The absorption of water vapor by proteins

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THE ABSORPTION OF WATER VAPOR BY PROTEINS

A Thesis Presented to the Faculty of the Department of Chemistry University of Southern California

In Partial Fulfillment of the Requirements for the Degree Master of Science

by Robert Walter Zwanzig May .1950

UMI Number: EP41594

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c, 'tr/ z 97 T h is thesis, w ritte n by

HO BERT W. ZWANZIG under the guidance o f tik§.~. F a c u lty C o m m ittee, and ap p ro ved by a ll its members, has been presented to and accepted by the C o u n c il on G ra d u ate S tu d y and Research in p a r tia l f u lf i ll ­ m ent o f the requirem ents f o r the degree o f

MASTER OF SCIENCE

Date.

May 31. 1950

Faculty Committee

Chairman & L . LAS,

ACKNOWLEDGMENT This work was carried out with the aid of a grant from the Office of Naval Research, under contract N6-onr-67900, I wish to express my extreme indebtedness to Professor Sidney W. Benson for his advice and encouragement.

?ifithout

his help, these experiments could not have been completed.

TABLE OF CONTENTS CHAPTER

PAGE

I.

STATEBJEENT OF THE P R O B L E M .....................

1

II.

SURVEY OF THE L I T E R A T U R E .....................

2

Sources of Data

..........

Theories of absorption

2

..........

Absorption and other properties

...........

Rate of a b s o r p t i o n ......................... III.

IV.

V.

4 11 18

EXPERIMENTAL P R O C E D U R E .......................

21

Introduction ................................

21

Preparation of the S a m p l e s .................

22

Description of the a p p a r a t u s ...............

24

Preparation and conduct ofa r u n ............

32

The temperature e f f e c t .....................

34

D I S C U S S I O N ....................................

37

Isotherms....................................

37

Rate measurements............................

48

CONCLUSIONS.

. .............. .. ..............

BIBLIOGRAPHY .............

. . . . . . . .

A P P E N D I X ...............................

60 6l 64

LIST OF TABLES TABLE I* II. III. IV. V. VI. VII.

PAGE Rate of Absorption on Egg A l b u m e n ............65 Rate of Absorption on Bovine PlasmaAlbumen V

. 66

Temperature Variation During Absorption . . . .

67

Effect of Aluminum C e l l .................... .

68

Absorption Isotherm A .

........................ 69

Desorption Isotherm A .................... .. Absorption and Desorption

Isotherm B

. . . . .

70 71

LIST OF FIGURES FIGURE

PAGE

1. Rate of Absorption on Egg Albumen:Bull . . . .

19

2.

Sample C e l l ....................................

25

3.

High Vacuum

L i n e ...........................

29

4.

Water Vapor

Supply

I

....................

30

5.

Water Vapor

Supply

I I ....................

31

.....................

41

6.

Isotherm:

Bov 5 cr •

7.

Isotherm:

Bov 5 lyo

8.

Isotherm:

Bov 5 s f ............................

43

9.

Comparison of

I s o t h e r m s .......................

44

....................

42

10.

BET Plots of Isotherms

......................

45

11.

Rate of Absorption: Egg Albumen C r u d e .........

49

12.

Rate

of Absorption: Egg Albumen Spray Frozen.

13.

Rate

of Absorption: First Order Plot

14.

Rate

of Absorption: Second Order Plot I . . .

.

53

15*

Rate

of Absorption: Second Order Plot II. . .

.

54

16,

Rate of Absorption: log(W0-W) — ^ t ...........

55

17*

Temperature Effect

............................

57

18.

Effect-of

Aluminum C e l l .......................

59

19.

BET

Plot: Nitrogen on Bov 5 sf D E ..............

72

20.

BET

Plot: Nitrogen on Bov 5 sfRZ . . . . . .

.

73

21.

BET

Plot: Nitrogen on Bov 5 l y o ...............

74

.

. . . . .

50 51

1

CHAPTER I STATEMENT OF THE PROBLEM Experimental studies of the interaction between gases and solid proteins have shown that some correlation exists between the measured absorption isotherms and the chemical structure of the proteins, and that the absorption is not always a surface effect,

In all published work in this field,

the tacit assumption is made that the absorption of gases like water vapor is independent of the state of subdivision of the protein particles.

This study was undertaken to pro­

vide accurate experimental data on this point.

The data

has shown that the assumption is justified. It has been known for some time that water vapor is absorbed on proteins far more slowly than seems reasonable. Data obtained during this investigation has shown that with proper experimental technique, it is possible to increase absorption rates by a factor of about one hundred. important both experimentally and theoretically.

This is

2 CHAPTER TWO SURVEY OF THE LITERATURE I

SOURCES OF DATA

The literature on the absorption of gases on proteins is concerned mostly with the absorption of water vapor. However, some investigations have been on the absorption of ammonia and hydrogen chloride, and of the relatively inert gases like argon, nitrogen, oxygen, and some hydro­ carbons.

Shaw (1) measured the nitrogen absorption isotherm

of egg albumin at -183° C.

Using the Brunauer, Emmet, and

Teller theory (hereafter abbreviated BET), which will be discussed in a later section, he showed that the apparent surface area was 2.4 square meters per gram.

Rowen and

Blaine (2) have carried out the same measurements on a series of textile fibers, including wool and nylon, both polypeptides. Benson and Ellis (3) have obtained isotherms for several proteins at different temperatures with nitrogen, argon, oxygen, methane, butane, and neopentane.

Their results indicate

(1) Shaw, J. Chem. Phys. 12, 391

(1944)

(2) Rowen and Blaine, Ind. Eng. Chem. (3) Benson and Ellis, J. Am. Chem. Soc. , ibid. 72, 2095 (1950)

1&59

(194-7)

3563 (1948)

3

that chemically inert gases behave in much the same way in absorption on proteins.

In particular, the surface areas

calculated with the BET theory all agree fairly well. Bancroft and Barnett (4), Belden (5), Parks and Melaven (6)

and Seehof (7) have studied the absorption of ammonia

and hydrogen chloride.

They found that these isotherms are

discontinuous, and cannot be explained by the BET theory or any other existing absorption theory.

Seehof has estimated,

using the Langmuir theory of absorption on the curved parts of the isotherm, that the '*surface area" could be of the order of 1000 square meters per gram- a physically meaning­ less result, if interpreted as a physical area. In spite of the extensive literature on the theory of absorption of water by proteins, the accurate experimental data is scarce.

Ellis (8) has an extensive bibliography of

these references in his Doctoral dissertation.

We will refer

only to those which have any bearing on this research.

(4) Bancroft and Barnett, Proc. Nat. Acad. Sci. 16, 118, 135 (1930) (5) Belden, J. Phys. Chem. ^5, 2164 (193D (6) Parks and Melaven, J. Phys. Chem. 41, 1101 (1937) (7) Seehof, M. S. thesis, Univ. of Southern Cal. 1950 (8) Ellis, Ph.D. dissertation, Univ. of Southern Cal. 1949

4

II THEORIES OF ABSORPTION The theory of gas absorption in general has received a considerable amount of attention, and its applications to proteins have constituted a major test of the theory.

The

problem has been attacked from a kinetic, a statistical, and a thermodynamic point of view. The kinetic theory is based on an extension of the well known Langmuir treatment of physical adsorption, which was made by Brunauer, Emmet, and Teller (9).

Their kinetic model

is open to criticism, and since their final equations can be derived using a clearer model with statistical thermodynamics, their derivation will not be discussed.

The equation they

developed for the absorption isotherm is

where v is the volume of gas absorbed, vD is the volume of gas that completely covers the surface with a monolayer, c is a function of the heat of absorption E^_ - E l and of tempera­ ture, p is the pressure of the gas, and pD is the vapor pressure of the gas at the same temperature.

This equation

is usually used in the form

p 'V'Oo-p)

/ ■ -

-



Vo C

±

po

(9) Brunauer, Emmet, and Teller, Physical Adsorption, Princeton University press, 1943

5

Therefore, a plot of the expression on the left against p/pQ should give a straight line.

Usually, the fit is good from

p/pQ = .05 to .35 with marked deviations outside of this inter­ val.

Several attampts have been made to improve the fit by

making various modifications in the original model used by Brunauer.-

The successful ones (10) extend the upper limit of

validity to about p/pQ = .7 > but the corrections have lost all theoretical significance, and some of them involve added para­ meters. The BET equation has been derived by Cassie (11) and by Hill (12) using statistical mechanical methods.

The model

used is described by Cassiej The absorption is assumed to take place in the form of clusters around the localized sites; molecules will first be absorbed singly onto the sites. Each molecule absorbed onto this monolayer will have an amount of energy less than that of a molecule in the bulk liquid. Clusters will begin to form as the number of absorbed molecules increases. A cluster forms around the mole­ cule absorbed on the localized site, and it will be assumed that the molecules in the cluster apart from the one absorbed on the localized site, are identical in energy and partition function with those in the bulk liquid. The derivation of Hill used essentially the same model, with a slightly different method.

To the extent of validity of this

model, the parameter vQ can be interpreted as the number of

(10) Anderson, J. Am. Chem. Soc. j68, 686 (194-6) (11) Cassie, Trans. Fara. Soc. 41, 450, 458; 43 , 6l5 (1945-7) (12) Hill, J. Chem. Phys. 14* 263, 441 (1946)

6 sites on the material that are capable of absorbing the gas. These sites need not be localized on a surface but may be spread throughout the volume. More accurate isotherm equations can be developed in a statistical mechanical theory only by improving the model. The three basic assumptions that must be modified are1) The absorption sites all have the same energy. 2) The partition function of the absorbed molecules in the clusters is that of the liquid. 3) The absorbate does not change in structure during absorption. Probably the first assumption causes failure of the BET theory in the low pressure region, and the second and third are respon­ sible for the failure at high pressures.

It is certain that

the third assumption is not valid for the absorption of water by proteins because the proteins swell appreciably.

Eyring

and White (13) have tried to correct for this latter effect, but they introduce a third parameter which is essentially empirical, and which makes the absorption equation unwieldly. A thermodynamic study of gas absorption is hampered by two factors; a lack of good experimental data, from which one could calculate the important thermodynamic quantities, and the phenomenon of hysteresis (one of the causes of the lack

(13) Eyring and White, J. Textile Res. r£, 523

(194-7)

7

of data).

It is observed that the absorption isotherm

often lies below the desorption isotherm, and that the trans­ ition from one to the other at constant pressure is veryslow.

This makes a definition of the equilibrium state,

and hence a thermodynamic treatment, difficult.

This dif­

ficulty- has rendered valueless the extensive work of Bull (14) who determined the water absorbtion isotherms of 18 proteins at two temperatures.

He reported only the average of the

absorption and.desorption isotherms, which undoubtedly differed significantly.

Another possible fault of Bull's data is his

criterion for initial dryness.

He dried the proteins in.an

oven at 70° C., which resulted in considerable browning, and denatured the samples to an unknown extent.

Dole and

MacLaren (15) and Davis and MacLaren (16) have discussed the thermodynamics of water absorption, and have calculated the important thermodynamic quantities.

They used Bull's data.

The calculations are very tedious, and no one has performed them on such a scale for other systems, where the data is more accurate or reliable. Mellon, Korn, and Hoover (17) studied the hysteresis

(14) Bull, J. Am. Chem. Soc. 66, 1499 (1944) (15) Dole and MacLaren, ibid, 62, 651 (1947) (16) Davis and MacLaren, J. Polymer Science, 3, 16 (1948) (17) Mellon, Korn, and Hoover, J. Am. Chem. Soc. 7 0 * 1144 (1948)

8 effects in the absorption of water by casein.

They were

able to reproduce their isotherms to within experimental error.

By using samples which had been selectively ben-

zoylated in order to cover varying fractions of the free amino groups,- they found that the degree of hysteresis (defined roughly as the difference between the absorption and desorption capacity at a given pressure) did not depend upon the amount of free amino groups present.

By plotting

the amount of water present at a given relative humidity on absorption against the amount present at the same humidity on desorption, straight line was obtained.

If the desorption

is started at below 60 percent relative humidity, the slope of the line is unity and its intercept is positive.

This

indicates that the degree of hysteresis is independent of the amount of water present, and that the hysteresis is due to a constant amount of tightly held water.

This also agrees

well with the observations made by others that the BET plots for absorption and desorption are parallel.

If the desorp­

tion is started at a higher relative humidity, the slope of the line is 1.12, which is not explainable by experimental error.

This means that the degree of hysteresis is a function

of the amount of water present.

Eyring and White (18) beleive

that the hysteresis is intimately connected with the swelling phenomenon observed in these systems.

(18) Eyring and White, op. cit

S.

E. Smith (19) has proposed a somewhat different

treatment for the absorption of water vapor on polymers. He assumes that the water absorbed can be divided into two portions, one of which is "condensed with a normal heat of condensation" while the other is "bound by excessive forces".

He derives, using some doubtful assumptions, the

equation

where w is the weight fraction of water in the protein, and is the weight fraction of tightly bound water.

This

equation is apparently successful for some polymers when p/pQ is greater than .5.

He shows that the absorption and

desorption isotherms for water on cellophane are parallel, when plotted according to his equation.

For cellophane,

nylon, and cotton, the surface areas calculated from the BET theory agree well with the average of the surface covered with tightly bound water and the surface covered with the first normally condensed layer, both for absorp­ tion and desorption. worded thermodynamics.

His arguments involve mostly loosely Mellon, Korn, and Hoover (20)

observe that their data does not fit Smith's equation. has not been checked with Bull's data.

(19) S. E. Smith, J. Am. Chem. Soc. 6§, 64-6 (1947) (20) Mellon, Korn, and Hoover, op. cit.

It

10 Attempts have been made by Dole (21) and by Rowen and Simha (22) to relate water absorption to the theory of solu­ tions of high polymers, by considering the system as a very dilute solution of vapor in the solid polymer.

As one might

expect, they have been moderately successful in the high pressure region, but not at low pressures.

The simplified

equation of Rowen and Simha contains two parameters, one of which is related to the heat and entropy of solution or mixing, and the other is a measure of the swelling effect.

(21) Dole, Annals of the N.Y. Acad, of Sci. 21, 705 (1949) (22) Rowen and Simha, J. Am. Chem. Soc. £Q, 1663

(1948)

11 III

ABSORPTION AND OTHER PROPERTIES

The results of a BET analysis of an absorption isotherm are contained in the parameters vm and Ej - E^, the "surface area" and the heat of absorption.

Many attempts have been

made to correlate these quantities with the other physical and chemical properties of proteins.

They are concerned

mainly with the relation of the water surface area to the nitrogen area, with the degree of crystallinity and the lat­ tice structure, and with the specific chemical groups on the molecule. Shaw (23) determined the nitrogen and water surface areas of egg albumin, using BET theory.

He observed that the water

area was about 200 square meters per gram of protein, while the nitrogen area was 2.4 square meters per gram.

Frey and

Moore (24) studied the absorption of krypton and water on glycine, leucine, diglycyl-glycine, and diketopiperazine. They found that except for diglycyl-glycine, the water area was smaller than the nitrogen area.

Using the crystal struc­

tures, they showed that there was a correlation of the water area with the fraction of the crystal surface covered by polar groups.

For the one exception, the water area was

six times the nitrogen area.

This indicates that the water

in some way penetrated the crystal structure.

(23) Shaw, op. cit. (24) Frey and Moore, J. Am. Chem. Soc. 7 0 . 3&44

(1948)

12 Rowen and Blaine (25) measured the nitrogen and water surface areas of a series of textile fibers.

They observed that in

all cases the water area was much larger than the nitrogen area.

They enumerated four possible reasons for this differ­

ence— 1) The absorbing sites are not restricted to a surface. 2) If the absorbing sites are restricted to a surface, • there may be an additional internal surface specific to certain absorbates as well as an external surface. 3) The internal surface within the fibrous structure exists only in the presence of a swelling agent such as water. 4) The smaller diameter and the polarity of the water molecule enable it to penetrate into capillaries not accessible to the nitrogen molecule. X-ray diffraction studies on many proteins have been made.

In general, the diffraction patterns are either hope­

lessly complicated or too diffuse to be of much use.

However,

they have shown that in many cases, the material, either as a fiber

or as a powder, consists of domains which have vary­

ing degrees of order, from the completely crystalline and ordered to the amorphous and disordered. are usually called crystallites.

The ordered domains

These domains can exist in

a single particle, and are not separated by sharp boundaries, but in general by domains with intermediate degrees of order. There is a qualitative correlation between the sharpness and complexity of an x-ray diffraction pattern and the degree of order, or fraction of crystallite content. X-ray diffraction data appear to show that the crystallites

(25) Rowen and Blaine, op. cit.

13

in textiles and proteins usually absorb less water than the amorphous regions.

Katz and Berksen (26) have observed that

for gelatin the distance between peptide chains, in the dir­ ection of the side chains, increases with the amount of water absorbed.

Since only the crystallites can give strong dif­

fraction patterns, this indicates that the crystallites are capable of absorbing at least some water.

Palmer, Shaw, and

Ballantyne (27) have studied the water absorption isotherms and lattice spacings (of the 200 plane) of sodium pectate. The absorption isotherm shows a strong hysteresis, and the graph of lattice spacing against moisture content also shows a hysteresis loop.

On desorption, the spacing remains con­

stant from 65 percent water content to about 20 percent, and then decreases rapidly to the spacing of the dry protein. On absorption, the change is not so sharp, and the constant lattice spacing appears at about 30 percent water content. In the low humidity region, the spacing approximates a linear function of the relative humidity.

Y/hen the isotherm data

is plotted with the BET equation, parallel straight lines are obtained for absorption and desorption. differ by about ten percent.

The surface areas

The x-ray pictures show that at

about 14 percent relative humidity, the crystallinity of the pro­ tein increases, but also depends upon the past history of the

(26) Katz and Derksen, Rec. Trav. Chim. j?l, 513 (1932) (27) Palmer, Shaw, and Ballantyne, J. Poly. Sci. 2, 318 (1947)

14

sample.

The data is not sufficient to decide whether the

absorption isotherm depends on the degree of crystallinity. However, the isotherm data fits the BET equation well in the usual region. Frilette, Hanle, and Mark (28) have studied the rate of exchange of cellulose with heavy water.

They conclude

that the amorphous regions in cellulose are capable of exchanging hydroxyl ions very rapidly, but that the crystal­ lites exchange slowly, and then only the surface groups react.

If the exchange reactivity is related to its hydro­

gen bonding strength and accessibility, one might expect that the crystallites do not appreciably contribute to the absorption of water by cellulose. Pauling (29)> Bull, and Shaw believe that the absorption of water by proteins occurs in sheets which are formed between the polar groups on parallel sheets of protein.

Their analyses

did not consider the possible effects of order, but assumed that all parts of the protein molecule absorbed equally well. Dole and MacLaren (30) calculated from density measure­ ments that the nylon samples used by Bull were 37 and 29 per­ cent amorphous, and that 39 and 27 percent respectively of the total polar groups were saturated at 100 percent relative humidity.

They suggested that the crystallites did not absorb,

(28) Frilette, Hanle, and Mark, J. Am. Chem. Soc. 7 0 . 1107 (1948) (29) Pauling, ibid. 62, 555

(1945)

(30) Dole and MacLaren, op. cit.

15

and that in the amorphous nylon each polar group absorbed one molecule of water at saturation.

This would involve

discarding entirely the model of absorption suggested by Cassie. Mellon, Korn, and Hoover (31) made absorption measure­ ments on protein samples which had been drastically treated to alter their degree of crystallinity or order.

They ob­

served that certain treatments do not appreciably alter the water absorption isotherm, and concluded that the amount of water absorbed does not depend upon the degree of order. From the point of view of the physical chemist and bio­ chemist, the most interesting aspect of the absorption of water by proteins is its dependence on the chemical consti­ tution of the protein particle.

Most of the literature on

this subject contains the tacit assumption that the absorp­ tion does not depend upon the physical state of the protein particle, but only on its molecular structure.

Pauling (32)

has compared the data of Bull and Shaw on several proteins with their amino acid analyses.

His concluding remarks are—

The data published by Bull and other investigators on the absorption of water by proteins can be in consi­ derable degree interpreted on the assumption that the initial process is the attachment of one water molecule to each polar amino acid side chain. The data also indicates that peptide carbonyl and iraido groups usually do not bond water, because of their mutual

(31) Mellon, Korn, and Hoover, J. Am. Chem. Soc. 7 1 . 2761 (1949) (32) Pauling, op. cit.

16 interaction by hydrogen bond formation, but that water is bound by carbonyl groups which are not coupled by hydrogen bonds with imido groups. Frey and Moore (33) show that the absorption of water by crystallin amino acids is primarily a function of their physical states.

They found, that with diglycyl-glycine,

the "chemical" effect becomes important, and believe that this is an intermediate case between the crystalline amino, acids and the proteins. Mellon, Korn, and Hoover (34) have published several papers in which the effect of chemical structure has been explored.

In their first paper, they have calculated the

fraction of water absorbed by casein that is absorbed by free amino groups.

They accomplished this by preparing a

series of casein derivatives, in which the free amino groups had been selectively benzoylated to differing extents. They assumed that the benzoyl groups and amino groups that had been substituted would not absorb water.

They concluded

that in casein, about 25 to 30 percent of the absorption is due to free amino groups. not particularly good—

Their selective benzoylation was

in some samples, analysis, showed that

about 20 percent of the benzoyl groups that were introduced did not react with free amino groups.

With all these assump­

tions, the results are of only qualitative significance.

(33) Frey and Moore, op. cit. (34) Mellon, Korn, and Hoover, J. Am. Chem. Soc. 6 9 . 827; 20, 1144; 20, 3040 (1947,1948)

17

It is clear, however, that the free amino group is respon­ sible for a large part of the water absorption. In another paper, Mellon, Korn, and Hoover studied the effect of the peptide group on water absorption.

They ob­

tained water absorption'isotherms for di-, tri-, tetra-, and penta-glycine, and two polyglycines, about 40 and 50 units long.

Glycine, diglycine., and triglyeine showed no appre­

ciable absorption.

Tetraglycine gave some absorption, which

was not due to the terminal groups.

(They showed this by

benzoylating the end groups and repeating the measurements.) Penta glycine gave more absorption, and the polyglycines still more.

The polyglycines gave the same absorption

isotherm, when plotted in terms of grams of water per gram of sample, indicating that a limit had been reached on the effect of chain length on absorption.

The BET parameter

gave a value of about .16 moles of water per mole of peptide nitrogen for the "monolayer" coverage on the polyglycines. They did not give any explanation for the size of this value.

18

IV

RATE OF ABSORPTION

All of the workers in the field of water absorption on proteins except King and Cassie observed that the absorption took a rather long time.

For example, Bull allowed from 18

to 30 days on each point*

Rowen and Blaine allowed one or

two days with textile fibers.

Mellon, Korn, and Hoover

allowed six days for absorption and 18 days for desorption. Frey and Moore, working with amino acids, needed 12 hours in a high vacuum apparatus.

Palmer, Shaw, and Ballantyne

took two or three weeks for absorption and four to six weeks for desorption.

Of all of these, only Bull reported actual

rate data for proteins.

This is plotted in Figure 1 and

shows that after a short initial period, the data can be represented by the equation

* * } ( ' - Sz)

=

13

This can be interpreted by assuming that the rate determining step is the diffusion of water vapor through an air film at the surface of the protein particle. King and Cassie (35) measured the rate of absorption of water vapor by wool, and proposed a satisfactory explanation of their results.

They worked in a high vacuum apparatus,

thus eliminating the effects of air films.

The absorption

was measured directly by placing the wool on a quartz fiber

(35) King and Cassie, Trans. Fara. Soc. 36 . 445 (1940)

100

FIGURE 1

00

10

RATE OF ABSORPTION ON EGG ALBUMEN > BULL

19

balance, (McBain Balance), which was kept in a glass tube connected to the high vacuum line.

This was maintained at

constant temperature in an air bath.

The system was eva­

cuated until the weight of the wool became constant.

Then,

water vapor, at constant pressure from an external source, was admitted and the extension of the quartz spiral was measured at intervals of time. about an hour.

Equilibrium was reached in

.

Then they wrapped the wool with a fine plat­

inum wire, and repeated the experiment, using the platinum wire as a resistance thermometer.

They observed a sharp

initial rise in the temperature of the wool of about 4-0° C., and then a gradual cooling, which took about an hour.

The

heating effect can be shown by a simple calculation to be due to the large initial heat of absorption and the low specific heat of the wool, and to the low heat conductivity of the system.

By setting up a heat balance, they derived

the differential equation

where c is the specific heat of the wool, H is the heat of absorption per gram of water, M is the amount of water ab­ sorbed at time t, and k is the heat transfer coefficient of the whole system.

TQ is the external temperature.

The

equation can be integrated numerically, by assuming that the absorption instantaneously reaches the equilibrium value characteristic of the pressure of the water vapor, assumed to be constant, and the temperature of the wool, which is a

20

function of the time.

In other words, the rate determing

step is assumed to he the rate of cooling of the system. The data fits the theory very well, and leaves no doubt that this is the correct explanation of the experimentally observed rate.

By assuming a diffusion mechanism as the

ultimate limiting process, and taking a reasonable diffusion coefficient, it can be shown that the system should reach equilibrium in less than one second. port of the proposed explanation.

This is further sup­

21

CHAPTER III EXPERIMENTAL PROCEDURE I

INTRODUCTION

The experiments to be discussed were designed to give accurate information about the correlation between water ab­ sorption and nitrogen adsorption on the same protein sample. Because previous workers had reported such long times of absorption,- some attention was focussed on the determination of the rate of absorption.

When the problem of the rates had

been satisfactorily settled, the same apparatus and technique were used to obtain the absorption and desorption isotherms. Several samples of Bovine Plasma Albumen were prepared, and were lyophilized or spray frozen to give particles of varying sizes.

The surface areas of these particles were

measured using nitrogen absorption and the BET theory.

In

the case of the Bovine Albumen, the areas differed by a factor of one hundred. The apparatus used for isotherm and rate measurements was designed so that the samples could be evacuated and kept out of contact with the air during the whole series of measure­ ments.

Nearly identical procedures were used to obtain the

isotherms and to measure the rates; therefore, the procedure used in getting the rates will be discussed, since the equi­ librium values are naturally obtained at the same time.

22

II PREPARATION OF THE SAMPLES 1)

Bovine Plasma Albumen, fraction 5 was obtained

from Armour, control number G10106.

This was used without

further preparation, and is hereafter denoted by Bov.5 cr. The surface area, measured by Ellis (1) was .26 square meters per gram. 2)

The preceding protein was dissolved in de-ionized

water to make a three percent solution, and then lyophilized according to the procedure of Ellis. to as Bov.5 lyo.

This sample is referred

The surface area, determined by Zwanzig,

was 3.86 square meters per gram. 3)

The crude material was also dissolved to give a one

percent solution, filtered, and then spray frozen according to the procedure of Ellis. Bov.5 sf R Z .

This sample is referred to as

The surface area, determined by Zwanzig, was

25.1 square meters per gram.

(This is apparently the highest

nitrogen area ever obtained for a solid protein.) 4)

A sample of the same protein, spray frozen by Ellis

from a two percent solution, was also used. referred to as Bov. 5 sf D E .

This sample is

The surface area, determined

by Zwanzig, was 14.1 square meters per gram. 5)

Egg albumen was prepared by Ellis and referred to

by him as the 5/4-8 sample.

A parton of this was lyophilized

by Ellis from a 5.6 percent solution.

This is denoted by

(1) Ellis, Ph.D. Dissertation, Univ. of Southern Cal. 1949

23

E.A. cr.

The surface area, determined by Ellis, was six

square meters per gram. 6)

A parton of the egg albumen was spray frozen by

Ellis from a two percent solution. by E.A. sf.

This sample is denoted

The surface area, determined by Ellis, was

twelve square meters per gram. The surface areas of all the proteins used were obtain­ ed with the apparatus and using the technique described by Ellis.

Since only the surface areas were desired, a simpli­

fication was m a d e , in that only four or five points were taken on the adsorption isotherm, and none on the desorption isotherm.

The points were taken in the range in which the

BET equation applies, and gave good straight lines in the BET plots.

Since there is no hysteresis with nitrogen on

proteins, the desorption isotherm is not necessary.

The

BET plots for the proteins used, with the exception of those which have already been reported by Ellis, are given in the appendix.

24 Ill

DESCRIPTION OF THE APPARATUS

Balance and Weighing - All weighings were performed on a Christian Becker Projection Reading A&alytical Balance #911. The weighings were made to the nearest tenth of a milligram. After the balance had been in use for some time, it was discovered that the balance could not be relied on for this degree of accuracy.

The’ probable error of each weighing is

about three-tenths of a milligram.

Whenever the sample cells

were weighed, they were supported on the balance pans by a small wire hook.

The weight of the hook was always considered

a part of the tare weight of the cell. Sample Cell - Figure 2 is a diagram of the sample "cell. It can be sealed onto the manifold through the small ground glass joint.

High vacuum stopcock grease did not give a

vacuum tight seal with these joints, so a low melting resin was used.

The resin, called Shawanigan Resin, is insoluble

in water, but very soluble in acetone, and can easily be removed.

It is applied by warming the male part of the joint

and rubbing it with a block of the resin.

Apiezon L stop­

cock grease was used on the high vacuum stopcock.

During

the earlier runs, the other cell joint was sealed with Sha­ wanigan Resin.

The necessitated warming the sample bulb

slightly, and made possible a slight denaturation of the pro­ tein sample.

In the later runs, and during the isotherm

measurements, Apiezon L grease was used instead.

The dis­

advantage in using grease is that if too much is used, so

25

$ 7/15

W 19/22

FIGUHE 2 SAMPLE CELL

■HOLLOW GLASS PLUG

26 that it flows out of the joint, it may cause trouble when the cells are cleaned before weighings, and if not enough is used, some water will get into the crack of the joint from the con­ stant temperature bath, and evaporate slowly during the weighing, making it difficult to get a reproducible weight. In spite of these faults, the grease was used for the iso­ therm measuremerfts because, it-was felt that any denaturation would be undesirable. Since the proteins always contain some absorbed water fr.om the atmosphere, and because all air had to be removed, the samples were pumped on for several days.

During the ini­

tial evacuation, they have a tendancy to spurt out of the cell.

Therefore, a hollo?/ glass plug was used to decrease

this possibility.

Also, a small plug of glass wool, usually

about ten milligrams, was placed in the constriction beneath the stopcock. occurred.

With these in place, very little spurting

If any had occurred, the run was discarded.

The following procedure was used to load the cells.

The

cell parts were cleaned and dried, and the stopcock plug was greased and inserted.

The glass wool was put into place.

All the parts were now weighed.

The protein was loaded into

the sample bulb and the parts were again weighed.

The dif­

ference between these weights is the amount of protein added. Then the bulb was sealed to the cap of the cell, and the cell was again weighed.

When the weight of the protein that had

been loaded is subtracted from this, the remainder is the tare weight of the sealed cell.

27

Temperature Controls - The temperature of the samples was maintained by a water bath set at the desired tempera­ ture.

The temperature was obtained with a Beckman Thermome­

ter, which had been calibrated against a National Bureau of Standards thermometer, number 101824, with an accuracy of - .01° C.

A 500 watt Cenco knife heater, connected to a

Variac, was used for a heat source, and a copper coil was immersed in the bath in case circulating cold water should be needed to maintain the temperature.

A mercury thermo­

regulator, of conventional design, was connected to an electronic relay which controlled the heater. stirrer provided sufficient agitation.

A motor driven

The temperature of

this bath usually did not fluctuate more than a few hundredths of a degree. The temperature of the water vapor supply was maintained during the rate measurements by an ice bath, kept in a large Dewar flask. degree.

The fluctuation was less than a tenth of a

For the isotherm measurements, a water bath was con­

structed like the one described above, with a few changes. A large Dewar flask was used as the container, and a 150 watt knife heater was used.

The temperature was read on a thermo­

meter calibrated in tenths of a degree, and was estimated to one hundredth.

The thermometer was calibrated at the ice

point, and against the Beckman thermometer on the other bath. Fluctuations were usually less than two hundredths of a degree.

28 High Vacutun Line and Water Supply - Figure 3 is a dia­ gram of the essential parts of the high vacuum line.

The

vacuum was obtained with a Welch Duoseal pump and a mercury diffusion pump. gauge.

The pressure was determined with a McLeod

In general, no work was done unless the vacuum was

better than 10“3 mm of mercury.

When necessary, liquid

nitrogen was used to trap out condensible gases. Two water vapor supplies were used.

One consisted of

a bulb maintained at 0° C., and was used for the rate measure­ ments; the other was a flask containing a sulfuric acid solu­ tion at 25°«

The first is shown in Figure 4.

connected it to the manifold.

Stopcock B

The water supply bulb G con­

tained a glass stirrer, with a small piece of soft iron sealed in its upper portion. small permanent magnets.

This could be moved with two

The water was always stirred during

and before a run, so that possible thermal gradients due to evaporation could be eliminated.

The water was distilled

from a 200 ml flask F to the bulb G under vacuum, and then stopcock D was closed.

The water placed in flask F was al­

ready doubly distilled. The other water vapor supply is shown in Figure 5*

This

was constructed so that the flasks H containing sulfuric acid solutions could be replaced without difficulty. operated stirrer was built in.

A magnetically

This is definitely needed here

because of possible concentration gradients in the solutions,

TO MANIFOLD

7/15

FIGURE 3 HIGH VACUUM LINE

30

SZ?

TO

MANIFOLD

i ' MAGNETIC STIRRER

FIGURE 4 WATER VAPOR SUPPLY I

6

.7

44

SO COMPARISON OF ISOTHERMS ■ ABSORPTION A AND B

15

LYO & SF

.10 W/grara

CRUDE

05

•1

•S

5

•3 *7*0 FIGURE

9

6

.7

45

WATER ON BOV 5 SUNS A AND B 15

05

-ABSORPTION DESORPTION

V

(Length of arrow Indieat spread of three samples

FIGURE

10

46

The isotherms for the three samples are separated by less than .005 grams.

Therefore the isotherms are to with­

in experimental error identical.

The nitrogen areas of the

three samples differed by the ratio of one to fifteen to one hundred, and cover the whole experimentally available range.

There is no correlation between nitrogen absorption

and water absorption on Bovine Plasma Albumen V, to within the observed limits.

These limits are just as good, and

possibly better than comparable data in the literature. Undoubtedly there should be some ultimate difference in the isotherms, because the surface areas are different.

However,

the amount of water corresponding to the nitrogen area is less than .005 grams even in the case of the largest area. The isotherms obtained here are considerably flatter than those reported by Bull.

In fact, in the middle of the

isotherm, the points may be fitted quite well by a straight line.

The BBT plots are conventional; the fit is good in

the usual region, and deviations occur in the usual regions. The BET parameters are not very valuable, in view of all the approximations involved in the application of the theory to water absorption.

Average values of these constants to two

significant figures (they are not any better than that) are— Absorption- vm = 6.3 milligrams of water/ gram of protein c Desorption

= 9.0

vm = 7.0 milligrams c

= 16.0

"

4-7 It is a general property of BET plots that the desorp­ tion line is parallel to the absorption line, and lies below it.

This is borne out in this case.

Since the surface area

and heat of absorption depend on both the slope and the inter­ cept, both parameters will be different.

These isotherms

show that the desorption area is larger than the absorption area, and that the heat of absorption is much larger.

In a

qualitative way, one could say that it is harder to get the water out than it is to put it on. The identity of the isotherms implies either that there is no dependence of water absorption on the degree of crystallinity, or that the degree of crystallinity is the same. bably, the samples used were highly amorphous.

Pro­

Electron

microscope pictures obtained by Ellis show that protein par­ ticles prepared by lyophilization do not look particularly crystalline.

One would expect the absorption of more "chem­

ical" gases like hydrogen chloride to show the same lack of dependence on physical state.

48

II

RATE MEASUREMENTS

The data gathered during the rate measurements is given in the appendix: typical curves are shown in Figures 11 and 12.

The data is quite reproducible, as long as the geometry

of the sample cell is not disturbed -- when the protein is removed and put back in again, or when the s a m e .protein is put into a different sample cell, the results are different. Because later evidence showed that the numerical data has no quantitative interest, only representative rate runs are given here.

The errors of this type of absorption measure­

ment have been discussed in the section on the isotherms. The data seems to be of about average accuracy, for rate measurements in general. In order to contrast this data with that of Bull, the same type of semi-log plot was made. figure 13. here.

This is shown in

It is obvious that the situation is different

In earlier experiments, with small amounts of air

present in the system, longer absorption times were observed, and were more in accord with Bull's data.

If the air pres­

sure is greater than 10~3 mm Qf mercury, the absorption process is likely to be slowed very much.

Therefore, Bull

measured the rate of diffusion of water vapor through a film of air surrounding the protein particles.

The shape of his

curve is typical of film diffusion kinetics. Some calculations were made on run 5 to determine whether bulk diffusion could explain the data.

Solutions for the bulk

01 FBQEEIH / OMM GRA.MS mEES

Ok

M S 01 A.BSORPTIGH? EGO 03

02

01

0 0

1

2 HOUSE 11

/ 0B4M OF PBQT2JK SB&MS mTEE

m ® o fa b so h m a n ? boo albomest spr a t f rozbr

03

02

01

0 FIGUHS 12

51

HATE OF ABSORPTION EGG ALBUMEN CRUDE

O

TEMP. BEL. HUM

©

52 diffusion equation, for diffusion into a sphere from a medium of constant concentration, have been tabulated by Boyd, Adam­ son, and Myers (1), and proved to be very useful. did not fit the equation.

The data

Moreover, by guessing at the approx­

imate value of the diffusion coefficient, using this equation, the result is of the order of magnitude of ten Angstroms per day.

This is remarkably small for a system of this type*

Therefore, it is reasonable to discard film or bulk diffusion as the limiting process. An attempt was made to find out whether the data could be represented by some simple equation of chemical kinetics. The first order rate law was tested by a semi-log plot, and was not fitted. against 1/t.

The second order was tested by plotting 1/W

If the process is second order, this should

give the straight line equation W

where W is the weight of water absorbed by one gram of dry protein at time t:j and W Q is the equilibrium concentration. This follows from the differential equation

The results are shown in Figures 14 and 15; the fit is excellent. Figure 16 shows the result of a test on another equation, which has no theoretical significance, but which is fitted remarkably

(1) Boyd, Adamson, and Myers, J. Am, Ghem. Soc. ,62, 2848

53

HATE OF ABSORPTION EGG ALBUMEN CHUDE TEMP. HEL. HIM.

30*0 C 0*144

3.8

sg|H'

3.4

3.0

2.2 10 (hours )“■*■

FIGURE

14

12

54

SATE OF ABSORPTION EGG ALBUMEN S.F. 50 .0 ■ C 0.144 5.8

3*6

k

3.0

0

2

4

8

6 (hours)

FIGUHE

••I

15

10

12

55 FIGURE

16

RATE OF ABSORPTION EGG ALBUMEN CRUDE

JL 2

56

well.

The equation is

jS'H (\Kj0~ J) ~ A ypc

-f-S

The second order equation is very convenient for comparing rate of absorption curves, but it turns out that the agree­ ment is fortuitous.

It seems rather strange that two such

different equations should fit the same data equally well, and yet have no theoretical meaning, when applied to that data. The correct mechanism for the absorption was discussed by King and Cassie, in the paper referred to in Chapter II. Unfortunately, this paper was not discovered until some work had already been done, because of its unusual title Rate of Propagation of Temperature Changes in Textile Fibers. After the paper was read, their interpretation was tested on Bovine Plasma Albumen V with essentailly identical results. The data is given in the appendix, and in Figure 17*

In this

case, the initial temperature rise was much less than the rise observed by King and Cassie, but still large enough to cause trouble.

The gradual drop of temperature took roughly

the same length of time as is required for the sample to come to absorption equilibrium.

Parallel weight measure­

ments were not made because of the general procedure, which allowed the sample to undergo temperature fluctuations during the handling and weighing.

However, another check

was made on the hypothesis, which King and Cassie could not carry out because of their procedure.

A rate measurement

MIBB9 ES

57

to

jt

Q

to

C\J

ESI H E&Q28H3M2i

o

58

was made in an aluminum cell.

This increased the thermal

conductivity, and the absorption went about four times as fast.

This is shown in figure 18, using a second order rate

equation plot to compare data obtained with a glass cell and with an aluminum cell. Seehof (2) has used a sample cell with a sealed-in thermocouple to check this theory in the case of the ab­ sorption of hydrogen chloride on egg albumen, and observed a large temperature rise. When a protein that contains water vapor is evacuated, the temperature falls several degrees.

This is the reverse

of the effect observed during absorption, and may be of im­ portance in the practical application of vacuum drying to protein systems. The theory of King and Cassie can be applied in prin­ ciple to any gas-solid reaction where large quantities of heat are involved.

The success of the second order rate

law is certainly accidental, and suggests that false conclu­ sions may easily be drawn when working out the kinetics of any such reaction.

(2) Seehof, private communication.

m

EFFECT

OB ALUMINUM

CELL

20

10 (hours)

FIGUBE IB

60

CHAPTER V CONCLUSIONS Water absorption on Bovine Plasma Albumen V is inde­ pendent of the surface area of the protein particle to with­ in experimental error.

This implies that water absorption

is qualitatively different from nitrogen adsorption and probably takes place at specific sites on the protein mole­ cule. The rate of absorption of water is limited by two factors; in the presence of even small amounts of air, the absorption is enormously inhibited; in the absence of air, the absorption is immeasurably fast, the observed rate merely being limited by the rate of heat loss to the bath. The procedure described for getting the isotherms is capable of giving results as accurate as any heretofore ob­ tained, in much less time. The findings suggest that all kinetic processes in heterogeneous systems which involve even small heat changes are inherently limited by the thermal transport properties of the media and that such data be scrutinized very care­ fully.

BIBLIOGRAPHY

62 Anderson, J. Am. Chem. Soc. 68, 686 (1946) Bancroft and Barnett, Proc. Nat. Acad. Sci. 16, 118, 135 (1930) Belden, J. Phys. Chem. 41, 1101 (193D Benson and Ellis, J. Am. Chem. £0, 3563 (1948) Boyd, Adamson, and Myers, ibid. 6£, 2848 (1947) Brunauer, Physical Adsorption. Princeton University Press (1943) Bull, J. Am. Chem. Soc. 66, 1499 (1944) Cassie, Trans. Fara. Soc. 41, 450, 458 (1945) . . . . , ibid. 43 , 615 (1946) Davis and MacLaren, J. Polymer Science, 3» 16 (1948) Dole and MacLaren, J. Am. Chem. Soc. 69 , 651 (194?) Dole, Annals of the New York Acad, of Sci. j[l, 705 (1949) Ellis, Ph.D. Dissertation, Univ. of Southern Calif. 1949 Eyring and White, J. Textile Research 32, 523 (1947) Frey and Moore, J. Am. Chem. Soc. 22? 3644 (1948) Frilette, Hanle, and Mark, ibid. 2Q, 1107 (1948) Hill, J. Chem. Phys. 14, 263 , 441 (1946) Katz and Derksen, Rec. Trav. Chim. j[l, 513 (1932) King and Cassie, Trans. Fara. Soc. 3 6 , 445 (1940) Mellon, Korn, and Hoover, J. Am. Chem. Soc. 65?, 827 (1947) ibid. 2Q, 1144, 3040 (1948) . . . . , ibid. 21 2761, (1949) Palmer, Shaw, and Ballantyne, J. Polymer Sci. 2, 318 (1947) Parks and Melaven, J. Phys. Chem. 41, 1101 (1937) Pauling, J. Am. Chem. Soc. 62, 555 (1945)

Rowen and Blaine, Ind. Eng. Chem. 32 5 1^59 (1947) Rowen and Simha, J. Am. Chem. Soc. £0, 1663 (1948) Seehof, M. S. Theses, Univ. of Southern Calif.

(1950)

Shanfcman and Gordon, J. Am. Chem. Soc. 6l, 2370 (1939) Shaw, J. Chem. Phys. 12, 391 (1944)

APPENDIX

65

TABLE I RATE OF ABSORPTION ON EGG ALBUMEN Bath temperature . 3O.O 0 C. Relative humidity. . 0.144 E.A. crude, Run 4 Time (hours)

0.0 0.2 0.7 2.0 3.0 11.3

Absorption (grams water per gram of protein)

0.0 .0334 .0424 .0441 .0453 .0451

E.A. crude, Run 5 0.0

0.1

0.0

.0255 .0363

0.3 0.5 0.7 1.0

.0391 .0408 .0420

1.5

.0432

2.5

.0443

Sample weighed .620 grams.

E.A. spray frozen, Run 4 Time (hours)

Absorption (grams of water per gram of protein)

0.0 0.2

0.0 .0275

0.7 2.0 3.0 11.3

.0400 .0438 .0446 .0447

E.A. spray frozen, Run 5

0.0 0.1 0.3 0.5 0.7

1.0

1.5 2.5

0.0 .0200 .0339 .0383 .0392 .0407 .0418 .0435

Sample weighed .545 grams.

66

TABLE II RATE OF ABSORPTION ON BOVINE PLASMA ALBUMENTV Bath temperature . 30.0° C. Relative humidity. . 0.144 Run 11 Time (hours)

Run 12

Absorption (grams water per gram of protein) Bov 5 cr.

Time (hours)

Absorption (grams water per gram of protein) Bov 5 cr.

0.0 0.1

0.0 .0287

0.0 0.1

0.0 .0318

0.2

.0380

0.5 1.0 2.0 21.0

.0435 .0465 .0475 .0482

0.2 0.5

.0395 .0435

1.0

.0452

3.0 20.1

.0467 .0470

Bov 0.0 0.1

0.0 .0173

0.2 0.5

.0267 .0386

1.0 2.0 21.0

.0428 .0462 .0483 Bov

0.0

0.0 .0328

0.2 0.5

.0404 .0455

1.0

.0501

2.0 21.0

-----.0509

0.0

0.1

.0181

0.2 0.5 1.0 3 .0---------20.1 Bov

5 sf.

0.0 0.1

The Bov The Bov The Bov

Bov .5 lyo.

5 «lyo.

0.0 0.1 0.2 0.5 1.0 3.0 20.1

5 cr sample weighed .600 grams. 5 lyo sample weighed .693 grams. 5 sf sample weighed .739 grams.

.0267 .0383 .0428 -----.0472

5 sf. 0.0 .0339 .0411 .0448 .0462 .0489 .0505

67

TABLE III TEMPERATURE VARIATION DURING ABSORPTION Sample . . . . . . Bov 5 lyo Bath Temperature . 30*0 C. Relative Humidity. . 0.144 RUN 14 Time (rain.)

Milliv.

RUN 15 T° C.

Time (min.)

Absorption

0.0 2.0

0.0 .372

4.0

.'276

8.0 12.0 16.0 23.0 30.0

.144 .077 .043

40.0

60.0

.020 .010 .006 .006

Milliv.

T° C

Absorption

0.0 9.1 6.7 3.5 1.9

1.0 0.5

0.2 0.1 0.1

00.0 . 1.0 2.0 3.0 5.0 7.0

11.0 16.0 20.0 36.0

0.0 .326 *368 .328 .240

.170 .093 .046

.026 .000

0.0 7.95 8.98

8.00 5.86 4.15 2.27

1.12 O .63 0.00

Desorption under vacuum - 0.0 -0.0 - .108 - 2.6 4. -3.0 - .123 6. - .110 -2.7 - .096 9. -2.3 - .036 -0.9 25.

0.0 2.

NOTE -- For run 14, the reference junction was at room temperature. For run 15* it was immersed in the constant temperature bath at 30.0 .

68

TABLE IV EFFECT OF ALUMINUM CELL Sample • . . . . Bov 5_lyo Bath temperature . 30.0 C. Relative humidity. * 0.144 Run 13— Time (minutes)

0.0 3-0 6.5 ..12,3

30.0

.

960

glass cell

Run 17—

aluminum cell

Absorption (grams water per grams of protein)

Time (minutes)

0.0 .0172

0.0 3.0

.0255 .0333 .0415 .0458

12.0 30.0

Cell contained .641 grams,

Absorption (grams water per gram of protein)

0.0



.0315 .0428 .0450

Cell contained .614 grams,

69

TABLE V ABSORPTION ISOTHERM A Bovine Plasma Albumen at 3O.O0 C.

20

d ^ H 2SO4

1.514-6

1.4694

1.417

p/Po

0.085

0.155

0.23

Sample

Grams water

cr lyo sf

0.0340

cr lyo sf

0.0460

cr lyo sf

0.0587 .06 25

.0382 .0376 .0508

.0493

.0639

1.3667

O .308

cr lyo sf

0.0725 .0734 .0776

1.2862

0.451

cr

0,1014

lyo sf 1.1907

0.599

cr

lyo sf 1 .0 8 3 0

O .703

cr lyo

sf Sample weights were

cr 1.013 5 grams. lyc 0.5313 sf 0.5552

.1073

.1081 O.I396

.1532 .1518

0.158 .171 .169

70

TABLE VI DES0RPTI0E: ISOTHERM A

Bovine Plasma Albumen at 30.0 ° c . d2!

k 2s c 4

P/P 0

Sample

■Grams wa1

1 .1 9 7 6

0 .5 8 6

cr lyo sf

0.1490 .1455 .1430

1.2848

0 .4 4 7

cr lyo sf

0.1187 .1148

cr

0.0936 . 0894

1.3636

c .3 0 7

lyo *

0 .2 3

sf

.0911

cr

0 .0 8 0 1

Ivo sf

1.5298

0 .0 8 6

.II38

cr lyo sf

.0757 .0774 0.0466 .0439 .0441

*Note- This solution was lost before its density could be obtained; however, it certainly did not change ouch from its value in the preceding absorption run*

71

TABLE VII ABSORPTION ISOTHERM B Bovine Plasma Albumen at 30 .1 0° C. d2!

h 2S0 4

p/pg

Sample

Grams water

1.5322

0.084

cr lyo sf

0.0336 .0348 .0339

1.4680

0.130

cr lyo sf

0.0465 .0476 .0470

1.4200

0.217

cr lyo sf

0.0593

.0602 .0630 *

1.3652

0.304

cr lyo sf

0.0746 .0757 .0737 *

1.2867

0.445

cr lyo sf

0.0992 .1013

* Note'* These points are somewhat less reliabl< than the others. DESORPTION ISOTHERM B

1.2920

0.448

cr

0.1166

1.4233

0.217

cr

0.0697

1.5322

0.084

cr

0.0364

Note- The densities were measured at 20°.

72

FIGURE

20

19

NITROGEN ON BOV 5 SF DE

15

.10

s 42 .0

» 10.4 am

05

0

.1

3 */*