Extractive Titration Technique for Kinetic Studies of Fast B ro m i n at i o n Reactions

R. K . Baker and Quintus Fernando Department of Chemistry, University of Arizona, Tucson, Ariz. 85721

Bromine was generated coulometrically at a constant rate and monitored amperometrically in an aqueous phase which was in equilibrium with an organic phase such as chloroform. Because the bromine was rap- idly distributed and equilibrium was almost instan- taneously established in the stirred two-phase system, the method could be used to investigate the kinetics of bromination of compounds with finite distribution coefficients or compounds that are insoluble in the aqueous phase but soluble in chloroform. The rate constants for the monobromination of phenol were determined in the aqueous as well as the chloroform phases. The rates of bromination of a series of metal chelates of 5-chloro-8-quinolinol were studied in chloro- form. Owing to the complexity of the bromination reactions, only qualitative information could be ob- tained on the effect of metal ion on the rate of bro- mination of the metal chelate.

THE KINETICS of bromination of phenolic compounds ( I , 2 ) in aqueous solutions have been followed by the coulometric generation of bromine at a fixed rate in the presence of the phenol in a well stirred solution; the unreacted bromine was monitored amperometrically as a function of time. This method has been used by several workers for the in- vestigation of the kinetics of a variety of fast halogenation reactions (3-5). Although the method can be used for studying the reactivity of coordinated ligands (6-8), it has several drawbacks which arise from the insolubility as well as the partial dissociation of most metal complexes in aqueous or alcoholic media. To obviate these difficulties, we have devised an extractive procedure in which bromine is gen- erated and monitored in the aqueous phase, and the bro- mination reaction takes place in an organic phase, such as chloroform, in which most metal complexes dissolve to a measurable extent. The success of the method depends on several factors that have to be carefully controlled, and the validity of the technique has been established by measuring the known rate constant for the reaction of bromine with phenol to form monobromophenol in an aqueous solution.

EXPERIMENTAL

Apparatus. The apparatus employed was similar to that described in previous kinetic studies ( I , 2) . A titration cell of approximately 150-ml capacity was connected to two auxiliary cells by means of side arms with glass frits and the connecting arms were filled with polyacrylamide gel saturated

(1 ) G. S. Kozak and Q. Fernando, J. Phys. Chem., 67, 81 1 (1963). (2) G. O'Dom and Q. Fernando, ANAL. CHEM., 37,893 (1965). (3) J. Janata and 0. Schmidt, J. Electroanal. Chtm., 11, 224

(4) J. Janata and J. Zyka, Collect. Czech. Chem. Commun., 30,

( 5 ) L. Riccoboni, G. Pilloni, G. Plazzogna, and G. Togliavini,

(6) C. Bostic, Q. Fernando, and H. Freiser, Inorg. Chem., 2, 232

(7) Ibid., 4,602 (1965). (8) N. K . Chawla, D. G . Lambert, and M. M. Jones, J. Amrr.

(1 966).

1723 (1965).

J. Elecrroanal. Chsm., 11, 3 4 0 (1966).

(1963).

Chem. SOC., 89, 557 (1967).

with 0.1M NaCl. The generating electrodes consisted of a platinum foil anode (6 cm2) at which bromine was electro- generated, and a silver foil cathode (40 cmZ) immersed in one of the auxilary cells filled with 0.lOM HC10,. The detection system consisted of a rotating platinum micro- electrode (600 rpm) which was maintained at a potential of 0.25 volt L'S. a saturated calomel electrode which was located in the second auxiliary cell. The solution in the titration cell was stirred with a variable speed magnetic stirring motor and stirring bar. A Sargent Model IV coulometer was em- ployed for the electrogeneration of bromine and a Sargent Model XV recording polarograph was used to maintain the potential of the indicator electrode cs. the SCE and to re- cord the limiting current as a function of time. The entire cell assembly was maintained at a temperature of 25.0 i 0.1 "C.

Reagents. All buffer components as well as the NaBr used in this work were free of impurities that reacted with bromine. The ligand, 5-chloro-8-quinolino1 (Pfalz and Bauer) was recrystallized from ethanol (mp 124-5 "C) and the Mn(II), Fe(II), Co(Il), Ni(II), and Cu(I1) chelates of the ligand were prepared from the respective metal perchlorates (G. F. Smith Chemical Co.). The metal chelates were washed thoroughly to remove traces of uncomplexed ligand and the purity of metal chelates was determined by bromometric titrations in acid solutions.

Partition of Bromine in the H20/CHC13 System. The titration vessel contained 25 ml of CHCl, and 50 ml of an aqueous solution which was 0.10M in HCIOl and 0.50M in NaBr and which contained sufficient NaN03 to make the ionic strength 1.0. The solutions were mixed with the stirring bar rotating at 500 rpm; the rate of rotation was measured with a stroboscope (General Radio Co. Type 1531- A). At this rate of stirring, the chloroform layer was uni- formly dispersed in the aqueous layer in the form of small drops. Approximately 5 minutes after the stirrer and the rotating platinum microelectrode were started, a small amount of bromine was generated and the limiting current in the indicator system recorded after the system was allowed to equilibrate for approximately 1 minute. The limiting current did pot change appreciably for periods as long as 15 minutes. Successive increments of bromine were generated in this manner until a complete range of bromine concentrations that would be encountered in the kinetic studies was obtained. The bromide ion concentration in the aqueous phase was varied between 0.10M and 0.90M and the above set of ex- periments was repeated. Straight line plots were obtained when values of the limiting current were plotted against the concentration of bromine generated. When a similar series of experiments was carried out in which the bromine was generated continuously and not interrupted periodically, linear plots of the limiting current L'S. concentration of bro- mine were obtained which were essentially identical with the straight line plots that were obtained in the first set of ex- periments. Hence it may be concluded that the rate of ex- traction of bromine into the chloroform phase is extremely rapid,

Monobrornination of Phenol. The reaction that was selected for the evaluation of the extractive coulometric technique was the reaction of bromine with phenol, which has been extensively studied in aqueous solutions and shown to have a second-order rate constant of 2 X l o 5 l./mole .

ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971 1561

sec (9, 10). An added advantage is that phenol distributes itself between the chloroform phase and the water phase, and the bromination reaction takes place in both phases.

A stock solution of phenol (0.01M) was prepared and standardized by a bromometric titration (9). Fifty milli- liters of an aqueous solution 5.0 X lO-'M in phenol, 0.10M in HCIOl and containing varying amounts of NaBr (0.30 to 0.70M) and NaN03 to adjust the ionic strength to 1.0, together with 25 ml of chloroform were introduced into the titration cell which was maintained at a temperature of 25 "C. The solutions were stirred with a magnetic stirrer (500 rpm) and the rotating platinum microelectrode was started. After about 5 minutes had elapsed, bromine was generated at the lowest current setting in the coulometer (4.849 mA) and the current in the indicator circuit was recorded (at 2.5 PA full scale deflection) as a function of time.

RESULTS

Response of the Amperometric Indicator Electrode. In all the kinetic experiments the solution in the titration cell consisted of an almost uniform dispersion of chloroform droplets in the aqueous phase, but the rotating platinum microelectrode responded to the total concentration of bromine in the aqueous phase only. This was established conclusively by a calculation, based on this assumption, of the equilibrium constant, K , for the formation of Br3- in the aqueous phase and the distribution coefficient, K D of Brz between the water and chloroform phases.

(3)

The terms in square brackets represent concentrations in moles/l., the subscripts o and w refer to the organic and aqueous phases, respectively and D in Equation 3 is the distribution ratio which can be expressed as a function of the bromide ion concentration in the aqueous phase and the constants K and KD.

1 1 K - = - + -- [Br-1, D KO K D (4)

The total concentration of bromine generated can be calcu- lated from the number of coulombs of electricity used. On the assumption that the limiting current in the indicator circuit is proportional to the concentration of bromine in the aqueous phase only, it is possible to calculate the values of the distribution ratio D for various values of the bromide ion concentration, [Br-I, in the aqueous phase. From a linear least squares treatment of the experimental data (Equation 4), values of the constants K and K D that were obtained were 16.3 and 42, respectively. The former agrees well with the values 16.1, 16.0, and 16.5 that have been re- ported by other workers (9, 11, 12). The value of K D de- termined independently by equilibration of a standard soh- tion of bromine between HzO, (containing 1.OM NaBr) and CHC18, followed by an iodometric titration of the bromine in the aqueous phase, was 39 f 2.

(9) D. B. Scaife and H. J. V. Tyrrell, J. Chem. Soc., 1958, 386. (10) G. S. Kozak and Q. Fernando, Anal. Chim. Acta, 26, 541

(1 1) G. N, Lewis and H. Storch, ibid., 39, 2544 (1917). (12) G. Jones and S. Baeckstrom, ibid., 56, 1517 (1934).

( 1962).

Kinetics of Bromination of Phenol. The shapes of the current-time curves obtained for the phenol bromination were similar to those obtained in previous studies ( I , 2 ,5 ) , and were used to calculate the rate constants for the monobro- mination of phenol in the aqueous phase and in the chloro- form phase with the aid of the following equations.

The distribution coefficient of phenol is

( 5 ) [CsHsOHL [CsHaOHlw

If the volumes of the organic and aqueous phases are V,

Ks =

and V, ml, respectively,

where mo and m, represent the number of millimoles of phenol in the organic and aqueous phases, respectively.

At time t sec after the generation of bromine was started, the number of millimoles of bromine that reacted must be equal to the number of millimoles of phenol that reacted, if the monobromination reaction is the only reaction that is of importance in the time interval, t sec. Hence:

where i is the generating current in mA, and mT is the total number of millimoles of phenol that was added to the system. Rearrangement of Equation 7 and substitution for m, and [CeHsOHI, from Equations 5 and 6 and D from Equation 3 give Equation 8.

The decrease in the concentration of phenol in the organic phase can be calculated as a function of time from Equation 8. The rate of the reaction in the organic phase, - d[CsHsOH],/dt, can be calculated by differentiating Equation 8 with respect to t and measuring d([Brz], + [Br*-],)/dt, the slope of the current-time curve, at barious values of t . The current in the amperometric indicator system is proportional to ([Brz], + [Br3-],), the total con- centration of unreacted bromine in the organic phase, and its value can be obtained from a calibration curve. The distribution ratio, D , which is a function of the bromide ion concentration in the aqueous phase (Equation 4), is essentially a constant since the concentration of phenol is severaI powers of ten smaller than the concentration of the bromide ion and the electrogeneration of bromine for com- plete reaction would not deplete the concentration of bro- mide ion to any appreciable extent. The distribution co- efficient of phenol when equilibrated in the two phase system, and the concentration of phenol in each phase were determined by a bromometric titration. The value of KS obtained in this manner was 2.50. This value was confirmed by a spec- trophotometric method in which the concentration of phenol in the organic phase was calculated from the measurement of the absorbance of the organic phase at 279 nm and a Beer's law plot. Hence, the second order rate constant in the organic phase, kohs, can be calculated from Equation 9.

d dt

(9) -- [CsHsOHL = koob* [CsHsOHLIBr&

1562 ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971

Table I. Rate Data for the Moaobromination of Phenol in H20/CHCls System (Generating current = 4.849 mA. Volume of aqueous phase = 50.00 ml. Volume of organic phase = 25.00 ml.

Total amount of phenol present in both phases initially = 3.936 mmoles.)

0.63 0 .80 1.01 1.11 1.29 1.42 1.70 1.85

1.75 1.94 2.30 2.40 2.68 2.95 3.41 3.97

8.22 2.68 9.08 2.46

10.8 2.18 11.2 2.05 12.5 1.81 13.8 1.64 16.0 1.27 18.6 1.08

6.70 6.14 5.45 5.12 4.53 4.10 3.18 2.70

0.94 4 . 7 1.21 4.7 1.36 4 .4 1.27 4.5 1.92 4.5 2.02 4 . 0 2.76 5.0 3.32 5 . 0

10 9.9 9.4 9 . 6 9 . 7 9 .7

11 11

The decrease in the concentration of phenol in the aqueous phase can be calculated as a function of time from Equation 9 which is obtained from Equations 5 and 8.

The reaction rate in the aqueous phase is also a function of (d/dt)([Brz], + [Bra%), the slope of the current-time curve. The monobromination of phenol in the aqueous phase was shown to be an overall second-order reaction, first-order with respect to phenol and first-order with respect to the total bromine. The second-order rate constant, kl, for the monobromination of phenol is given by the equation:

kl = kwobs (1 + K[Br-I) (11)

where Lobs is the experimentally determined reaction rate constant in the aqueous phase in the presence of various concentrations of bromide ion. The average value of kl that was obtained was 4 X lo6 l./mole . sec which is of the same order of magnitude as the value, 2 X 106 l./mole sec (10, 13), that has been reported previously. A typical set of experimental data is shown in Table I.

The rate of monobromination of phenol in the chloroform phase was determined in an analogous manner and was shown to be an overall second-order reaction, first-order with respect to bromine and first-order with respect to phenol. The observed rate constant was 1 X lo4 l./mole sec, and as expected, was independent of the bromide ion concentration in the aqueous phase.

Kinetics of Bromination of Metal Chelates of 5-Chloro-8- quinolinol. One of the objectives in this study was to de- termine whether it would be possible to carry out electro- philic substitution reactions of metal chelates with bromine under conditions that would ensure that the intact metal chelate would react and not the free ligand. The bis chelates of Mn(II), Co(II), Ni(II), and Cu(I1) with 5-chloro-8-quino- linol were selected for this investigation. The complexity of the kinetics would be reduced since only the 5-position in each of the 8-quinolinol rings would be available for elec- trophilic attack by bromine. It was important to control the pH of the aqueous phase within narrow limits. Too low a pH would disrupt the metal chelate rings and the free ligand would distribute itself between the aqueous and

(1 3) R. P. Bell and D. J. Rawlinson, J. Chem. Soc., 1%1,66.

organic phases and react with bromine. Too high a pH would tend to form undesirable electroactive species such as HOBr which would complicate the kinetics. It was neces- sary, therefore, to control the pH in the aqueous phase at the optimum value of 6.87 by means of a phosphate buffer (0.025M Na2HP04, 0.025M NaH2P04, and NaBr together with NaN03 to maintain an ionic strength of 1). The buffer constituents did not react with bromine and the ex- tent of HOBr formation was less than 0.6%. A comparison of the calibration curves in acid solution and in the phosphate buffer showed that the current efficiency was essentially the same in both solutions.

Kinetic studies were carried out in the two-phase system, consisting of a chloroform solution of the metal chelate (10-4M) and an aqueous solution containing a phosphate buffer (pH = 6.87), NaBr (OSOM), and sufficient NaN03 to maintain an ionic strength of 1.0. The experimental pro- cedure was the same as that used for determining the rate of monobromination of phenol. Since the metal chelate is almost completely insoluble in the aqueous phase, the value of the distribution coefficient is infinitely large and Equation 12 that is analogous to Equation 8 can be derived for the calculation of the decrease in the concentration of the unreacted metal chelate, (ma/Vo), as a function of time.

It may be seen from Equation 12, (as well as Equation 8), that the rate of decrease in the concentration of bromine in the aqueous phase is a measure of the rate of the reaction. Hence, the method used for the determination of the rate of bromination of phenol could also be used for following the kinetics of bromination of the metal chelates. A related method that has been described by Janata and coworkers (3) was used to confirm the results that were obtained. The rates of bromination of the Mn(II), Fe(II), Ni(II), and Cu(I1) chelates showed a first-order dependence on the concentration of bromine in the organic phase but showed a fractional-order dependence on the concentration of the metal chelates, whereas the bromination rate of the Co(I1) chelate showed a first-order dependence on the chelate con- centration and a fractional-order dependence on the bromine concentration. The validity of the assumption that no free ligand was formed in the system was checked by an examina- tion of the absorption spectra of the two phases at various stages of the reaction. The absorbance of the aqueous phase at 244 nm showed no evidence for the presence of

ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971 1563

PA

21

148 - d B - F- 1.5 a a 0 1-2

2 2 0,9 t; n

K

K

0.6

0.3

I I I

60 120 180 T I M E ISECI

Figure 1. Current us. time curves for bromination of metal chelates of 5-chloro-8-quinolino1

1. Blank 2. Bis(5-chloro-8-quinolinolato)cobalt(II) 3. 5-chloro-8-quinolinol 4. Bis(S-chloro-8-quinolinolato)iron(II) 5. Bis(5-chloro-8-quinolinolato)manganese(II) 6. Bis(5-chloro-8-quinolinolato)nickel(II) 7. Bis(5-chloro-8-quinolinolato)copper(II)

Volume of aqueous phase = 50 ml. Volume of chloroform phase = 25 ml. [Br-] = 0.50M. Total ionic strength = 1.0. pH = 6.88. Initial concentration of metal chelate in both phases = 1.00 X lO-*M. Initial concentration of 5-chloro-8-quinolinol in both phases = 2.00X 10 -'M

the free ligand in the aqueous phase at all stages of the reaction. At the conclusion of a kinetic experiment, the ab- sorption spectrum of the chloroform phase closely resembled the absorption spectrum of a chloroform solution of the di- brominated metal chelate. An extensive series of spectro- photometric measurements in the aqueous as well as in the chloroform phase confirmed the prediction that the integrity of the metal chelate ring system was preserved throughout the course of the bromination reactions of all five transition metal chelates that were investigated.

DISCUSSION

The success of the extractive technique depends on several factors: the volume and geometry of the cell compartment in which the bromine is electrogenerated and monitored amperometrically, the relative volumes of the aqueous and organic phases and the rate of mixing of the two phases. The rate of mixing should be sufficiently rapid to form a uni- form dispersion of droplets of the organic phase in the aqueous phase, without forming an emulsion. If optimum conditions for a given system are used, the current-time curve that is obtained in the absence of a compound that con- sumes bromine should be a straight line that passes through the origin. The slope of this straight line is governed by the rate of stirring, the rate of equilibration of bromine between the two phases, the rate of generation of bromine, and the bromide ion concentration in the aqueous phase. Blank experiments in which bromine was continuously gen- erated and in which the bromine generation was interrupted and allowed sufficient time to reach equilibrium showed

that even in the former case, equilibrium was established instantaneously. Furthermore, these experiments showed that the response of the rotating platinum microelectrode was proportional to the concentration of electroactive species that were present only in the aqueous phase. The current efficiency was checked by the amperometric titration of known amounts of allyl alcohol with couIometrically generated bromine, in an aqueous system as well as in the stirred two- phase system. The bromination of allyl alcohol is so rapid that the current-time curve resembles an amperometric titration curve. The results obtained confirmed that the current efficiency was 100% in both systems. The assump- tion that the distribution of bromine between the two phases is not a rate-limiting step was confirmed by the determination of the distribution coefficient of Brz between chloroform and water and the verification of the value for the known con- stant for the formation of the tribromide ion from bromine and bromide ions in the aqueous phase.

One of the most stringent tests that the extractive coulo- metric method could be subjected to is the determination of the rate of monobromination of phenol in the aqueous phase as well as in the organic phase. The rate of monobromina- tion of phenol in water is about the fastest reaction, (with a second-order rate constant of 105 l./mole . sec), that can be investigated by the combined coulometric-amperometric technique (9). Reactions that are faster give current-time curves which resemble amperometric titration curves that usually consist of two straight line segments which intersect at the end point of the titration. No information about reaction rates can be obtained from such current-time curves. Since the dibromination reaction is much slower than the monobromination reaction, the rate constant for the latter reaction could be obtained on the assumption that the extent of dibromination is negligible. This assumption was confirmed experimentally from the current-time curves. The amount of bromine electrogenerated up to the point at which a rapid increase in the current was observed, cor- responded to almost complete monobromination of the phenol in the system. The reproducibility of the rate con- stant for the monobromination of phenol in water, (4.+ 1) X 106 l./mole . sec, indicated that the phenol as well as the bromine were in equilibrium between the two phases through- out the course of the reaction. The rate constants obtained in this study are not strictly comparable with the rate con- stants obtained previously in water or in organic solvents. In this work the aqueous phase is saturated with chloroform and the chloroform phase is saturated with water, and the effect of this unavoidable complication on the rate constants cannot be predicted.

This extractive technique was devised primarily for the investigation of the rates of electrophilic substitution of a series of related metal chelates in which the effects of metal ion variation could be evaluated. A comparison of the rate constants for the bromination of the metal chelates would be meaningless since the rate equations have fractional orders with respect to the metal chelate or bromine, and differ from one complex to another. An estimation of the relative rates of reaction, however, can be obtained from a comparison of the current-time curves obtained under identical experi- mental conditions. When the total metal chelate concen- tration was 1.00 X lO-4M and the bromide ion concentra- tion 0.50 M, the current-time curves obtained are shown in Figure 1. There is little difference in the rates of bromina- tion of the Cu(II), Mn(II), and Ni(I1) chelates; the Fe(I1) chelate reacts somewhat slower and the Co(I1) chelate is sub-

1564 * ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971

stantially less reactive than all the other chelates. It was con- clusively 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 photoiniti- ation 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 ex- tracted 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 to 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 lo3 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 to 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-D- glucose by a variety of spectrometric, chromatographic, and polarimetric techniques are presented and dis- cussed. 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 mag- netic resonance spectrometry. I t i s shown that, during melting, both a-D-gIUCOSe and p-D-glUCOSe anomerize rapidly to give an equilibrated mixture of these anomers.

FOR ABOUT 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 standardiza- tion 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 en- zymes are employed.

Thus, clinical chemists require a D-glUCOSe standard ref- erence 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 suc- cessfully 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. Neverthe- less, 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 to a-D-glUCOSe, where we anticipated that the im- purity total determined might represent the sum of the con- tents 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-D- glucose and in a commercial P-D-glucose have been deter- mined 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., Inter- science, 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).

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