by hydrogen-ion concentration^^ 2 - American Chemical Society

calculated from the streaming potential data by the formula for diaphragms developed by Briggs (2) in which 17 is the viscosity of the liquid, K~ the ...
1 downloads 3 Views 960KB Size
ELECTROKINETIC PROPERTIES OF PROTEINS. I1 ADSORPTION OF GLIADINAT GLASS-LIQUID INTERFACE AS

INFLUENCED BY HYDROGEN-ION CONCENTRATION^^ 2 WILLIAM McK. MARTIN

Agricultural Experiment Station, M o n t a n a State College, Bozeman, Montana Received February 21, 1933

In the streaming potential studies reported in an earlier paper (ll),the isoelectric points of some of the wheat proteins were interpolated from the values for the r-potential of the protein-liquid interface. The latter were calculated from the streaming potential data by the formula for diaphragms developed by Briggs (2)

in which 17 is the viscosity of the liquid, K~ the electrical conductance across the diaphragm, H the streaming potential, P the hydrostatic pressure, and e the dielectric constant of the liquid. At the isoelectric point the value for is zero, and since the properties of the liquid represented by 7,K,, and e, and the hydrostatic pressure P have definite values, the streaming potential H is also equal to zero. The isoelectric point is thus the hydrogen-ion concentration a t which no electromotive force is produced by streaming the solution through a diaphragm on which the protein is adsorbed. Inasmuch as it was desired to use the isoelectric point as a differentiating property in the study of proteins, an attempt was made in this investigation to devise a convenient and rapid method for determining the electroneutral point of a protein by adsorbing it on a fritted glass diaphragm and then varying the hydrogen-ion concentration of the solution until its streaming potential fell to aero. Preliminary experiments showed, however, that the hydrogen-ion ccncentration of the solution could not be experimentally adjusted to maintain the streaming potential at zero, but that a positive potential was gradfially

r

Presented before the Division of Physical and Inorganic Chemistry a t t h e Eighty-fourth Meeting of the American Chemical Society a t Denver, Colorado, August 24, 1932. Paper No. 29, Journal Series, from Montana State College, Agricultural Experiment Station, Bozeman, Montana. 213

214

WILLIAM MCK. MARTIN

developed. It was evident from these experiments that either the adsorption equilibrium, or the chemical equilibrium, of the proteins was disturbed by changes in the hydrogen-ion concentration of the streaming solutions. The adsorption equilibrium would, obviously, be influenced by the magnitude and sign of the charge of the protein micelles in relation to that of the adsorbent (glass particles in diaphragm), and in consequence this phase of the problem has been studied in the present work. EXPERIMENTAL

Preparation of gliadin Two kilograms of patent flour, from which the fatty materials had been removed by extraction with anhydrous diethyl ether, was made into a pliable dough and allowed to stand under water for 1 hour. The starch was then washed from the gluten by kneading in a stream of tap water. It was then finely divided and added to sufficient pure ethyl alcohol to form a solution containing 60 per cent of alcohol and 40 per cent of water by volume. The mixture was then placed in a mechanical shaker for 10 hours, after which it was centrifuged, filtered, and the filtrate reduced to a sirupy consistency by evaporating under reduced pressure. The gliadin was precipitated by pouring the cooled sol into a 1 per cent sodium chloride solution a t OOC., filtered, and thoroughly washed with distilled water. The precipitate was redissolved in a 60 per cent alcohol solution, its volume reduced by evaporating under reduced pressure, and again precipitated by pouring into cold 1per cent sodium chloride solution. The re-solution and reprecipitation was repeated four times, the final precipitation being accomplished by pouring the concentrated sol into a mixture of 1 part of absolute alcohol and 1 part of anhydrous diethyl ether, after which the precipitate was washed with anhydrous ether and dried in a vacuum oven a t 80°C.

Streaming potential measurements The apparatus shown diagrammatically in figures 1 and 2 was designed to measure, by the streaming potential method, the progressive change in the potential across a solid-liquid interface when proteins, acids, alkalis, or other reagents were added to the liquid phase. Inasmuch as slight traces of electrolytes, or surface-active substances dissolved in the liquid or adsorbed a t the solid-liquid interface, were readily detected by a marked change in the streaming potential, it was necessary to eliminate every possible source of contamination. Atmospheric gases such as carbon dioxide, ammonia and acid fumes were excluded from the apparatus by a counter-current of air which had been purified by bubbling successively through sulfuric acid, sodium hydroxide, and distilled water. All parts of

ELECTROKINETIC PROPERTIES OF PROTEINS. I1

215

the apparatus coming in contact with the liquid were cleaned with hot chromic acid and rinsed with conductivity water. The streaming cell 2 shown in figures 1 and 2 consists of a fritted glass filter crucible (Jena Glass Works No. lbG4) with sheet platinum electrodes 1 cm. square on each side of the diaphragm. The crucible is attached a t the top to an outlet receptacle bearing the upper electrode and closed 'at the bottom with a two-hole rubber stopper carrying the inlet tube and lower electrode. The liquid in reservoir 1, the hydrogen-ion concentration of which is adjusted experimentally by adding acid or alkali from burettes 11 and 12,

FIG. 1. STREAMINQ POTENTIAL APPARATUS

is streamed through the diaphragm of cell 2 into flask 3 by suction. The air in flask 3 is exhausted through needle valve 6 into tank 5 which is evacuated by a water pump. The hydrostatic pressure under which the liquid streams through the diaphragm may be measured by either the mercury manometer 7 or the water manometer 8, or both, by adjusting the three-way stopcock 9, the pressure being maintained constant by the automatic regulator 10. Stopcock 4 is closed when it is desired to stop the streaming liquid without readjusting the hydrostatic pressure. The electromotive force produced by streaming the liquid through the fritted glass diaphragm in cell 2 is measured by connecting the electrodes in cir-

216

WILLIAM MCK. MARTIN

cuit with the potentiometer (28) and Compton electrometer (29). Switches 40 and 42 are set in contact with 36 and 39, respectively, and the reversing switch 41 is set so that the positive electrode of the streaming cell is connected to the positive binding post of the potentiometer. The electromotive force supplied to the potentiometer by the 2-volt lead storage cell (31) is balanced against that of the standard cadmium cell (32), thenull-point being indicated by the galvanometer (30). The electrometer, which is used to indicate the null-point in balancing the electromotive force from the potentiometer against the streaming potential, is operated on a charge of 90 volts by connecting two radio "B" batteries (33) in series between the needle and one pair of quadrants. After adjusting the electrometer to its zero-point, switch 43 connecting the two pairs of quadrants is opened and the liquid caused to stream

1 FIG.2. STREAMING-CELL AND HYDROGES ELECTRODE APPARATUS

through the diaphragm by opening needle valve 6. The hydrostatic pressure gradually increases to that automatically maintained by regulator 10, while at the same time the increasing potential across the diaphragm is balanced by adjusting the potentiometer experimentally. The applied hydrostatic pressure may be increased or decreased by changing the tension of the helical spring of the pressure regulator, after which it remains constant during the progress of the experiment. The effective hydrostatic pressure decreases slightly, however, owing to the lowering of the liquid in reservoir 1. This difference in level between the liquid in 1 and the end of the discharge tube in 3 is read directly from scale 13 in millimeters, divided by 13.6, and added to the pressure indicated by the manometers. If the diaphragm or the streaming liquid have not been contaminated in any way, the system will come to equilibrium in one or two minutes, and

ELECTROKINETIC PROPERTIES OF PROTEISS. I1

217

the streaming potential will remain constant except for a slight gradual decrease due to the lowering of the level of the liquid in the reservoir. The system being in equilibrium and the streaming potential constant, except for the slight change explained above, a measured volume of standard gliadin solution containing 0.002 g. of gliadin for each liter of streaming liquid is added and the solution quickly homogenized by the mechanical stirrer 14, the speed of which is regulated by rheostat 15. The time of adding the gliadin is recorded and the change in streaming potential measured at 2-minute intervals for 10 minutes, and at 5-minute intervals thereafter.

Electrical resistance across diaphragm With the liquid still flowing, switch 43 is cloedd and the streaming cell thrown in circuit with the conductivity apparatus by setting switches 40 and 42 in contact with 36 and 38, respectively. The resistance across the diaphragm is then balanced against the adjustable resistances 35 by means of the Kohlrausch slide wire 34, a pair of ear-phones being used to detect the null-point. With solutions of high resistance in the streaming cell, the slide wire could not be accurately balanced by the telephone detector, and in consequence three or more determinations were made and the average of these used in the calculations. Bull and Gortner (6) overcame this difficulty by using an alternating current galvanometer instead of the telephone detector. After completing the streaming potential, hydrogen-ion concentration, and electrical resistance measurements, the cell constant was determined by replacing the liquid in the cell with a standard solution (10) composed of 0.7476 g. of potassium chloride and 1000 g. of redistilled water. The resistance of the cell was measured with the standard solution streaming through the diaphragm under the same hydrostatic pressure used in making streaming potential measurements.

Hydrogen-ion concentration of streaming liquid Although the glass electrode might have been used advantageously in determining the hydrogen-ion concentration of the extremely dilute unbuffered solutions, it has not, perhaps, been investigated sufficiently to justify its use as a routine method. The better known hydrogen electrode was therefore used in the apparatus shown in figures 1 and 2. The hydrogen electrode vessel (18) is filled by closing stopcock 23, opening 24 and 27 and tilting it so that it i s filled with the liquid, thereby displacing the air upward and out through stopcock 27. Stopcock 24 is then closed, 23 opened and the vessel tilted down, after which 27 is turned to permit hydrogen gas to displace the liquid from the vessel into drain 19. These operations are repeated several times to free the system from oxygen.

218

WILLIAM MCK. MARTIN

All stopcocks are finally closed with the vessel about one-quarter filled with liquid. Stopcock 27 is then turned so that the hydrogen pressure from reservoir 21, which is equal to about 15 cm. of water above that of the atmosphere, is maintained within the hydrogen electrode vessel. This not only saturates the platinum electrodes more quickly, but prevents the atmospheric gases from diffusing into the system. It also causes the films of liquid in the ground glass joints of stopcocks 23 and 24 to move outward instead of inward, thus reducing error from this source. The vessel is then gently shaken by the geared mechanism 17 by connecting shaft 16 to the motor 14 with a short piece of rubber tubing, the speed of the motor being regulated by the rheostat 15. The system is saturated with hydrogen within four minutes, after which the motor is stopped with the liquid in the electrode end of the vessel. With the system still under pressure from the hydrogen reservoir, stopcock 23 is turned to discharge from the bore of the stopcock plug, and from the tube between the stopcock and the vessel, the solution which has not been saturated with hydrogen. The pressure within the vessel is then released through 27, after which 23 is carefully turned to connect the vessel with the calomel electrode 20. Stopcock 25 is then carefully opened and saturated potassium chloride solution flows from reservoir 22 and rises slowly in the tube above the stopcock, forming a well defined and easily reproducible liquid junction. When the liquid junction has risen to a mark on the tube midway between stopcock 23 and the vessel, 27 is closed to prevent the potassium chloride from rising too high and mixing with the liquid. The plug in 26 is greased only a t the ends t o facilitate electrical conductance when the stopcock is closed. The difference in potential between the hydrogen and the calomel electrodes is measured by throwing switches 40 and 42 in contact with 37 and 39, respectively. The electromotive force is then balanced with the potentiometer, using the quadrant electrometer as a nullpoint instrument, The potential of each of the twin hydrogen electrodes is measured and two complete determinations made on each solution.

Calculation of the electric moment of the double layer From the streaming potential data may be calculated either the {potential or the electric moment of the double layer. For the reasons set forth by Bull and Gortner (7) the electric moment is preferable, and accordingly the data obtained in the present work have been computed t o this basis. The formula for the electric moment given by Bull and Gortner is

where q is the charge per unit area of the double layer in electrostatic units, d the thickness of the double layer in centimeters, q the viscosity of the



ELECTROKINETIC PROPERTIES OF PROTEINS. I1

219

streaming liquid in poises, K* the electrical conductance across the diaphragm in reciprocal ohms, H the streaming potential in volts, and P the hydrostatic pressure in centimeters of mercury. The product qd (charge X distance) is the electric moment per square centimeter of the double layer and is represented by p . Remembering, however, that one C.G.S. electrostatic unit of potential is equal to 299.86 absolute volts, and that the C.G.S.electrostatic unit of conductance is equal to 1.112151 X ohm-’ cm.-‘ (9); and that the density of mercury is 13.6, and the force of gravity 981 dynes, the different quantities in the above equation are converted to C.G.S. units by evaluating the constants to give the simplified expression : p

K~H

= 224755.22 X 7 X -

P

(3)

For convenience in comparing the data presented in this paper with the more familiar {-potential in the literature, the relation of the two may be expressed by substituting the {-potential formula developed by Briggs (2). I

q

= 847,649,450 X - X

e

xsH P

(4)

in equation 3, which gives I=----3771.43 p €

(5)

or = 2.65

x

10-4 x

(6)

where E is the value for the dielectric constant used in the original calculations. Although the value for E generally used in calculating the {potential from streaming potential data is probably incorrect, as explained by Bull and Gortner (7), expressions 5 and 6 may be used to convert the data of this and other papers to a comparable basis. DISCUSSION

Fritted glass discs on filter crucibles are satisfactory diaphragms for streaming potential measurements, because they are composed of glass particles of uniform size and are of sufficient porosity to produce results not in error due to “back-pressure” effects. The importance of uniformity of size of particles in streaming potential diaphragms has been emphasized by Bull and Gortner (6), whose experiments showed that the streaming potential was a linear function of hydrostatic pressure only when the particles in the diaphragms were homogeneous in size. The effect of size of pores on the streaming potential has also been investigated by Bull (4),

220

WILLIAM M CK. MARTIN

whose experimental data showed that an appreciable “back-pressure” was developed by electroosmotic flow if the radius of the capillaries was 1 I* or smaller. The data in table 1 and the graphs in figures 3 and 4 show that the glass diaphragms were negatively charged in both acidic and basic solutions. The magnitude of the charge, however, was a function of the total ionic concentration rather than of the concentration of hydrogen ions. This is shown by the relation of the values for the electric moment to those for the specific electrical conductivity of the solution, a relationship similar to that observed by Bull and Gortner (5) for cellulose in aqueous solutions of inorganic salts. TABLE 1 Electric moment of the double layer at the solid-liquid interface of a fritted glass diaphragm as inJEuenced b y hydrogen-ion concentration STREAMING LIQUID

PH

Ys

x

-ohms-’

HC1 HC1 HC1 HC1 HC1 HC1 NaOH

NaOH NaOH NaOH NaOH NaOH NaOH

5.69 5.22 4.82 4.43 4.25 3.20 7.76 8.16 9.06 9.44 9.65 9.74 9.94



10-0

1.76 3.02 5.96 12.62 17.48 36.43 2.42 3.82 10.22 17.68 23.71 43.81 48.19

1

TEMPERATURE

HYDROSTATIC PRESSURE

BTREAMING POTENTIAL

ELECTRIC MOMENT

3F D O U B L E LAYER

~

degrees C

posses

0 0 0 0 0 0 0 0 0

0089 0084 0085 0087 0087 0093 0088 0086 0084

I

25 27 27 26 26 23 25 26 27

2 9 4 2 1 5 0 9 7

mm.

Tflg

124 124 125 124 125 125 125 125 126 124 123 124 125

millzrolts

- 1230

-874 -643 -386 -320 -257 - 1315 -1134 -724 - 524 -434 -237 -244

.s.u. x 10-4

-3.49 -4.02 -5.86 -7.69 -8.75 -15.67 -5.04 -6.70 -11.10 -14.12 -16.37 -16.76 -17.99

The results of the adsorption studies given in table 2 and presented

’ graphically in figures 5 and 6 show that the electrostatic charge of the

double layer at the glass-liquid interface is reversed upon adding protein to the streaming liquid if its hydrogen-ion concentration is on the acid side of the isoelectric point of the protein. The negatively charged glass surface apparently adsorbs a layer of positively charged protein and assumes its electrokinetic properties,. The behavior of the glass is thus similar to that of quartz particles used by Briggs (3), Freundlich and Abramson (8), and by Svedberg and Tiselius (12). Recently Abramson (1)has shown that the isoelectric point, the cataphoretic mobility, and the dissociation constant of serum albumin and egg albumin are very nearly the same when adsorbed on quartz as when dispersed in solution. The

ELECTROKINETIC PROPERTIES OF PROTEINS. I1

221

FIG. 3. ELECTRIC MOMEXTOF DOUBLE LAYERAT GLASS-LIQUID INTERFACE AS INFLUENCED BY HYDROGEN-ION CONCENTRATION

Electrical

Conductance in Ohm&

(Ksx

FIG. 4. ELECTRIC MOMENTOF DOUBLE LAYERAT GLASS-LIQCID INTERFACE .a I S F L U E N C E D BY TOTAL IOSICCOKCENTRATIOX (ELECTROLYTIC CONDUCTIVITY OF SOLUTION)

222

WILLIAM MCK. MARTIN

TABLE 2 Influence of hydrogen-ion concentration o n the rate of adsorption of gliadin at a glass-liquid interface a s indicated b y change in the electric moment of the double layer TIME

HYDROSTATIC PRESSURE

I

X

q =

ELECTRIC MOMENT OF D O U B L E LAYER

STREAMING POTENTIAL

,

.

mm. Hg

mv.

0 5 10 15 20

127 126 125 124 124

-1316 - 1304 - 1297 - 1290 - 1285

-3.33 -3.34 -3.35 -3.34

-920 -638 -390 - 195 -38

-2.41 -1.67 -1.03 -0.51 -0.10 $0. 87 $1.50 f2.00 $2.32 $2.61 $2.77 $2.92 +3.03 $3.08

$325 $558 $738 $862 $956 $1007 $1053 $1084 $1102

HC1 solution, pH = 5.69; xs = 1.76 X 10-e; q = 0.0089; T = 25.2%. 0 5 10 15 20

126 125 124 124 123

-1246 -1237 -1231 -1222 -1215

-3.49 -3.49 -3.50 -3.48 -3.49

0.002 g. gliadin per liter added 22 24 26 28 30 35 40 45

123 122 122 122 121 121 120 119

-809

-402

-51 $299 +538 $911 $1134 $1269

HYDROSTATIC PRESSURE

STREAMING POTENTIAL

ELECTRIC MOXENT OF D O U B L E LAYER

-

0.002 g. gliadin per liter added 123 123 122 122 121 120 120 119 118 118 117 116 115 115

TIME

0,0090; T = 24.9%.

minutes

22 24 26 28 30 35 40 45 50 55 60 65 70 75

1:

~

-2.32 -1,16 $0.86 $1.57 $2.66 $3.33 $3.76

I1

x

mm. Hg

mu.

60 65 70 75 80 85

118 117 117 116 115 114 113 112

$1355 +1410 $1453 $1494 $1526 $1528 4-1525

$4.05 $4.25 $4.38 $4.54 $4.65 $4.72 $4.77 +4.80

0 5 10 15 20

126 125 124 123 123

-884 -879 -874 -869 -863

-4.01 -4.02 -4.03 -4.04 -4.01

+1517

!.S.U.

10-4

0.002 g. gliadin per liter added

22 24 26 28 30 35 40 45 5o 55 6o 65 70 75 80

122 122 121 121 121 120 119 118 118 117 116 115 115 113 111

-208

-0.97

+154 $321 $401 $459 $530 $563 f593 $616 $631 $644 $657 $663 $669 $666

$0. 72

$1.52 $1.89 $2.17 +2.52 $2.70 $2.87 $2.98 $3.08 $3.17 $3.26 $3.29 +3.38 $3.43

HC1 solution; pH = 4.82; K~ = 5.96 x 10-6; = 0.0085; T = 2 7 . 4 ~ . 0 127 -651 -5.84 5 126 -648 -5.86 -5.86 10 125 -643 -5.87 15 124 -639 -635 -5.84 20 124

223

ELECTROKINETIC PROPERTIES O F PROTEINS. I1

TABLE 2-Continued HYDROBTATIC PRESSURE

TIME

BTREAMINQ POTENTIAL

,

I

ELECTRIC MOMENT OF D O U B L E LAYER

0.00: g. gliadin Der liter added minutes

mm. H g

mu.

22 24 26 28 30 35 40 45 50 55 60 65 70 75 80

123 123 122 122 121 121 120 119 118 117 117 116 115 114 113

-241 $63 $204 $264 $291 $322 $342 4-350 $356 $360 $364 +362 f367 $366 $363

-2.23 $0.58 $1.90 $2.47 $2.74 $3.03 $3.25 $3.35 $3.44 $3.51 $3.55 $3.56 +3.64 $3.66 $3.66

0 5 10 15 20

126 125 124 124 123

-392 -389 -386 -383 -382

-7.68 -7.68 -7.69 -7.63 -7.67

e.8.u.

X 10-

0.00 g. gliadin Der liter added

22 24 26 28 30 35

- 14 $97 $128 $169 $195 $204 $207 $210 $211 $215 +218 $217 $222 +225

123 123 122 122 122 121 121 120 119 119 118 117 117 116

-0.28 $1.95 $2.59 $3.42 $3.95 $4.16 40 $4.22 45 $4.32 50 $4.38 55 $4.46 60 $4.56 65 $4.58 70 $4.69 75 $4.79 HC1 solution; pH = 4.25; K # = 17.48 X 10-6; q = 0.0087; T = 26.1"C. 0 I 127 I -325 1 -8.76 5 126 -322 -8.74 10 125 -319 -8.73

1

1

1

1 I I

1

PRESSURE

1

i

HY;t$-

TIME

STREAMINO POTENTIAL

ELECTRIC MOMENT OF DOUBLE LAYER

HC1 solution; pH = 4.25; K~ = 17.48 X lo-*; q = 0.0087; T = 26.l0C.--Cont'd minutes

20 l5

mm. Hg

ii:

1

mu.

1

5.S.U~.

x

10-4

-8.75 -8.76

0.002 g. gliadin per liter added 22 123 -3 -0.08 122 24 $139 $3.90 122 26 $175 $4.91 122 28 $ 182 $5.10 121 30 $200 f5.66 121 35 $209 $5.91 $206 120 40 $5.87 45 $205 120 $5.85 50 $201 119 +5.78 55 118 $5.71 $197 60 $193 118 $5.60 65 117 $ 189 $5.53 70 116 $ 186 $5.49 75 115 $ 186 $5.53 HC1 solution; pH = 3.20; K~ = 36.43 X IO-*; q = 0.0093; T = 23.5"C. 0 I 127 I -260 I -15.61 5 126 -259 -15.67 125 -256 10 -15.61 15 125 -255 -15.55 124 20 -253 -15.55 0.00 g. gliadin Der liter 124 - 100

22 24 26 28 30 35 40 45 50 55 60 65 70 75 80 85

123 123 122 122 121 120 120 119 118 118 117

117 116 115 114

$46 $95 $106 $112 $113 $115 $115 $114 $117 f113 $115 $116 $115 $117 $115

Sded -6.15 $2.85 $5.89 $6.62 $7.00 +7.12 +7.30 $7.30 +7.30 $7.56 $7.30 $7.49 $7.56 $7.56 $7.75 $7.69

224

WILLIAM MCK. MARTIN

HYDROSTATIC PRESSURE

TIME

STREAMING POTENTIAL

I

I

TABLE 2-Continued

1 /I

ELECTRIC MOMENT OF DOUBLE LAYER

NaOH solution; pH = 7.53; K~ = 2.84 7 = 0.0089; T = 25.2"C. X minutes

HYDROSTATIC

ELECTRIC MOXENT OB DOUBLE LAYER

STREAMINO POTENTIAL

KaOH solution; pH = 7.76; K~ = 2.42 7 = 0.0088; T = 25.0"C. X

I

-1167 -5.27 125 0 -1158 -5.27 125 5 -1150 -5.27 124 10 -1144 -5.29 123 18 -1137 123 20 0.002 -5 89 122 I -1264 22 -6 41 - 1375 24 122 - 1451 -6 76 122 26 -7 14 - 1519 121 28 -7 28 - 1560 121 30 -1579 -7 48 120 35 -7 61 - 1593 119 40 -1596 -7 69 118 45 - 1590 -7 66 118 50 - 1581 -7 68 117 55 -7 65 -1561 116 60 -7 66 - 1549 115 65 -1538 -7 67 114 70 - 1531 -7 64 114 75 NaOH solution; pH = 7.14; K~ = 2.04 q = 0.0089; T = 25.4"C. X 0 1 124 I -1215 1 -4 00 123 -1207 -4.01 5 123 -1198 10 15 20 0.002 g. gliadin per liter lded 121 -1239 22 -4 29 121 - 1272 24 - 1285 -4.37 26 120 -4.41 - 1296 120 28 - 1302 -4.43 120 30 -4.47 119 - 1304 35 -4 48 - 1295 118 40 -4.50 - 1288 117 45 -4 47 - 1281 117 50 -4 50 116 - 1277 55 -4.52 115 - 1272 60 -4.54 114 - 1267 65 -4.54 - 1255 113 70 -4.53 - 1242 112 75

I

TIME

'

minutes

0 6

10 15

m m Hg

ma.

e S . U . X 10-4

122 126 125 124

- 1330 - 1323 - 1316 - 1307 - 1299

-5.02 -5,04 -5.05 -5.06 -5.03

1 I

0 002 g. gliadin per liter added 22 24

123 122

-1597 1 -6 23 (exceeded range of potentiometer)

NaOH solution; pH = 8 16; K~ = 3.82 q = 0 0086; T = 26 9°C. X 0 5 lo 15 20

126 125 125 124 124

-1138 -1135 -1135 -1132 -1130

-6 -6 -6 -6 -6

67 70 70 74 73

0.002 g. gliadin per liter added

1 1

22

:t

_.

28 30

45

55 60 65 70 75

-7.70 -8.54 -9.12 -9.33 -9.52 -9.76 -9.87 -9.91 -9.86 -9.75 -9.62 -9.53 -9.54 -9.51

- 1294 - 1423 - 1520 - 1555 -1573 - 1599 - 1604 - 1598 - 1589 - 1559 - 1525 -1511 - 1499 - 1468

124 123 123 123 122 121 120 119 119 118 117 117 116 114

NaOH solution; pH = 9.06; K~ = 10.22 7 = 0.0084; T = 27.7"C. X 0 5 10 15 20

I

127 127 126 126 125

I

-726 -725 -724 -724 -722

1

-11.04 -11.02 -11.10 -11.10 -11.15

225

ELECTROI