or @ anomers, possibly other ring or acyclic forms, and those nonglucose impurities insoluble in the solid, but soluble in the liquid phase. The relative proportions of a and p anomers in the a-Dglucose and in a commercial P-D-glucose have been determined either directly, by proton magnetic resonance (PMR) spectrometry of solutions in methyl sulfoxide-ds at 90 MHz, (4) G. J. Sloan in “Physics and Chemistry of the Organic Solid State,” D. Fox, M. M. Labes, and A. Weissberger, Ed., Interscience, New York, N. Y . , 1963, p 179. (5) W. J. Mader, in “Organic Analysis,” Vol. 2, J. Mitchell, Jr., I. M . Kolthoff, E. S. Proskauer, and A. Weissberger, Ed., Interscience, New York, N. Y . ,1954, p 253. (6) C. S. Hudson and E. Yanovsky, J. Amer. Chem. SOC.,39, 1013
(1917).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
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Table I. Effect of Duration of DSC Scan on Apparent Purity of a-lpGlucose Beginning temper- Extrapolated Duration of ature meltingscanning at Heat of Apparent of slow point, 0.625 OK/ fusion, purity, scan, OK OK min, min M/mol mol 41 1 420.7 15.5 34.7 98.75 414 422.7 13.9 32.9 99.03 417 422.5 8.8 34.7 99.40 417 422.0 8.0 34.4 99.41 418 422.7 7.5 33.9 99.47 420 423.8 6.1 33.9 99.43 Table 11. Anomerization of a-~GIumseduring Melting to T,,,, Determined by Gas-Liquid Chromatography of Trimethylsilyl Derivatives Other components, Tmax,OK U-D-G~UCOS~, fl-~Glucose, 298" 99.52 0.48b 0 418 97.8 2.2 0 420 55.2 43.5 1.3 421 45.3 52.3 2.4 Unmelted material. b Mean of 10 determinations, standard deviation of individual determinations (S),O.O6.
z
z
5
or indirectly, by gas chromatography of penta-0-(trimethylsilyl) derivatives. Supporting data were obtained from polarimetry of the sugars, particularly concerning the behavior of the anomers during melting (7). Characterization of the anomers by other chromatographic techniques is discussed briefly, and in the experimental section are given the results of analyses for trace metal contaminants and other impurities that are important from the point of view of the use of the standard material in enzymic chemistry. EXPERIMENTAL
Materials. Microcrystalline, anhydrous a-D-glUCOSe packaged in polyethylene bags was obtained from Pfanstiehl Laboratories, Inc., Waukegan, Ill., and was sieved before use (mesh size 40) to remove larger aggregates. The sieved material was homogeneous according to paper chromatography on Whatman No. 1 filter paper, performed by the descending method with butyl alcohol-pyridine-water [6 :4:3 (v/v)l, and to thin-layer chromatography conducted immediately after application of solutions of the D - ~ ~ U C O S ~ to layers (20 X 20 cm) of nonactivated Silica Gel G (Merck), with methanol-chloroform 15 :1 (v/v)l as eluent. The paper chromatography of massive amounts of a-Dglucose was also performed by application of solutions containing 50-300 mg of the sugar to seed-test paper (2) (45 X 60 cm), followed by descending development as before. Only a single, large spot, elongated for larger quantities, was observed. The rare disaccharide sophorose, which has been found (8) in some typical, "pure" commercial samples of D-glucose, was not detected (detection limit of 1 %). The Cr-D-glUCOSe was also homogeneous according to liquid chromatography in the Oak Ridge National Laboratory high-pressure, ionexchange, chromatography system. (7) Preliminary Report, B. Coxon and J. H. Thomas in Ref. 3, p 19.
(8) M. Mandels and E. T. Reese, Biochem. Biophys. Res. Commun., 1, 338 (1959). 1566
e
Anhydrous @ - D - ~ U C O S ~was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio, in the form of small, colorless rosettes that were homogeneous according to paper chromatography. Polarimetry. For measurements on mutarotated D-glucose in aqueous solution, the sugar (5 grams) was diluted to a volume of 20.0 ml at 20 "C, with the inclusion of one drop of concentrated ammonium hydroxide. Measurements on nonmutarotated, crystalline a- and @-D-glucose,and on melted samples of these sugars were made by dissolving the samples (2.5 grams) in dry methyl sulfoxide (total volume, 25.0 ml) at 20 "C. Ultrasonic vibration was employed to assist the dissolution of the syrups. The sample weights in air were corrected to weights in uacuo (9) by application of the factor 1 (k/1000), where, for D - ~ ~ U C O Sk~ ,= 0.64. This factor was assumed to be the same for a-and fi-D-ghCOSe, and the melts. Most of the measurements were made at five different wavelengths by using a Perkin-Elmer automatic polarimeter Model 141 with a 1-dm sample-cell jacketed at 20 "C. However, the optical rotations obtained from this instrument at 589 nm agreed within 0.1 Z with those measured on a manual Rudolph polarimeter. The accuracy of the automatic polarimeter was further confirmed by measurements of both dextro- and levo-rotatory standard quartz plates that agreed within 0.15 % with international standard values for these plates. Differential Scanning Calorimetry. A Perkin-Elmer differential scanning calorimeter Model DSC-1B was used that had been calibrated against indium metal [purity, 99.999 %, heat of fusion, 3.26 kJ/mol (IO)]according to the procedure of Plato and Glasgow (10). Sample weights of indium and of a-D-glucose were determined on a Cahn electrobalance. Portions of a-D-glucose (3 mg) were heated in open, aluminum sample-pans under nitrogen from 298 OK to various temperatures (T)just below the melting point, at 80 "K/min, and then from T to beyond the melting point at 0.625 "K/min. Thermograms were recorded by using the maximum sensitivity of the calorimeter, and were divided into 5-7 segments, the areas of which were measured by planimetry. The curved plot of the reciprocal of the fraction of the sample melted against its temperature was linearized (IO) by application to each segment of the thermogram curve of a correction equal to 17-22Z of the total area of it. The molar percentage of apparent, total impurity (see Table I) was calculated from the function 100AH,AT/RTo2, where AH,, the heat of fusion, was determined from the corrected, total area of the curve; AT, the depression of melting point, from the gradient of the linearized plot; and To,the extrapolated melting-point, from its intercept (10). Melting of a- and ~~-D-GIUCOW. Samples of a-D-glucose (-3 mg) in open, aluminum pans were flushed six times with nitrogen, and were heated in the differential scanning calorimeter from 380 "K to 414 OK at 80 "K/min, and then to a maximum temperature (Tmax)at 0.625 "K/min. One sample was heated to TmSx418 OK, corresponding to a small amount of pre-melting, a second sample to Tmsx420 OK, corresponding to the maximum rate of heat input on the DSC thermogram (melting -60% complete), and a third sample was melted completely (T,,, 421 OK). Purity was not determined in these experiments. At Tmsx the sample pan was rapidly removed from the calorimeter and allowed to cool to room temperature. Each melt was then analyzed by gas chromatography after stirring the sample pan and contents with trimethylsilylating reagent as described later (for results, see Table 11).
+
(9) F. J. Bates and Associates, "Polarimetry, Saccharimetry and the Sugars," Nat. Bur. Stand. (U.S,), Circ. C440, Washington, D. C., 1942, p 613. (10) C. Plato and A. R. Glasgow, Jr., ANAL. CHEM., 41, 330 (1969).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
In other experiments, larger portions (100 mg, or 2.5 grams) o ~ e placed in glass vials or volumetric of a-and f l - ~ - g l ~ c were flasks, respectively, and were melted completely by heating in an oven at 160 OC for 15 min. Under these conditions, a small amount of caramelization of the sugar occurred, as revealed by the odor and pale-yellow color of the melts. Paper chromatography indicated the formation of trace quantities of compounds having rates of migration less than that of D-glucose. The syrupy melts were cooled to room temperature and analyzed either by polarimetry (2.5-gram samples), or PMR spectrometry (100-mg samples). Gas Chromatography. CY- and p-D-Glucose were analyzed as their penta-0-(trimethylsilyl) derivatives, which were prepared by treatment of portions (5 mg) of C Y - D - ~ ~ U C OorS ~its melts with 1 ml of a solution of N-(trimethylsilyl)imidazole in pyridine (TriSil 2, Pierce Chemical Company) at 0 "C for 10 min. Aliquots (1 pl) of the solutions were injected into a Vprian Aerograph gas chromatograph Series 2100 equipped with a glass column (1.8 m) of 3 % of silicone rubber (SE-30) on Chromosorb (WAWS, 80-100 mesh) at 180 "C. The separated components of the mixtures were detected by means of a flame-ionization detector operated at 220 "C, and their proportions were determined by triangulation of the peaks recorded, with the assumption that per(trimethy1silyl) derivatives of sugars of similar structure have the same response factor. The proportions determined were not changed by trimethylsilylation of the sugar at 0 "C for 1 hr instead of for 10 min. However, trimethylsilylation of a-D-glucose at room temperature generally gave higher proportions of the j3-anomeric derivative, in the range of 1-2 %. Proton Magnetic Resonance Spectrometry. Solutions were prepared by dissolving samples (100 mg) of crystalline aor @-D-glucose,or of their syrupy melts, in portions (0.5 d) of methyl sulfoxide-& (Stohler Isotope Co., purity stated, 99.5% D) taken from freshly opened vials. Dissolution of the syrups was encouraged by ultrasonic vibration. Each solution was filtered in close proximity to a small magnet, and then tetramethylsilane (0.03 ml) was added. PMR spectra were recorded in the frequency-sweep mode at 90 MHz on a Bruker Scientific nuclear magnetic resonance spectrometer Model HFX-11 with internal, field-frequency stabilization on the signal of tetramethylsilane, automatic optimization of the y-magnetic field gradient, and audiofrequency modulation of the magnetic field at 4.17 kHz. For quantitative measurements, particular care was taken to ensure that the audiofrequency amplifier was not overdriven into a nonlinear mode of operation, and that the power of the observing radiofrequency was maintained well below the saturation level, for all resonances of interest. Typically, this power was a nominal 0.5 W attenuated by 52 dB. For each solution, and within the time period (1.5 hr) during which mutarotation was not significant, 16 scans of the region of anomeric hydroxyl signals were accumulated in 4096 channels of a Fabritek instrument computer Model 1074 (for example, from 25 to 68 min after dissolution). The sweep time for one scan was 164 sec, which corresponded to a sweep rate of 1.5 Hz/sec. After readout of the analog form of the time-averaged spectrum, it was integrated digitally within the computer, and the magnitudes of the integrals of interest were obtained from a numeric, oscilloscope display of the contents of the memory. Integrals were also determined by cutting out and weighing peaks recorded, after signal-averaging, on plain chart paper, and these were in good agreement with those obtained by numeric readout. Inorganic Analysis of c~-~-Glucose. The ash content of the a-D-glucose was determined to be 0.002x by ignition of samples (20 grams) of the sugar at 750 "C. Analysis of this ash by emission spectrometry showed the contents to be aluminum,
