RESEARCH
Instrument Determines Glucose Continuously Special differential filter-photometer measures rate of air oxidation of glucose catalyzed by glucose oxidase Clinically important substances can be determined continuously by a procedure that's based on measurement of enzyme-catalyzed reaction rates. The technique was developed by Dr. W. J. Blaedel and G. P. Hicks of the University of Wisconsin. A specially designed, sensitive differential filter-photometer for measuring the absorbance of a flowing solution is used, they told the Ninth Detroit Anachem Conference. The research technique has been tested by applying it to the well known enzymatic determination of glucose. The chemical basis of the glucose method is the air oxidation of glucose
—catalyzed by glucose oxidase—to gluconic acid and hydrogen peroxide. The rate of peroxide production is measured by the peroxidase-catalyzed oxidation of colorless o-tolidine to a benzidine blue derivative, giving an absorbance increase in the 635-millimicron region. By controlling the concentration of reagents, the rate of change of absorbance is proportional to the glucose concentration. The sample containing glucose is introduced through a one-channel peristaltic pump. The other reagents are introduced in a single solution by gravity flow. The reaction starts at the mixing point. Absorbance of the
CONTINUOUS. Dr. W. J. Blaedel (left) and G. P. Hicks examine a recorder chart from their filter-photometer which measures absorbance of a flowing solution. !n glucose determination, glucose is oxidized to gluconic acid and H202f which oxidizes o-tolidine to benzidine blue. The instrument determines glucose by measuring the absorbance increase due to the formation of benzidine blue
38
C&EN
OCT. 30,
1961
reaction mixture increases uniformly as the solution flows at a constant rate downstream through a pair of spectrophotometer cells. A fixed distance in the flowing stream separates the cells. The absorbance difference between the two cells is proportional to the rate of reaction, thus to the glucose concentration. The absorbance difference is measured directly on a differential recording filter-photometer. The unique feature of the photometer is its high sensitivity. This sensitivity is due to the measurement of the small absorbance difference as a small fluctuating intensity, superimposed on a high, steady-state illumination level. Full-scale on the recorder corresponds typically to a difference of 0.01 absorbance unit, or a 2% difference in transmittan.ee between the two cells. Stability is about 1% of fullscale, permitting differences of the order of 0.0001 absorbance unit to be detected between the cells. With this high sensitivity, the reaction doesn't have to proceed far to allow getting an estimate of its rate. The reaction rate is, therefore, an instantaneous or differential rate rather than an integral one. In determining glucose, for instance, the two cells are only 30 seconds apart in the flowing stream. Locating the first cell close to the mixing point makes the measured rate an initial rate. With glucose, a delay of about 30 seconds is deliberately imposed between the mixing point and the first cell to overcome an induction period (during which the rate builds up to its initial value). This delay isn't needed for all reactions. Because an initial rate is measured, the response is linear with amounts of glucose concentration in the region of clinical interest ( 0 to 400 mg. ). The average deviation on glucose standard solutions is 2 mg. of glucose per 100 ml. of solvent, or 2% relative, whichever is greater. By a simple calibration with a standard glucose solution,
the sensitivity may be adjusted to give direct readout. The number of chart divisions is numerically equal to the amount of glucose in the solution. Four Minutes. Dr. Blaedel's and Mr. Hicks' original instrument handles about 15 samples per hour with a readout time of four minutes for each sample. A second design is in progress which will double the sample throughout to 30 per hour, with a readout time of two minutes per sample. Blood samples give no mechanical or chemical difficulties in glucose determinations. Only 0.2 ml. of sample is needed for a determination. Accuracy is comparable to other accepted methods of glucose analysis, Mr. Hicks says. Because of rapid measurement near the start of the reaction, the technique is not subject to some interferences that occur in conventional methods which need longer incubation periods. This is shown by the use of otolidine, which some workers say is too unstable for the determination of glucose. Other applications being studied are the determination of serum enzymes such as isocitric dehydrogenase and lactic dehydrogenase. In principle, the method also seems applicable to alkaline phosphotase, transaminases, and other enzymes. Many other clinically important substrates (of which glucose is one) might also be candidates for this technique. Activators and inhibitors of enzyme reactions might also be determined. Isocitric dehydrogenase, for instance, activated by manganese or magnesium ions, has been studied as a possibility for measuring the activating metal. The continuous method is readily adaptable, and good linearity is observed between the reaction rate and the metal's concentration (for metals occurring in suboptimal concentrations) . The sensitivity is very high, 5 p.p.b. of manganese ion being readily detectable. But the specificity is not good. Calcium ion is antagonistic and so are several other metal ions to a lesser extent. As a result, direct application to aqueous analysis without first separating interfering agents isn't possible. If this nonspecificity is typical of activators and inhibitors, the greatest promise for this technique appears to be the determination of substrates and enzymes themselves, the Wisconsin scientists feel.
Thermometric Titrations Gain Favor Continuous recording of solution temperature, automatic reagent delivery help the technique New techniques and equipment should do much to help thermometric titrations gain favor as standard lab operations (C&EN, Oct. 23, page 4 9 ) . Not limited to solution titrations, the method can be used for titrations with gaseous titrants as well, Dr. David N. Hume of the Massachusetts Institute of Technology told the Ninth Detroit Anachem Conference. It's even possible to apply the technique to a gasgas reaction in the absence of a liquid phase. Once considered impractical because of the tedious measurements involved, a thermometric titration is now no more complicated than many other instrumental titration methods, Dr. Hume says. The development of sensitive thermistor sensing elements with rapid response, which allow continuous recording of solution temperature, helped this advance. Equally helpful is the use of an automatic device that delivers reagent at a constant rate. In thermometric titrations, temperature measurements follow the course of a reaction and locate the end point. A reagent is introduced into the system at a constant rate, while a sensitive thermistor in a bridge circuit (coupled with an automatic recording potentiometer) continuously measures temperature as a function of the reagent that's been added. Temperature changes of a few hundredths of a degree C. are enough to give satisfactory titration curves. These curves approach the form of straight line segments with breaks at the start and at the equivalence point of the titration. Thermometric titration offers the analyst several advantages. It is generally applicable to almost all analytical reactions. The method has been used in neutralization, complexation, reduction, redox, and precipitation reactions. Sensitive. The method is sensitive. It can be used to find end points between tandem reactions for which the free energy change is small. Examples are the titration of mixtures of weak acids in which the ionization constants are not well separated, or reactions which are appreciably incomplete at the end point (such as the titration of
very weak acids in which the salts show considerable hydrolysis). Free hydroxide, for instance, can be readily titrated in the presence of carbonate. A mixture of weak acids can be titrated by converting them to the corresponding salts, then titrating the salts with strong mineral acid. The heat of reaction in this case is due only to the ion association between the anion and the hydrogen ions. As many as five components have been determined in a single titration, Dr. Hume says. The method is not restricted by the solvent. Dielectric constant, optical density, and electrical conductivity of the solvent have no effect. This makes thermometric titrations particularly attractive for nonaqueous titrations in low dielectric media. For instance, bases as weak as acetamide and acetanilide can be titrated in glacial acetic acid. Also, Lewis acids and bases are readily titrated in aprotic solvents. Titration with Gases. The thermometric titration method can also be used for titrating with gaseous titrants. If the titrant gas is rapidly absorbed and reacts quickly with the substance being titrated, good curves are obtained. Such a reaction has the advantage of minimum heat of mixing and change in heat capacity due to increasing bulk of sample. Gaseous ammonia has been used to titrate a number of acids, both in aqueous and in benzene solutions. The characteristics of both these curves are better than those obtained with conventional liquid reagents, Dr. Hume says. Redox reactions are also feasible. For example, gaseous sulfur dioxide can be used to titrate iodine in aqueous solution. Thermometric titrations for gas-gas reactions without a solvent phase are also practical. The low heat capacity of such systems and the tendency for gas-gas reactions to be relatively slow make the technique more difficult to apply. However, ammonia and volatile aliphatic amines react rapidly and quantitatively with hydrogen chloride, and may be titrated to good end points. OCT. 3 0, 1 9 6 1 C&EN
39
