Potentiometric Method for Karl Fiseher Titrations F. L. J.
LAMOEN and
VAN
Vezelinrtitvt
H. BORSTEN
T.N.O., Delft, The Netherlands
Two p l a t i n u m electrodes, one b r i g h t andone platinized, permit p o t e n t i o m e t r i c titrations using the Karl Fischer-Johansson procedure with an aoeuraey equal t o the dead-stop technique.
T .
0 MINIMIZE the effects of the hygroscopicity of the Karl Fischer reagent ($), Johansson (6) suggested the use of two solutions essentially forming the reagent in situ (8). The sulfur dioxidepyridine-methanal component serve8 as sample solvent and a relatively stable, nouhygroscopic iodine solution as titrant. The methods that have been used for end-point detection in this titration have been described by Mitchell and Smith (7). For colored solutions visual indication is usually impractical and electrical methods are usually employed. The most commou of these include the dead-stop technique with polarized platinum electrodes a8 described by Faulk and Bawden ( 8 )or utilizing selfpolarizing electrode systems 8nch tbs that of platinum-tungsten as discussed by Almy, Griffin, and Wilcox ( I ) and Willard and Fenwick (9). Heinemsns (6) has used n platinum-glass electrode system for both direct and indirect titrations.
: L* , , , , 0
1
2
I
5
-added
6 7 m i d iodini
8
1
D
11
In the authors' lshoratory i t has been found convenient t o use two platinum electrodes, with a laboratvry pH meter, for titrations involving the Johitnsson technique. The bright platinum electrode to serve as reference is placed in a tube containing the sulfur dioxide solution. Electrical contact with the solution to be titrated is made through a thin asbestos fiber sealed through the tube wall. The platinized platinum indicator electrode is placed in a second glass tube, perforated a t the bottom, to minimize stirring effects. At the equivalent point an e.m.f. change of about 150 mv. occurs. This is compamble to the e.m.f. change occurring in titrating sodium bisulfite with O.IN iodine in water. Other reference electrode solutions may be used. Figure 1 illustrates titrations with ( a ) iodine-methanol; ( b ) sulfur dioxide-pyridine-methanol; ( e ) solution ( b ) just titrated to an end point with iodine; and ( d ) solution ( b ) with a large excess of iodine. Solution (a) was found most convenient t o use, as it is one of the components of the Johansson technique. Initial voltages with this reference solution are relatively constant. EXPERIMENTAL
Final design of the titration cell and electrodes used is shomm in Figure 2 and the completely assembled apparatus is pictured in Figure 3. The direct titration technique was used (no back titration). The following reagents were used. Solution A: dissolve 100 grams of dry sulfur dioxide in a mixture of 500 ml.
12
F i g u r e 1. E.m.f. change d u r i n g titration u s i n g different solutions in referenoe electrode
F i g u r e 2.
Design of t i t r a t i o n cell a n d electrodes used
,
1638
F i g u r e 3.
Completely assembled a p p a r a t u s
V O L U M E 27, N O . 10, O C T O B E R 1 9 5 5 Table I. HIO in Sample Taken, Mg.
29.8 32.2 33.1 38.6 39.7 40.1 41.0 42.6 43.5 4F.4 50.2 51.5 98.G
1639 Solution B is best standardized by titrating a known weight of a stable hydrate (4)and should he checked daily.
Titration of Water
Water Found, hlg.
Difference
29.7 32.4 32.9
3 8 , .5
39.8 40.0 41.2 42 5
L70
-0.1 f0.2 -0.2 -0.1 f0.1
-0.34 +O.G2 -0.60 -0.Zi f0.25 -0.25 fO.49 -0.24 -0.23 -0.43 0.00 +0.78 -0.51
-0.1 f0.2 -0.1 -0.1 -0.2 0.0 f0.4 -0 5
43.4 46.2 50.2 51.9 98 1
RESULTS
~-
Mg.
of anhydrous methanol and 500 ml. of anhydrous pvridine. Solution B: dissolve 50 grams of resublimed iodine in 1 liter of anhydrous methanol.
Procedure. Place 30 to 50 ml. of Solution A in the titration vessel, start the stirrer, and rapidly titrate with Solution B until a sharp inflection is noted on the vacuum tube voltmeter. Continue the titration dropwise until the increased voltage remains constant for a t least 1 minute. Add the sample to be tested and repeat the titration with Solution B, recording the volume used. This is equivalent to the moisture in the sample taken.
The precision attainable is evident from Table I. ACKNOWLEDGMENT
The authors wish to thank J. R. H. van Nouhuys, director of the Fibre Research Institute, T.N.O., for permission to publish the results of this investigation, and are indebted to Nicolaas de Hart of this institute for his assistance with the analytical work. LITERATURE CITED
(1) Almy, E. G., Griffin, W.C., and Wileox, C. 9 . . 1x1.ENG.CEmLf,, ANAL. ED., 12, 392 (1940). (2) Faulk, C. W.t and Rawden, d. T.. J . .4m. C'heru. ,Cot.. 48, 2045 (1926). (3) Fischer, K., Angew. Chem., 48, 394 (1935). (4) Frediani, H. A , , et al., ANAL. CHEM.,23, 1332 (1961). (5) Heinemans, B. J., J . Dairy Sei., 28, 845 (1945). (6) Johansson, A, Saensk Papperstidn., 50, 11B, 124 ( 1 9 4 i ) . (7) hIitehell, J., Jr., and Smith, D. &I.,"Aquametry, .4pplication of the Karl Fischer Reagent to Quantitative Analyses Involving Water." Interscience, London-New York, 1948. (8) Seaman, W. hI., McComas, W. H., and Allen, G . Ai.,.4iv.a. CHEM.,21, 510 (1949). (9) Willard, H. H., and Fenwick, F., J . Am. Chem. Sock, 44, 2504, 2516 (1922).
RECEIYIDD for review March 15, 1954. Accepted M a y 23, 1955,
Turbidimetric Method for the Determination of Yeast Mannan and Glycogen J. A. ClFONELLl
and
F.
SMITH
Department o f Agricultural Biochemistry, University o f Minnesota, St. Paul, M i n n .
The interaction of concanavalin-.&a globulin extracted from jack bean meal, with glycogen and with yeast mannan has been utilized for the determination of these two polysaccharides. It is suggested that the reaction between concanavalin-.4 and glycogen could be used for the determination of alpha-amylase activity.
D
URING a study of the purification of yeast invertase it n as desirable to have a method which would permit the estimation of small concentrations of yeast mannar (yeast gum). Although yeast mannan was generally detected by means of its precipitation with alkaline Fehling solution ( 7 ) , this method lacked the sensitivity necessary for the determination of w a l l amounts. Sumner and O'Kane (9) had reported that a jack bean globulin, concanavalin-A, was effective in precipitating not only invertase but also yeast mannan. This observation was utilized t o develop a simple method for the determination of yeast gum. The method was also found to he suitable for the determination of glycogen. METHODS
Preparation of Concanavalin-A Solution. A partially purified concanavalin-A solution suitable for determining yeast mannan and glycogen was prepared as follows (6, 9). Twenty grams of jack bean meal (Arlington Chemical Co., Yonkers, N. Y.) was stirred n;ith dilute sodium chloride (2%, 200 ml.) for about 10 minutes and then centrifuged for 10 minutes at 2000 r.p.m. The turbid supernatant solution was treated with 2 ml. of 2M acetate, p H 4.2, and after standing for an hour was centrifuged. The solution was warmed to 50" to 55" C. with
continual stirring, and after standing at room temperature for
0.5 hour was centrifuged. Generally, the solution was clear at this point, but occasionally it was slightly turbid, in which case 2 or 3 ml. of 0.1% glycogen in water was added, and after standing in the refrigerator overnight the solution was filtered or centrifuged. The clear solution was treated with 15 ml. of a 7% aqueous solution (15 ml.) of poly(viny1 alcohol) (Elvanol, Grade 71-24, Du Pont Co.) and the reagent kept in the refrigerator until ready for use. The solution (pH 6.0) is stable for several months when refrigerated. It is possible to re-use the concanavalin-h reagent several times ; it should be clarified before being re-used. With some solutions little change in absorbance was noted when they were treated with a standard glycogen solution even after the reagent had been used several times. Generally the reagent develops a slight sediment on standing longer than several days, hut this is much less in solutions prepared by acetate and heat treatment than in those prepared under neutral conditions. Lyophilization of the acetate- and heat-treated preparations gives products which are stable at room temperature and which dissolve completely in 1% saline to yield solutions which are stable v hen refrigerated. Preparation of Yeast Mannan and Standard Liver Glycogens. The yeast polysaccharide was obtained from baker's yeast autolyzate (4)by precipitation with alkaline Benedict solution and subsequent purification according to the method dexribed bv Haworth, Hirst, and Isherwood ( 5 ) . The product was dried in vacuo at 56" C. and n-as slightly green. The color, presumabljdue to bound copper, could not be removed by dialysis against distilled water or against 0.0LV acetic acid, but was removed by passage over a cation exchange resin (Amberlite IR-120, Rohm & Haas Co.). The addition of concanavalin-A reagent to a standard weight of the yeast mannan before and after passage through Bmberlite IR-120 produced the same absorbance. Hence traces of copper do not influence the analytical results. The specific rotation of the yeast mannan was +88' in water ( r , 1.0). in agreement with the value reported previously ( 5 ) .
