Lumometric End Point Detection for Nonaqueous Titrations MARTIN DIMBAT and G. A. HARLOW Shell Developmenf Co., Erneryville,
Calif.
b Many nonaqueous titrations are accompanied by low-level luminescence which can be detected with a sensitive scintillation counter. Very little light i s produced during a titration until the equivalence point i s reached and then a sharp peak in intensity occurs. The phenomenon has been studied with acid-base titrations and oxygenmetal alkyl titrations, both in nonaqueous solvents. There i s some evidence that the luminescence i s due to acidity-sensitive side reactions involving auto-oxidation products.
D
a recent investigation in which a liquid scintillation counter was used for the determination of radioactive carbon dioxide i t was found, quite unexpectedly, that the absorbing solution itself gave off measurable amounts of light. The absorbing solution in this case was toluenecontaining acetyldimethylbenzylammonium hydroxide, a strong base used as a n experimental titrant for nonaqueous acid-base titrations. The high blank was first presumed to be due to some inadvertent contamination with carbon14. A systematic search revealed, however, that the high background was due not t o contamination, but to luminescence associated with the quaternary ammonium titrant. This phenomenon was of such unusual nature that a general exploratory investigation was undertaken. It soon become apparent t h a t the phenomenon was not limited to solutions of quaternary ammonium bases but accompanied a wide variety of reactions. The present phase of study
,
1 . 0 0 rnl of O . I C 6
N HC1 (IP.41
URING
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Figure 1. Apparatus for lumometric titration of acids and bases
450
deals mainly with luminescence which accompanies acid-base and metal alkyloxygen titrations and the peculiar peak in intensity which occurs at the end point. A search of the literature has failed to reveal any previous report of acidbase titrations where the end point was detected by luminescence in the absence of any added indicator. The extensive
ANALYTICAL CHEMISTRY
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Figure 2. Lumometric titration of hydrochloric acid (0.106 meq.) in isopropyl alcohol with tetra-n-butylammonium hydroxide
work of Audubert (1-6), although concerned with low-level chemiluminescence (in aqueous solutions), makes no mention of such a phenomenon. This is not strange in light of the fact, uncovered by the present investigation, t h a t such end points are produced only in nonaqueous solutions. A similar lack of literature references exists in the case of metal alkyl-oxygen titrations.
used for stirring were introduced into the titration vial through stainless steel hypodermic tubing. Titrations were carried out as follows: One- or two-ml. samples of the acid or base solutions to be titrated were pipetted into the titration vialj the serum cap put into place, and the vial placed into position above the multiplier phototube. The three hypodermic tubes mere inserted, the light-tight covering replaced oyer the tube housing, and the nitrogen flow started. Several 1-minute counts were taken before the titration was started to establish the background. Titrant was then introduced in small, equal increments m d 1-minute counts taken until the rate subsided to about the background rate. This occurred within the first minute ewept near the equiralence point. The titration was continued until the end point peak had been passed or, as in the case of aqueous solutions, the calculated equivalence point had been passed. Titration Results. T h e lumometric titration curve obtained when 0.1 meq. of hydrochloric acid in isopropyl alcohol solution is titrated with a nonaqueous quaternary ammonium hydroxide is shown in Figure 2 . T h e titrant, tetra-n-butylammonium hydrovide (TRAH) in isopropyl alcohol, was prepared by a n ion exchange method (6) and delivered from a syringe microburet. Stirring was accomplished by bubbling nitrogen through the solution. The height of the peak was not reproducible, varying from about 3000 to 130,000 counts per 0.05-ml. increment. The location of the peak was, honever, very reproducible and it always occurred near the calculated equivalence point.
ACID-BASE TITRATIONS
Apparatus and Procedure. T h e apparatus used for t h e lumometric titration of acids a n d bases is shown in Figure 1. T h e light detection system consisted of a D u M o n t K 1448 multiplier phototube with a preamplifier a n d scaler, a n assembly very similar t o t h e liquid scintillation apparatus reported by Hodgson, Gordon, and Ackerman (7). KOmodification of the apparatus was required for lumometric titrations because they were carried out in a 3-dram vial of the type normally used with this apparatus. Titrant was introduced from a syringe microburet manufactured by the MicroMetric Instrument Co., Cleveland, Ohio. Both the titrant and the nitrogen gas
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Figure 3. Lumometric titration of sulfuric acid (0.0645 meq.) in pyridine with tetra-n-butylammoniumhydroxide
1
LSCO -
ZbCO
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Figure 5. Apparatus for lumometric titration of metal alkyls with oxygen o r air
Figure 4. Simultaneous lumometric and conductometric titration of 1 .O ml. of 0.1 059N HCI (IPA) with 0.0975N KOH (IPA)
Tlie titration of sulfuric acid in pyridine ITith the same quaternary ammonium titrant is shown in Figure 3. An interesting feature of this titration curve is the occurrence of a sniall maximum a t the point corresponding to the titration of the first hydrogen of sulfuric acid. A second much larger maximum occurs a t the equivalence point corresponding to both hydrogens. Similar inflections were obtained ryhen strong acids in nonaqueous solvents w r e titrated with a solut'ion of potassium hydroxide in isopropyl alcohol, although with this titrant the light emitted was less intense. An interesting esaiiiple of a combined luinometric and conductometric titration of hydrochloric acid in isopropyl alcohol solution with potassium hydroxide as titrant is shown in Figure 4. The conductometric apparatus used n-ss the simplest possible: tn.0 platinum wires with looped ends mounted on a rubber stopper and fitted to the titration vial. A battery was connected to the electrodes in series with a milliammeter. The vial was placed in position in tlie scintillation counter, titrant and nitrogen (for stirring) being introduced through hypodermic tubing. This lumometric cur\-e differs from that in Figure 3 in t h a t the emitted light does not fall to a very low value after tlie end point is passed. The residual luminescenee is due to the passage of the conductometric indicator current through the solution. This electroliiniiiiescence is very weak in scid solutions such as HCI, but considerably stronger in alkaline solutions such as the one obtained with a n excess of potassium hydroxide. When potassium isopropylate is substituted for potassium hydroxide a s the titrant, similar end point inflections are obtained. The addition of ethyl
alcohol, nater, and methanol to a sample of hydrochloric acid in isopropyl alcoliol reduces the end point luniinescence in the order indicated, ivith methanol having the greatest effect. No detectable end point peaks were obtained with aqueous solutions, even those as strong as 5 5 . To check the possibility that light of shorter n-avelength was produced in aqueous reactions, a n ultraviolet-sensitive phototube was used. A count only slightly above background was obtained in the titration of nitric acid n i t h 5 S sodium hydroxide, but even this slight increase in count was traced to the effect of temperature on the phototube. KO end point luminescence could be detected when the titration 11-as carried out with rengents prepared in a dry bo.\ (nitrogen atmosphere) froin isopropyl alcohol m-hich had been redistilled under nitrogen. However, titrations carried out under a n atmosphere of nitrogen yielded lumometric end points d i e n reagents, prepared in the normal manner, viere employed.
the niultiplier phototube and the hypodermic tubes inserted through its serum cap. The 5-mL syringe of the microburet n s filled nith titrant gas through stopcocks 2 and 3. With the silicone oil in the manometer pulled t o the top of the manometer, the gas in the syringe was passed through stopcocks 2 and 3 through the sample and back into the manometer. The unnhorbed gas n-as drawn back into the syringe and then recycled through the ssmple until no further absorption took place. When air was used as the titrant, tlie unabsorbed nitrogen mas discharged into the atmosphere through stopcocks 2 and 1. The total count obtained during the complete absorption of the oxygen in each 5-ml. increment of gas \vas recorded. Titration Results. K h e n metal alkyls are titrated n i t h air or oxygen in inert solvents lumometric curves are obtained which are similar to those obtained in acid-base titrations. An example of such a curve is s h o n n in Figure 6. The sample in this case was 2.12 nimoles of aluminum triisobutvl dissolved in octane and t h e titran; was d r y air. T h e ueak in light emission occurs a t tl;e point -
I - - -
-
METAL ALKYL-OXYGEN TITRATIONS
Apparatus a n d Procedure. T h e apparatus shown in Figure 5 was used in the titrations involving air and oxygen. T h e components are much the same as those used in the acid-base titrations except for t h e addition of the manometer and associated stopcocks. T h e syringe microburet in this case serves the twofold purpose of metering out the gaseous titrant and serving as a circulating p u m p for recycling the gas through t h e sample. The titration procedure consisted of the following: d vial containing a known amount of metal alkyl (in hydrocarbon solution) was placed in position above
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Figure 6. Lumometric titration of 2.1 2 mmoles of aluminum triisobutyl with dry air VOL. 34, NO. 4, APRIL 1962
e
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where one a t o m of oxygen has been introduced for each atom of aluminum in t h e sample. A similar curve is obtained when aluminum diethylmonochloride is titrated with dry air. The scatter of the points is, however, much greater in this case. The purity of the sample was not sufficient to determine the stoichiometry with any degree of accuracy. Lithium n-butyl reacted in somewhat different manner. When a 2% solution in toluene was titrated with air, a very sharp increase in light intensity occurred a t a point corresponding to one atom of oxygen for two atoms of lithium. Beyond this point no more oxygen was absorbed but light emission continued, a t a gradually decreasing rate, for more than 24 hours. DISCUSSION
To be completely satisfactory, any explanation of the lumometric end point must account for the following experimental observations : The peak in luminescence occurs during titrations which are very different in nature; Le., acid-base titrations and metal alkyl-oxygen titrations; the lumometric phenomenon is not displayed in aqueous solutions; acid-base titrations carried out with reagents prepared in the nornial manner exhibit the phenomenon even when a n inert gas atmosphere is used;
the acid-base lumometric end point is not obtained when solutions are used which have been prepared and stored in a n inert atmosphere; and the light produced lies in the visible region of the spectrum. This experimental evidence leads to the belief that the source of the light, even in the case of the acid-base titrations, is due to a n oxidation-reduction reaction. Atmospheric oxygen is probably not involved directly in the chemiluminescence but rather indirectly through oxidation products which slowly build up when strongly basic nonaqueous titrant is exposed to air. These oxidation products may be a complex series of peroxides, olefins, and free radicals arising from auto-oxidation of the basic isopropyl alcohol solution. The light-producing reaction must in some way be sensitive to the acidity of its environment, perhaps because some of the oxidation products are stable in both acid and alkaline solution but decompose rapidly in neutral solution with the emission of light. T o explain the end point peaks in the case of the reaction between metal alkyls and oxygen, one might assume that an intermediate compound is first formed which is stable in the presence of excess metal alkyl. At the equivalence point, where all of the alkyl has reacted, the intermediate oxidation product
reacts with additional oxygen to produce a n unstable product which then decomposes with luminescence. The lumometric method of detecting end points does not appear to have many practical applications. I n the case of acid-base reactions, i t cannot compete in terms of speed, selectivity, or simplicity of apparatus with existing techniques such as potentiometric and color-indicator titrations. I n the case of metal alkyl-oxygen titrations, there may be some analytical applications, but a clarification of the mechanism involved is necessary before they can be exploited fully. LITERATURE CITED
(1) Audubert, R., J. chim. phys. 33, 507
(1936).
(21 Audubert, R., T r a n s . Faraday SOC. 35. _ _ 197 f1939’I. \ - - - - ,
(3) Audubert, R., Prost, RI., Compt. rend. 202, 1047 (1936). (4) . . Audubert, R., Van Doormaal, Ibid., 196, 1883 (‘1936). (5) Audubert. R.. Viktorin, 0.. Ibid.,. 202,. .
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(6) Harlow, G. .4.,Noble, C. M., Wyld, Garrard E. A , AXAL. CHEM.28, 787
(1956). (7) Hodgson, T. S , Gordon, B. E., Ackerman, M. E., Yucleonics 16, 89 (1958). RECEIVED for review Sovember 2, 1961. Accepted February 1, 1962. Gordon Research Conference (preliminary report), -4uguet 1961.
A N e w Automatic Spectrophotometric Rate Method for Selective Determination of Glucose in Serum, Plasma, or Blood H. V. MALMSTADT and S. 1. HADJHOANNOU Departmenf of Chemisfry and Chemical Engineering, University o f Illinois, Urbana, Ill.
b A new method i s described for the determination of glucose in serum, plasma, or blood by a rapid automatic spectrophotometric rate procedure. Glucose i s oxidized selectively in the presence of glucose oxidase and the hydrogen peroxide produced reacts immediately with iodide in the presence of molybdate catalyst to form triiodide which absorbs strongly at about 360 mp. Near the start of the reaction, the rate of change of the triiodide concentration i s proportional to the glucose concentration and the time required for a small preset change in tri-iodide concentration i s related easily to the glucose in the sample. Glucose i s determined in 0.08 ml. of serum, plasma, or blood with relative errors within 270 and measurement times are about 1 to 3 minutes. 452
ANALYTICAL CHEMISTRY
E
have been used widely for the specific determination of glucose in biological fluids. I n recent procedures glucose is oxidized in the presence of glucose oxidase, forming hydrogen peroxide, which in turn oxidizes a dye in the presence of horseradish peroyidase to forni a colored reaction product proportional to the glucose concentration (1-3,8). The absorbance of the colored product is measured after the reaction has approached completion (1, 8) or after the reaction has been stopped a t a preset time ( 7 ) . The above procedures require a long time per determination and the peroxidase enzyme is expensive. hlalmstadt and Hicks ( 4 ) described a method for the determination of glucose in blood serum based on the above coupled enzyme reaction but obtained K Z Y ~ I S T I CMETHODS
the quantitative data 17-itliinthe first few minutes of the reaction by an automatic spectrophotometric system This method reduces greatly the total technician time but still employs the peroxidase enzyme and is not as sensitive as desired. 3Ialnistadt and Pardue (3presented a potentiometric reaction-rate method for aqueous glucose samples in nhich the hydrogen perohide formed in the oxidation of glucose reacts immediately with excess iodide, in the presence of molybdate catalyst, to produce an equivalent amount of iodine. Readout data are obtained nithin the first minute of the reaction and are related to sample glucose Concentration. The method is rapid, precise, sensitire, and substitutes the less expensive molybdate catalyst for the peroxidase enzyme.
