A COhIPARISON O F COLORIMETRIC AND ELECTROMETRIC METHODS I N T H E DETERMINATION OF p H VALUES O F VARIOUS GELATIN SOLUTIONS* BY R. J . HARTMAS' A X D I. F. FLEISCHER
Introduction The colorimetric methods have been resorted to almost entirely by investigators and by manufacturers in determining t,he p H value of various gelatin solutions. Walter S. Hughes,' however, measures the potential of a 1% gelatin solution by means of a glass electrode and a hydrogen electrode. The potential of this solut'ion is changed by the addition of sodium hydroxide and hydrochloric acid solutions. A curve is then obtained by plotting the potential measured by the hydrogen electrode against that of the glass electrode. A similar curve is constructed from measurements of a solution where no gelatin is present. The former curve assumes the general shape of the latter but the potential measured by the glass electrode gives values lower than in the case where gelatin was not present. This phenomenon is explained by stating that the presence of gelatin produces substances other than hydrogen-ions which affect the glass surface potential. I t is further asserted that before any acid or alkali was added to the gelatin solution the glass surface potential was 0.531 and the hydrogen potent,ial 0.560. This difference is accounted for by saying that the presence of electrolytes has some influence on the adsorption of gelatin on the glass surface. It was further found that the potential measured by the glass surface electrode depends upon both the nature of the solution and the time of contact. Allen E. Stearn2 has studied the nature of isolectric gelatin in solution. I t was found that when a solution of a certain pH value was mixed wit'h a basic dye of the same p H value, there was a very significant decrease in pH. The opposite was noted with acid dyes. I n explanation two apparent mechanisms are presented. DC1+ HGe = DGe HC1 or better
D+
+ HGe =
+ DGE + H+
where Dfis the dye cation and Ge the gelatin radical. An adsorption mechanism depending on the equilibrium is the other explanation offered. DCl
+ HOH = DOH + HCl
It is definitely shown by Stearn that, when an acid dye reacts with a gelatin solution, the hydrogen-ions from the protein molecule are used up or, in the * From the Laboratory of Colloid Chemistry, Indiana University. Hughes: J. Am. Chem. SOC., 44,2860 (1922). Chem., 34,973 (1930).
* Stearn: J. Phys.
COLORIYETR[C AND ELECTROYETRIC METHODS FOR pH
I I 3 i
case of a basic dye, hydroxyl-ions are used up. The disappearance of ions from a gelatin solution upon the addition of crystal violet (basic dye) is shown by a decrease in the conductivity of such s ~ l u t i o n .I~t would have been indeed interesting had the two investigators above mentioned compared the pH values they obtained by potentiometric methods with values obt,ained from colorimetric indicators. Stearn has conclusively shown that a gelatin molecule in solution combines chemically with either an acid or basic dye. From this it is reasonable to assume that in the colorimetric determination of the pH value of a gelatin solution the indicator chemically unites with t,he gelatin molecule arrest,ing any free acid or basic ions due to the hydrolysis of a gelatin in solution. The work of Stearn indicates the existence of an ampholyte ion in gelatin solutions. This ion would, accordingly, prevent equilibrium in potentiometric determinations since it would not be combined chemically and would be free to react with other ions in solution. This investigation was undertaken to compare the pH values obtained from two colorimetric met hods with those obtained from three potentiometric methods and to study the variation of values yielded from the potentiometric methods with time, or, the time necessary for equilibrium.
Experimental Material used. The hide glue used in this investigation was furnished through the courtesy of Conrad-Kammerer Glue Company, New Albany, Indiana, the bone glue through the courtesy of Armour Glue Company, Chicago, Illinois. The casein glue was prepared in this laboratory by the grain-curd process. The vegetable glue was prepared in this laboratory by digesting on a boiling water bath 50 grams of tapioca flour with 150 cubic centimeters of water containing 5.6 grams of caustic soda and then adding j-roo cc. portions of water. This material was dried in an oven between 40-50' C . over night. The dry product was granulated for use. The solutions to be tested were prepared in the following manner: Two grams of the gelatin was allowed to soak in water over night and then heated to 62%. a t which temperature the solution became homogeneous after a 15 minute period. The solution was then permitted to come to room temperature. Ten cubic centimeter portions were used in all colorimetric determinations while fifty cubic centimeter portions were used in all potentiometric determinations with readings at definite t,ime intervals. Redistilled wat,er was used throughout the entire investigation. Equipment. The p H of each gelat'in solution was first determined using Clark and Lubs standard colorimetric indicators. The indicators were obtained from Coleman and Bell Company, Norwood, Ohio. The Hellige Colorimetric Comparator was also used in determining the p H value. This comparator and the standard indicator solutions were obtained from t,he Hellige :I
Reference
2, p.
981.
T 138
R. J. HARTMAN AND I. F. FLEISCHER
Klett Company, Inc., New York, New York. In each instance the two colorimetric methods gave the same pH value. The three electrodes used in the potentiometric methods were the hydrogen, quinhydrone and glass, each in combination with a saturated calomelhalf cell. With the hydrogen and glass electrodes Leeds and Northrup hydrogen-ion potentiometer (no. 7665) was used. Leeds and Northrup quinhydrone p H indicator (no. 7654) was used in making determinations with the quinhydrone electrode. The quinhydrone and hydrogen electrodes were obtained from the same company. The glass electrode was prepared by welding a very thin sheet of glass to a piece of glass tubing about I O centimeters long and 1.5 centimeterszin diameter as shown in Fig. I . The thin sheet of glass was prepared by blowing a large thin-walled bulb from a piece of soda glass tubing. The lower end of the glass was heated to dull red heat when it was pressed against the thin sheet of glass, which was placed on a piece of clean asbestos slate. Tests were made for leakage by filling the tube with saturated potassium chloride solution. The hydrogen used with the hydrogen electrode was electrolytic hydrogen purified by passing through a train consisting of hot copper, concentrated sulFIG.I phuric acid, glass wool and caustic soda sticks. A Copper wire. A convenient and inexpensive saturated calomelB Glass tube. C Cork. half cell was devised by using a small clean wide D Groove in cork C to prevent pressure in- mouth bottle arranged as shown in Fig. 2. side electrode. Data. Table I shows the values of E.M.F. and E Mercury. pH obtained by the various methods with the F Platinum wire. G Saturated KCI so- different gelatins. In carrying out the potentiometric lution. H Glass tube I O cm. long determinations, readings were taken at various time and about 1.5 cm. in intervals in an attempt to determine the length of diameter. time for equilibrium to take place in the solution. I Thin sheet of glass. Horiaontallv is listed the various gelatins studied while vertically the E.M.F. and p H values are given with the time of reading. All of the readings were taken over a temperature range of 24 to 30°C. Correction for same was made in the calculation of each pH value. From the following table it is noted that the two colorimetric methods, Clark and Lubs, and Hellige, gave identical pH values. The results obtained from the hydrogen electrode of each gelatin can in no way be compared with the value obtained from the colorimetric methods. At the expiration of 60 minutes in the potentiometric determination of the hide gelatin, a value
11\39
FIG 7 A Copper wire. B Cork. C Groove in cork B to prevent pressure inside cell. D I O O cc. \vide mouth bottle. E Glass tube. F Solution saturated with respect to KC1 and calomel. G Mercury. H Platinum wire. I Agar gel or crushed filter paper. J Calomel paste K Mercury. L Saturated KC1 solution. M IOO cc. beaker. N Saturated KCI bridge. 0 Solutiori to be tested. P Opposite electrode.
similar to that received with the colorimetric indicators is obtained. Since, however, the values received prior to the expiration of 60 minutes were much less than 6.6, and, since those values received after expiration of 60 minutes were greater than 6.6, this fact is coincidental. In general, the pH values obtained with the quinhydrone electrode did not vary with time to the same extent as the values obtained from the hydrogen electrode. Apparent equilibrium was reached in the quinhydrone determinations of the hide and casein gelatins after about 2 5 minutes. The values obtained at this point, however, are quite different from those obtained by the colorimetric indicators. The pH vslue obtained from the quinhydrone electrode with bone gelatin at the expiration of 40 minutes compared with that obtained with the two colorimetric methods. Since, however, the pH values continued to vary before and after the expiration of 40 minutes, this again may be considered coincidental. The pH values obtained from the glass electrode did not vary with time to the same extent as the Talues obtained from the hydrogen electrode and in each case the pH values obtained from this method were very much lower than those obtained by the colorimetric methods.
1140
R . J . HARTMAN A N D I. F. FLEISCHER
TABLE I Comparison of E M F and p H Values of Different Gelatin Solutions as measured by Various Methods Gelatin Colorimetric pH values by Clark and Lubs, and Helliee Comparator .'
Bone
6.6
5.6
Time in Min-
Hydrogen Electrode
Hide
Utes
EMF Volts
PH Calculated
15
,0707
-
I9
.oz 24
_.
28
,2360
37
.4477 ,5356 ,5410 ,5476 '5439 5449 ,4144
53
65 83 103 123 280 Quinhydrone Electrode
- ,126 - ,123 - ,118 - .I20 - .I22
Time in Minutes
EMF Volts
CEU-
5
,2513
0.
IO
'5427
5.007
-
15
3.55
30
'5980 ,6120 ,6180 ,6220 ,6418
5 930 6 160 6.260 6.580 6.920
,6473
7.020
4.89
45
4.98
60
5.28
130 23 5
5.01
5
I4 26
,3299 . 3 141 .3268
5.35
5
5.45
12
5.60
23
5
50
44
5.55
85
'
-
'
,1210
-.123j -.1235 -,122 - ,1210 - ,1210
>
,1150
20
,1263
40
,1336 ,1350 ,1336 , I324 ' 1297 ,1289
55
Electrode Fractured
'43
5.04 2.86
I20
Glass Electrode
lated
75 IO0 I1j I20
~
j.60 5.65 5,6j 5.60 5.60 j .60 (2)
COLORIMETRIC A X D ELECTROMETRIC METHODS FOR pH
1141
TABLE I (Continued) Comparison of EMF and pH Values of Different Gelatin Solutions as measured by Various Methods Gelatin Colorimetric pH values by Clark and Lubs, and Hellige Comparator
-
40
Time in Miriutes
Hydrogen Electrode
IO 20
25
35 50 60
75 90
105 I20
180 Quinhydrone Electrode
5 IO 25
46
62 108 128
Glass Electrode
Casein
pH
EMF Volts
Calculated
,4101 2 . 78 ,4173 2.91 ,420' 2.95 ,4242 3 . 0 1 ,4244 3.02 , 4 2 5 0 3.03 ,4253 3.04 ,4256 3.05 ,4258 3.0; , 4 2 7 0 3.07 , 4 2 7 4 3.19 -.3240 -.3290 -.3320 - ,3330 - ,3330 -.333s - ,3335
Vegetahle 9.0 (Phenol phthalein) (Hellige comparator not used) Time pH in Min- EMF Calcuutes Volts lated 1 , s
1000
5 .o
48.0 80 o
4243 6843 8087 8185 818; 8170
100.0
8167
120.0
8162
10.0
15 .o 30.0
--
2.05
9s
9 63 9 60 9 58 9 jj
2.02
I20
13
2.02 2.02 2.02
7
5
IO
IO
'7
I5 30 Electrode Fractured
9 62
28
2.07
2.15
3 I9 7 38 9 46
,0867 . I320 '1419
(2)
.I221 I301 139'
45 60 80
,1574
105
,1382
120
,1426
,
( I ) I t is known that the quinhydrone electrode will not give correct values for pH when the alkalinity is pH = 9 or greater. (2) The pH values were not calculated in this case due to the fact that sufficient information is not available.
1142
R. J. HARTMAN AXD I. F. FLEISCHER
The accuracy of the pH determinations does not seem to vary with gelatins made from different materials. I n view of the fact that the readings obtained potentiometrically in no case reached a constant value, it is apparent that equilibrium in solution is not obtained. This quite likely could be due to the existence of an ampholyte ion in solution. This ampholyte ion may form inner salts or react with other ions which are no doubt in solution as impurities. I n the colorimetric determination this ampholytic ion is probably united chemically with the organic indicator, thus yielding pH values more correct and constant. If the variations of the pH values with time is due to an ampholytic ion, which is probably the case, equilibrium would not be expected in unbuffered solutions such as those tested. Values of pH constant with time clso agreeing with the colorimetric values could be probably obtained if the solutions could be buffered. Further work along this line conducted by the authors will appear in a later issue of t,his journal.
Conclusion The pH values obtained from the glass, quinhydrone and hydrogen electrodes in the potentiometric determination of the various gelatin solutions do not agree with the pH values obtained colorimetrically. 2. Of the methods employed in this investigation, only the colorimetric methods can be relied upon. 3 . There was no equilibrium reached in the potentiometric determination of the gelatin solution over the period of time studied. 4. The data indicates the existence of ampholyte ions in each of the gelatin solutions studied as first pointed out by Stearn. 5 . Of the three electrodes studied, the pH values obtained with the quinhydrone electrode seemed to give readings most constant with time, but these values did not agree with the colorimetric determinations. I.
