oxalate concentration, iodine liberation by dichlomate is not instantaneous. Fui ther, the iodine-starch blue color appears when only 9570 of thiosulfateis oxidized, although the blue color gradually fades. Thus, returning and uncertain end points are obtained. nxenthe concentration of oxalate is greater, higher titlers are obtained, ProbablY because Of over-oxidation of thiosulfate I
LITERATURE CITED
(8) Sully, B. D., J . Chem. SOC. 1942,
0. H., Baty, AI., ISD. EXG.CHEV.ANAL.ED. 13, 442 (1941). ( 2 ) Hahn, F. L., J . Am. f3em. 57, 614 (1935). (3) Kolthoff, I. AI., Pharm. Weekblad 5 6 , 644 (1919). ( 4 ) Ibzd., 5 9 , 66 (1922). (5) Kolthoff, I. 11.) 2. AnaE. Chem. 59, 401 (1920). (6) Ibzd., p. 411. ( 7 ) Kolthoff, I. >I., van Berk, L. H., J . d m . Chem. SOC.48, 2799 (1926).
( 9 ) Treadwell, F. P., Hall, ITr. T., “An-
(1) Gaebler,
366.
alytical Chemistry,” 9th Ed., Tol. 11, p. 588, Wiley, New York, 1942. (10) Tan Dame, H. C., J . Assoc. Ofic. Agr. Chemists 30, 502 (1947). RECEIVED for review November 24, 1964. Accepted J ~ l y20, 1965. One of LIS (B.V.S.S.) has received an award of a research fellowship from the Council of Scientific and Industrial Research (India).
Rapid Removal of Alkali Metals from Quaternary Ammonium Bases by Ion Exc ha nge EUGENE D. OLSEN and ROBERT L. POOLE, JR. Department o f Chemistry, University o f South Florida, Tampa, Fla. The contamination of quaternary ammonium bases with alkali metal ions i s difficult to avoid. A simple procedure for removing sodium ion and other alkali metals from tetramethylammonium hydroxide, tetraethylammonium hydroxiide, tetrapropylammonium hydroxide, and tetrabutylammonium hydroxide on Dowex 50 W-X16 cation exchange resin i s described. It has been demonstrated that a sodium ion content in the bases of even 0.05M or more can be rapidly decreased to less than 1 p.p.m., with breakthrough capacities of about 20% of the total exchanige capacity, and yields of all four bases greater than 80%. Conditions necessary for optimum efficiency and yields were studied in detail. Breakthrough capacity data given should be useful in estimating the amount of resin that should b e used in purifying a given amount of quaternary ammonium base of known alkali metal content. Resins can b e regenerated and reused.
Q
bases have numerous important applications in analytical chemistry. Sonaqueous solutions of these bases are excellent titrants for the deterrnination of acids in organic solvents. They are superior to the alkali metal hydroxides and alcoholates because they can be used with the gla53 electrode without introducing the “alkali erior” and because they form salts which are more soluble in nonaqueous solvents than are those of the alkali metals (10). Similarly, tetramethylammonium h;,-droxide (TILIAiH) is a very useful titrant in potentiometric stability constant determinations, especially when there’ is a possibility of the ligand forming complexes with alkali metal ions (17). And while a UATERNARY AUMONUU
number of ligands will complex with alkali metals in aqueous solutions (W), this tendency to complex is accentuated in partially nonaqueous solvents (15). Other important applications of the bases or their salts include their use upporting electrolytes in the polarographic determination of the alkali metals (la),and their use in ion exchange separations involving the alkali metals (4,1 4 ) . I n all of these applications it is highly deqirable or even imperative that the alkali metal content of the quaternary ammonium base be low. I n potentiometric titrations in aqueous solution with the glass electrode, the “alkali error” a t high p H is well known (1). I n nonaqueous solutions, the deleterious and unusual effects of sodium and potassium on the response of the glass electrode is especially serious (8). I n polarographic analysis for the alkali metals, and in ion exchange separations of trace amounts of alkali metals, the presence of alkali metal impurities in the quaternary ammonium bases is intolerable. Unfortunately, reagmt or analyzed grade quaternary ammonium bases are not commercially available a t present. Harlo\T summarizes three widely used methods for the preparation of quaternary ammonium baces, and points out that all three are subject to alkali ion contamination (8). I n the classical preparation of quaternary ammonium bases a solution of a quaternary ammonium halide is reacted with a suspension of silver oxide (19). This procedure is sometimes used commercially ( 7 ) , and involves treating silver nitrate with sodium hydroxide to form the silver oxide precipitate. The precipitated silver hydroxide is thoroughly washed with distilled water before reaction with the quaternary
ammonium halide, but sodium hydroxide tends to adhere to the silver hydroxide, and sodium ion contamination of the final base is difficult to avoid. Peracchio and Meloche found it necessary to wash the precipitated silver oxide 12 to 15 times in preparing tetramethylanimonium hydroxide for polarographic studies of the alkali metals (16). Cundiff and Narkunas recently reported a procedure involving washing silver oxide with boiling water and methanol that resulted in sodiumfree tetrabutylammonium hydroxide (TBAH) ( 5 ) . Purified silver oxide is commercially available and can be used to prepare alkali-free base ( 1 4 ) , but even with this shortcut the silver oxide method is laborious and time-consuming. The other two widely used methods of preparing quaternary ammonium bases are likewise subject to alkali metal contamination. The potassium hydroxide method of preparing nonaqueous quaternary ammonium titrants yields bases containing 100 to 200 p.p.m. potassium ion (10), and the anion exchange resin preparation of Harlow, Xoble, and Wyld may also result in potassium ion contamination (9). An electrolysis method of preparing pure T M X H for polarographic studies has been reported by Supin (18), but has not been tried for other quaternary ammonium bases. Because the thermal instability of the free bases precludes purification by ordinary distillation methods (12 ) , another means of removing alkali metals is needed. I n an earlier article it was shown to be feasible to selectively retain alkali metals on Dowex 50 W-X8 in the tetramethylammonium ion form, even when the solution contained a fairly high concentration of tetramethylammonium ion (14). A deVOL. 37, NO. l l , OCTOBER 1965
* 1375
pendence on the degree of resin crosslinkage was also indicated. I n this paper a procedure is presented for the rapid removal of alkali metals from TALIH, tetraethylammonium hydroxide (TEAH), tetrapropylammonium hydroxide (TPAH), and TBAH on Dowex 50 K-Xl6. Conditions necessary for optimum efficiency and yields were studied in detail. EXPERIMENTAL
Apparatus and Reagents. T h e ion exchange resin used in most of this 1 1ork was Bio-Rad Laboratories Dowex 50 W - X l 6 , 20- t o SO-mesh, having a total exchange capacity of 2.3 meq./ ml. in the water-swollen hydrogen ion form. T h e breakthrough capacity of the resin (the number of milliequivalents of alkali metal ions t h a t can be taken u p quantitatively during column operation) varies with experimental conditions, and is expressed in this paper as t h e percentage of total exchange capacity. Resins used in preliminary studies included Baker Analyzed Dowex 50 R-X8, 100- to 200-mesh; Doirex 50 W-Xl2, 50- to 100-mesh; arid Xmberlite IR-124, 12% divinglbenzene (D.V.B.) , 20- to 50-mesh. The resins were cleaned in large lots as described previously ( 3 ) . Glass columns used for T M X H and T E A H were 21 em. long by 0.97-cm. i d . , closed a t the lower end by a coarse sintered-glass disk upon which the resin bed rested, and fitted a t the top with a b o d 5 em. in diameter to hold influents. For T P X H and TBXH, a Kimax 10-ml. buret 42 cm. long by 0.63-em. i.d. was used, and a glass wool plug supported the resin bed. Removable porous glass disks, furnished with some commercial columns, introduced sodium ion into the bases and could not be used. Fractions of column effluents were collected in borosilicate glass test tubes calibrated to indicate 2 ml. Brooks Instrument Co. flow meters (size R-2-15XA) were used to monitor all column flow rates. Over a n interval of one year, four different lots of T M A H , four different lots of TEAH, three different lots of TPXH, and one lot of TBAH, all as highest purity solutions (the first three as 10% aqueous solutions, with the fourth being 25% in methanol) were obtained from Eastman Organic Chemicals Co. I n addition, one lot each of T M A H and TEAH, as highest (feasible) purity, 25% aqueous solutions, were obtained from Matheson Coleman & Bell. All lots as received gave significant qualitative flame tests for sodium ion with a platinum wire, and flame photometric analysis indicated that the sodium ion content was over 0.05W in all five lots of TMAH, with the mole yo sodium ranging from 6 to 8% of total cation content. The sodium content of the five T E A H lots was much more variable, ranging from about 5 X 10-4M to 0.02X sodium ion (two lots had sodium ion contents of about 5 X lO-*M, and three lots were around 0.02M), with the mole % 1376
e
ANALYTICAL CHEMISTRY
sodium ranging from 0.1 to 3.5%. The sodium ion contents of the three T P A H lots ranged from about 5 x 10-4.11 to 5 X 10-3V, with the mole yo sodium ranging from 0.1 to 1%. The single TBAH lot had a sodium ion content of about 10-3JfJ or about 0.1 mole % sodium. The halide content of all bases was negligible, as determined by spot testing with XgNOa Flame after acidification with "0,. photometric analyses for potassium ion were negative in all cases except in one lot of T1LIH in which about l o - 3 x J or 0.1 mole % potassium was found. Highest purity tetramethylammonium chloride (T1IACl) (Eastman Organic Chemical Co.) had negligible alkali metal contamination. The nitrate salts of sodium and potassium, used to prepare flame photometric standards, mere reagent grade. Procedure for Removal of Alkali Metals from TMAH. T e n milliliters of Dotvex 50 TV-XI6 in the hydrogen ion form were partially converted (about SOY0) to the tetramethylammonium ion form by passing 25 ml. of 2.5.11 T l I A C l through the column a t a flow rate of about 4-5 ml. per minute. Then 5y0T M A H was passed through the column a t a flow rate of 3.0 ml. per minute and rejected until the effluent was basic to litmus. Thereafter 2.0-ml. fractions of effluent were collected. Procedure for Removal of Alkali Metals from TEAH, TPAH, and TBAH. T o purify T E A H , 6 ml. of Dowex 50 W - X l 6 in the hydrogen ion form irere subjected to 10% TE.IH a t a flow rate of 2.5 ml. per minute. To purify T P h H , 2 ml. of Dowex 50 W-Xl6 in the hydrogen ion form were subjected to 10% TPAIH at a flow rate of 2.3 ml. per minute. T o purify TBAH, 2 ml. of Donex 50 WX16 in the hydrogen ion form were washed with 10 ml. of reagent grade methanol and then subjected to 10% TBXH (in methanol) a t a flow rate of 2.3 ml. per minute. I n all cases the effluent was rejected until it was basic to litmus, after which 2-ml. fractions were collected. Analysis. With a platinum wire, qualitative flame tests for sodium ion were conducted on the effluent as it emerged from t h e column. I n addition, t h e effluent fractions were analyzed with a Beckman Model D U flame spectrophotometer equipped with a photomultiplier detector. An oxygen-acetylene flame was used. ilt high concentrations, dilutions were performed t o minimize self-absorption. The sodium ion detection limit by the qualitative flame test was about 3 p.p.m. sodium, whereas the flame photometer was operated a t a sensitivity sufficient to accurately detect 0.1 p.p.m. sodium. Procedure for Regeneration of Resin. After breakthrough of sodium ion occurred, the resins could be regenerated by washing with three 10-ml. portions of water (or, in t h e case of T B A H in methanol, with three 10-ml. portions of methanol), and then passing 5-41 HCl through until t h e effluent was free of sodium ion
as indicated by a flame test. Removal of sodium ion was slow, and took 200 to 300 ml. of 5 M HCl a t a flow rate of about 10 ml. per minute to completely remove all the sodium ion from 10 ml. of resin. RESULTS AND DISCUSSION
Table I gives the yields and sodium ion breakthrough capacity of the various quaternary ammonium bases by the above procedure. In all cases in this study, in order for the effluent bases to be considered free of sodium, the sodium ion content had to be less than 1 p.p.m. Thus, the breakthrough volume was taken as the volume of effluent in which the sodium ion content increased to 1 p.p.m. or above, and the sodium ion breakthrough capacity is thus a measure of the efficiency with which sodium ion is retained on the resin in the presence of the competing quaternary ammonium ions. The breakthrough capacity percentage is useful for estimating the amount of resin that should be used to purify a given volume of quaternary ammonium base of known sodium ion content. In testing the purification procedure 011 T P A H and TBAH, the sodium ion tontent of the influent was increased to 250 p.p.m. to decrease the time and volume to breakthrough. For example, under the conditions specified for TPAH, 114 ml. of the 10% T P A H were passed before sodium ion broke through. If the sodium ion content has been left a t 40 p.p.m., calculations indicate that about 700 nil. would have been required to reach breakthrough. For the same reason, the volume of resin used was decreased as higher molecular weight bases were purified. Because the percentage yield and the percentage of the total capacity at which sodium ion broke through was relatively independent of the amount of resin used, it would have been necessary to collect and analyze large volumes of solution for the higher molecular weight bases if 10 ml. of resin had been used for all of the bases. The yield, or percentage of the total base that was recovered free of sodium ion was less than 100% by the amount of base used to convert the resin from the original hydrogen ion form to the respective quaternary ammonium ion form. TBAH gives a lower yield and sodium ion breakthrough capacity than the trend with the other bases would predict, but this is probably because of lower exchange efficiency or slower rates of exchange in the methanolic solution (11). Other conditions affecting yields and breakthrough capacity are discussed in the next section. Conditions Affecting Yields and Sodium Ion Breakthrough Capacity. CONCENTRATION OF BASES. Table I1 summarizes the effect of t h e con-
centration of T M A H a n d T B A H on t h e yield and sodium ion breakthrough capacity t h a t can be expected. I n general as the concentration of t h e base is decreased, t h e yields and sodium ion breakthrough capacities increase, indicating more efficient exchange as t h e solution is diluted. Thus, in choosing t h e optimum concentration conditions for t h e purification procedure it is necessary t o compromise between high yield and high concentration. Because it is troublesome to reconcentrate a diluted base, unnecessary dilution was to be avoided. And because with TXAH twofold dilution of the 10% solution served to increase the yield from 50 to 83%, while another fivefold dilution increased the yield only moderately, a concentration of 5% T X i H was selected as the optimum concentration for purification. With the other three quaternary ammonium bases, good yields were obtained with 10% solutions. RESIN CROSS-LIIVKAGE. It could be predicted that the selective retention of sodium ion in the presence of bulkier quaternary ammonium ions would be improved the higher the degree of resin cross-linkage. Some preliminary investigations were made with 8 and 12% cross-linkage resins. The selective retention of sodium ion was improved as the degree of cross-linkage increased. For example, resins with 8% D.V.B. were unsuitable for the purification of T L A H even when TAILAHwas diluted to 1%; were poor for T E A H unless the TEhH was diluted to 1% (and even then the effluent contained up to 4 p.p.m. sodium) ; but worked reasonably ne11 for T P A H if the flow rate was kept to less than 3 ml. per minute. T B h H was not tried. Resins with l2Tc D.V.B. n ere unsuitable for the purification of 10% TXAH, but could purify 5% TXAH down to 9 p.p.m. sodium, and 1% TJLAH. seemed to work w ~ l with l T E A H , 1070, could also be purified, but TPhH and T13dH were not tried. I t was necessary to use resins with 16% D.V.B. to purify T M A H in concentrations of 5y0or greater. FLOWRATEANI) LENGTHOF RESIN BED, TXAH, \vhich proved to be the most difficult of the quaternary ammonium bases to purify, was chosen to test the dependence of the yield and sodium ion break1 hrough capacity on flow rate. Whereas the purification of 5y0TXAH at 3 ml. per minute gives the sodium ion breakthrough capacity and yields indicated in Table I, increasing the flow rate to 6 ml. per minute drops the sodium ion breakthrough capacity to 9.0% and the yield of TRIAH to 69%. At even faster flow rates the efficiency and yields fall off sharply. If the flow rate is decreased below 3 ml. per minute, little improvement in sodium ion breakthrough capacity and
Table 1.
Yields and Sodium Ion Breakthrough Capacity of Quaternary Ammonium Bases by Purification Procedure
Base ThIAH TEAH TPAH TBAH In methanol. Table II.
Influent Base concn. sodium ion ( % by wt./vol.) concn. (p.p.m.) 5.0 10 10 1o a
9*50 560 250 250
Yield,
70
Sodium ion breakthrough capacity ( % of total capacity) 20 22 27 19
83 84 92 85
Effect of Concentration of Quaternary Ammonium Bases on Yield and Sodium Ion Breakthrough Capacity
Base
ThIAH ThIAH TAIAH TBAH TBAH I n methanol.
Base concn. ( % by wt./vol.)
Influent sodium ion concn. (p.p.m.)
10 5.0 1.0 100 5.05
1,900 950 190 250 125
yield is realized, indicating 3 ml. per minute is close to exchange equilibrium. As the length of the resin bed was decreased, it was necessary to decrease the flow rate roughly proportionately to keep the yield and sodium ion breakthrough capacity relatively constant. Conversely, lengthening the resin bed permitted proportionately faster flow rates to be used with no significant change in the sodium ion breakthrough capacity or yield. USE OF TXAC1 IN RESIN CoxVERSION. I n preliminary studies with 10% TXAH, before use of TXACI was adopted, yields greater than 40% could not be obtained, even with long columns and low flow rates, because of the large amount of base used in the initial conversion of the column from the hydrogen ion to the tetramethylammonium ion form. For example, using 10 nil. of Dowex 50 W-X16 in the hydrogen ion form and a flow rate of about 2.0 ml. per minute, direct addition of 10% T M A H resulted in only about 28% yield, with a 6% sodium ion breakthrough capacity. If instead the resin were first treated with 25 ml. of 2 . 5 X TKACl, the 50% yield cited in Table I1 mas obtained. Twenty-five milliliters of the 2 . 5 X ThIACl was chosen as the resin pretreatment based on preliminary experiments in which 2.551 TAIhCl was passed through 10 ml. of resin a t flow rates of 4 to 5 ml. per minute and 2-ml. effluent fractions were collected and titrated with standard 0.1-11 KaOH. These studies indicated that the efficiency of displacement of hydrogen ion by tetramethylammonium ion is high
Yield,
70
Sodium ion breakt hSough capacity (5of total capacity)
50 83
85 85 93
7 3 20 22
19 41
a t first, but decreases almost exponentially, reaching 507, conversion of exchange sites a t about 20 ml. T l I A C l passed. I t was impractical to pass more than 25 ml. of the T1\I.ACl. The inefficiency of conversion with nonbasic quaternary ammonium salts is in agreement with the findings of Hale, Packham, and Pepper (6). I n the purification of the other quaternary ammonium bases it was not necessary to use the salts of the bases in the resin conversion because better than 80% yields could be obtained without the added step. Furthermore, it would be expected that the salts of higher molecular weight quaternary ammonium bases would not be as effective as was T l I A C l in displacing hydrogen ion from the resin (6). Reproducibility and Stability of Resin. Hale, Packham, and Pepper noted changes in certain properties of their l5Y0 D.V.B. resin after complete conversion t o t h e tetraethylammonium ion form, and presented data indicating a decrease in degree of cross-linkage (6). Therefore, it was of considerable interest to determine if the 16% D.V.B. resins used in this study could be regenerated and used over again without deleterious effects. Repeat runs were made with each of the bases after regeneration of the resin n i t h 5J1 HCI, and in all cases the breakthrough capacities and yields remained the same within the accuracy of nieasuring breakthrough volumes (about + 3 ml.). Likewise, several repeat runs with new portions of resin gave reproducible results. This may mean VOL. 37, NO. 1 1 , OCTOBER 1965
1377
contained as much as 3 to 4% carbonate as determined by potentiometric pH titration. Where applications necessitated the removal of carbonate, the anion exchange procedure was satisfactory.
that the commercial resins used in this study were more stable then the resins prepared by Hale, Packham, and Pepper, or possibly that any alteration of the resin which does occur does so on first contact with the base, with no further changes occurring with successive treatments. This possibility was not investigated. Removal of Other Alkali Metals. I n t h e one base solution t h a t contained potassium ion in addition to sodium ion, the purification procedure completely removed t h e potassium as well as the sodium ions. S o other attempts were made t o test t h e procedure for the removal of other alkali metals, b u t earlier studies with lithium, sodium, and potassium, plus some studies including rubidium and cesium, indicated sodium is consistently the first of the alkali metals to breakthrough on tetramethylammonium ion forms of Dowex 50 W-X8 and 12 (14). Thus, it can be predicted that alkali metals other than sodium should be removed with an efficiency comparable to or even greater than the efficiency of removing sodium ion. Removal of Carbonate Ion. T h e absorption of C 0 2 from t h e atmosphere is a well known problem with strong bases, b u t a n anion exchange resin procedure can be used effectively to remove this impurity ( I S ) . Commercial bases supplied in plastic bottles
Preliminary Polarographic Studies.
Preliminary attempts were made t o use the above-purified TLIAH, TEAH, and T P X H solutions as supporting electrolytes for polarographic analysis. T E A H seemed to work well, in agreement with the findings of Zlotowski and Kolthoff (20), permitting about 1 p.p.m. sodium ion to be detected. T P A H allowed about 5 p.p.m. sodium ion to be detected. T M A H gave undesirably high background currents, but no further polarographic investigations were made. ACKNOWLEDGMENT
The help of Charles E. Forbes with some of the preliminary experiments is acknowledged. LITERATURE CITED
(1) BgFes, R. G., “Determination
of
pH, Wiley, New York, 1964. (2) Bjerrum, J., Schwarzenbach, G., Sillen, L. G., “Stability Constants, Part I, Organic Ligands,” The Chemical Society, London, 1957. (3) Blaedel, W. J., Olsen, E. D., BLIchanan, R. F., ANAL. CHEM.32, 1866 (1960). (4) Buser, W., Helv. Chim. Acta 34, 1635 (1951).
(5) Cundiff, R. H., AIarkunas, P. C., ANAL.CHEM.34, 584 (1962). (6) Hale, D. K., Packham, D. I., Pepper, K. W., J . Chem. SOC.1953, p. 844. (7) Hapeman, R. C., Distillation Products Industries, Eastman Organic Chemicals Department, Rochester 3, N. Y., private communication, 1964. (8) Harlow, G. A., i l N A L . CHEY. 34, 148 (1962). (9) Harlow, G. A., Noble, C. XI., Wyld, G. E. A., Ibid., 28, 787 (1956). (10) Harlow, G. A., Wyld, G. E. A., Ibid., 34, 172 (1962). (11) Helfferich, F., “Ion Exchange,” McGraw-Hill, Yew York, 1962. (12) Kolthoff, I. RI., Lingane, J. J., “Polarography,” 2nd ed., 1-01, 11, p. 423, Interscience, Sew York, 1952 (13) Marple, L. W., Fritz, J. S.,. ~ N A L . CHEM.34, 796 (1962). (14) Olsen, E. D., Sobel, H. R., Talanta 12, 81 (1965). (15) Olsen, E. D., Sobel, H. R., Franklin and Marshall College, Lancaster, Pa., unpublished data, 1962. (16) Peracchio, E. S., Neloche, V. W., J . Am. Chem. SOC.60, 1770 (1938). (17) Rossotti, F. J . C., Rossotti, H., (‘The Determination of Stability Constants,” p. 61, llcGraw-Hillj New York, 1961. (18) Supin, G. S.,Zh. Fiz. Khim. 34, 924 (1960); C.A. 57, 44669 (1952). (19) Walker. J., Johnson, J., J . Chem. SOC.87, 957 (1905). (20) Zlotowski, I., Kolthoff, I. M.,IYD. ENG.CHEM.,ANAL.ED. 14, 473 (1942). RECEIVEDfor review May 14, 1965. Accepted July 15, 1965. Work supported in part by the National Science Foundation (NSF-GP-3482). Division of Analytical Chemistry, 150th Meeting, ACS, Atlantic City, N. J., September 1965.
Preparation of Immobilized Cholinesterase for Use in Analytical Chemistry E. K. BAUMAN AND L. H. GOODSON Midwest Research Institute, Kansas City, Mo.
GEORGE G. GUILBAULT AND D. N. KRAMER Defensive Research Division, Edgewood Arsenal, Md.
b This report describes the preparation of an immobilized (insolubilized) cholinesterase. The enzyme, immobilized by the use of a starch matrix and placed on a urethane foam pad, is stable and active for 12 hours. The substrate solution used, butyrylthiocholine iodide, is stable a t room temperature for two weeks. The activity of the enzyme is monitored electrochemically, using two platinum electrodes and an applied current of 2 pa. As long as the enzyme is active, the electrochemical system will indicate a low potential, ca. 150 mv., because of oxidation of the thiocholine (produced by enzymatic action) to the disulfide at the platinum anode. 1378
ANALYTICAL CHEMISTRY
As the activity of cholinesterase is decreased, the potential will rise to a higher value, 350 to 400 mv. (the potential of oxidation of iodide in the original substrate to iodine).
0
disadvantages of (objections to) the use of enzymes in analysis is the high cost of materials. A continuous or semicontinuous routine analysis using enzymes would require large amounts of these materials, quantities considerably greater than can be reasonably supplied, and quantities that would represent a prohibitive expenditure in many cases. If, however, the enzyme could NE OF THE PRIMARY
be prepared in a n insolubilized (immobilized) form without loss of activity so that one sample could be used continuously for many hours a considerable advantage would be realized. A search of the literature revealed that two primary techniques could be used to insolubilize an enzyme, such as horse serum cholinesterase: N i t z (8) and Bar-Eli and Katchalski (I) have chemically modified the enzymes chymotrypsin, trypsin, and urease by the introduction of insolubilizing groups. McLaren ( 7 ) , Xikolaev (9) and Barnett ( 2 ) , among others, have attempted physical entrapment of the enzymes asparaginase, ribonuclease, and chymotrypsin by adsorption, absorption, or
