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Volumetric method for the study of swelling and syneresis and its application to protein gels
Volumetric method for study of swelling and syneresis
54t
REFERENCES 1. B. GROTEN, Analyt. Chem. 36: 1206, 1964 2. J. MASHIKO, A. FOSHIZEN and A. NAKAO, J. Chem. Soc. Japan, Industr. Chem. Sec. 68: 7, 1206, 1965 3. C. E. R. JONES and G. E. J. REYNOLDS, J. Gas. Chromatogr. 5: 251, 1967 4. M. DRESSLER and M. KREJI~I, Chelnicke list s~ 61: 1571, 1967 5. B. G. BELEN'KII, Yu. V. ZHURAVLEV, T. V. PETROVA ct al,. Gazuvaya khrom~ttograliya (Gas Chromatography). VII, p. 97, N I I T E K h I M , 1967 6. T. S. WEST, Information Bull. IUPAC 26: 39, 1966 7. L. A. WALL, Sbornik: AnMiticheskaya khimiya polimerov (A~Laly~icalChemistry of Polymers). Ed. by G. Klein, Pyrolysis, vol. I[, "Mir", p. 152, 1965
METHODS OF INVESTIGATION VOLUMETRIC METHOD FOR THE STUDY OF SWELLING AND SYNERESIS AND ITS APPLICATION TO PROTEIN GELS* ~rE. YE. I~I~AUI)O ~md V. B. TOLSTOGUZOV Institute of tt(,I.ero-organie Compound~, U.S.S.R. Aca(b~my of Sciences
(Received 12 February 1969) THJ,: problem of dev,qopit~g a simple and. accurate method for det.crminmg t h,' velum,'trie d.,grce of swelling of subsl.ane~,s with diffewnt mechanical propcrt.ics up Io now r~,mains tll~ urgent problmn. A critical revi~,w is given of s¢,vera] method~ [1, 2]. Yermolellke and. Maz(,l' Ill dev,qepcd, two m~t.hods for determining vohnnetrie swelling. A shortcoming of the first, m,,l~hod, as a,lso of eth,~r well-l¢nowlx methods [3-6], is the distortion of rosult~s of measur(,nu,l~|s owing to ent.rapping of solvent, drops by the sample. In add, itiom tlmse methods are mtsuitable for |he investigation o1" swelling of soft, uustaMe or, (ut the eoutrary, fairly hard specimens. W}wn determinilxg swelling accord.ing to She sceoi~d method [1], the swelling specimen is under the pressure of the mereuIT colmrm, which obviously affects the results of the measuremetits. This shorteomixtg is also observed, with other methods based on l,he principle of measurements proposed, by Logtermoser and. l~adestoek [7-10]. The metho4 proposed b y Przheborovskii and Tile [2] is free from these drawbacks and involves weighing samples before and after swelling in two liquids in which they do not swell. To obtain fairly accurate results it is essential tba~t the liquid.s are of very different densities and consequently of d.ifferent chemical natures. However, it will be pointed out that this conflition is not always feasible. We developed a simple method, for determitfing swelling of substances with different, mechanical properties, which is based on the d.ireet determination of the specimen volume before and after swelling b y immersion in a liquid which is inert in relation to the specimen. The specimen is placed in a perforated, dish (Fig. 1) provided, with a stopper; the dish is made of a materiM which does not swell in the liquids used. * A thin steel wire passes • Vysokomol. soyed. A12: No. 2, 474-476, 1970. • For fluorocarbon and fluoroehloroearbou liquids it is recommended to use organie glass.
542
Y~.. YE. BRAUDO and V. B. TOLSTOGUZOV
through the openings in the walls of the dish t~ form a loop. The dimensions and shape of the specimen are selected so t h a t during swelling the specimen is not in contact with the walls of the dish. To determine the volume of the specimens, a modified apparatus is used [5, 6]
i ¢ IlO
:l,-I
FIG. 1. The wails and base of the dish are perforated (not shown in the Figure). The diameter of the openings is 1 ram. (Fig. 2). On the central tube of 3"5 cm a a scale is used with divisions corresponding to 0.05 cm s (1). The dimensions shown in Figs. 1 and 2 correspond to a n initial specimen volume of 0.2-0.5 cm s a n d a m a x i m u m degree of swelling of I0. The cross section of the device is sealed with graphite lubricant. The device is placed in a n air oven for the time of measurement. The zero reading of the device is previously determined; therefore, it is arranged vertically so that the stopper is in the upper position, the liquid allowed to flow in a large container, the device turned through 180 ° and the position of the meniscus recorded at certain intervaIs. The time required for achieving a constant level is determined empirically for each t 2
20
-j
~ m . 2, Device for the measurement of specimen volmne: I-- scale (one division =0.05 cma); 2 - - N S h 14.5.
liquid. Thus, for the fluorocarbon liquid we used. it was 15 min; for octane, 5 min. The liquid volume in the device should be such that the meniscus with zero reading is in the lower part of the scale. At least two measurements are made for determining the position of the meniscus. After establishing the zero reading the dish containing the specimen is placed in the apparatus, the dish is fastened by a loop to the hook on the stopper of the apparatus and the
V o h n n e t r i e metb.od for s t u d y of swellir~g and syneresis
543
m e a s u r e m e n t r e p e a t e d as described. Tlte v o h u u e of the e m p t y dish wittl the lid and loop is p r , viously d,'t,ermit~ed by a similar method. Swelling of the sample is effeeged in tim dish used. for d e t e r m i n i n g the volume, or separately, if t.he specimen is fairly strong. A f t e r swelling, the '.~peeimen or dish w i t h the specimen al'~ freed from free solvent residues b y a. filter paper or b y light, agitation. The v o l u m e of the swollm~ spectral,it is t h e n determiIled as described. "Whei~ select, ixtsa tt~e inert liquid to d e t e r m i n e the specimen v o l u m e air o p t i m u m eombinaLieu of volatility rout. viscosity should be a i m e d at. The use of tfighly velar, lie liquids involw,s errors of lnoasul',?lnt'nt; witlt highly viscous liquids a e o n s t a n t position of the Ineniseus is slowly ~mhiow'd, Wll,,n w a t e r or m m t h e r liquid ~aqt]t a leith surface tension is used for tire vohun,' nleasurem~'nt, to facilitate overflow from one eontainer into another, addition of surt'~m,~ act, ire substmmes is r e c o m m e n d e d . V~hel~. working wittt protein ~els t h e seh:el.,ion of llte iluq't liquid involves certain dittieulties sil~.ee these, st~})slane('s ttavt~ a solubilizing ability in relation to tmn-polar compounds, ineluding parafl3nie h y d r o c a r b o n s [11]. I t is kImwn glint m i x t u r e s of fluorocarbons and hydrocarbons show considerable posit|w> doviation~ from the R a o u l t law [12, 13]; fluorocarbons are v e r y sparingly soluble in w a t e r [12 I. W e tltorefore assumed t h a t fluorocarbons and possibly fluoroeltloroearbons are n o t sohttfilized, by proteins in praetiee. Tim a s s u m p t i o n was eol~firmt'd ext)oriment.ally b y th,, absence of ~lny ~Vei~llt v a r i a t i o u of dishes containing the gelatin gel after r e t e n t i o n for a wt~ek in a thtorocarbon liqui(l and in F r e o n - l l 3 . Therelbre, to determi~w (.lu! d(,gt'e(~ of swelling of protei~ gt.is w~' used a fluorocarbon liquid of tim following properties: r~0: : 10.6 eS; p~0 := 1"98; the iniiial |)oiling 1)oint aa 10 m m H g was tfigher t h a n 70 °, solid|fleet, ion 1)oint below --52:'. W e not(, tllai, waL(,r solubility its. tltese liquids is also \'t,ry low: 0-004 [~'4 for I~'t'('on - 1 [3 aL 20 ° [14], of the order of 0.001 }{ for fluoroearlion liquids [121. As aI~ e x a m p l e of the m e t h o d we deseribe resttlts of doi.erminillg the degree of swelling in w a t e r of p h o t o g e l a t i n e gels purified b y L~q)us. Prior to doLernfination the gel specimens we're l'e~airted at 10 :[_ 1.5 ~for 5 days. Swellil~g in 200-fold \vat.or excess was carried, out at, the siuno t e m p e r a t u r e for 6 da.ys. In|tin,1 eo~eentration, '~{; ])egree of swelling,
2"5 5'11 7"5 10"0 0.57:! 0.03 0.86 ! 0.02 0 . 9 8 ± 0 . 0 2 1 ' 1 1 ~ 0 - 0 1
3~he t].~wiee described can also be ust.d to invest,|gab; ~he syuer~,sis kitmt, ies of gels. The liquid in wlfieh syirert~sis is carried out s}}.otll~l not dissolve the sytieretie liquid a n d should be h e a v i e r ttmlt t]fis liquid. The speeilnen v o l u m e is d~termined as described, in a separat, e device, or in a d,~vice in which syneresis is efl'eeted if the liquid contained, is fairly inert in relatimi l,o the gel. The. liquid in the device should be sat, u r a t e d wit.It tim solvent c o n t a i n e d by the g~d. Syueresis is o b s e r v e d b y measuring tt~e v o l u m e of the synoretie liquid s e p a r a t e d and (if the liquid in wtfieh the lneasuremel}.t is earn'led out is not volatile) the gel volume. I t is only essential to eI~sttre t h a t the drops of the synoretie liquid do not adtmre to the specimen, dish, or t h e wails of the device. W e used F r e o n - i i 3 to investig~tte syn,r~sis oI( prot, eiu gels. It.s a d v a n t a g e over fire ttuorocarbon liquid is it,s lower viscosity; however, owing to l.tt, increased volatility of F r e o n (b.p. 48 °) the resull, s o[' d e t e r m i n i n g the v o h u n e of g,,1 samples are less reliable,
CONCLUSIONS (1) A mel~hod was d~velep~,d 1,o del,ermine tht~ volumel,rie dogree of swollilxg of substanee~ wit,h differm~t nleehal~iea.i prol)erties. (2) I t is p o i n t e d o u t ttl~t.l~ ftuoroeal'boltS or tluoroehloroear|mns should be used as i~ort liquids for t h e s t u d y of prot, ebz gels. (3) A met,hod is proposed for the invcst.igatiou of sy~ercsis of gels.
Translated by E . SEMERE
544
Y z . YE. BHAUDO and V. B. TOLSTOGVZOV
REFERENCES 1. 2. 3. 4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14.
I. N. YERMOLENKO and M. I. MAZEL', Kolloid. zh. 14: 390, 1952 Ya. S. PRZHEBOROVSKII and V. :K. TILE, Kolloid. zh. 16: 304, 1954 B. DOGADKIN, K. PECHKOVSKAYA ~nd L. CHERNIKINA, Kolloid. zh. 8: 31, 1946 R. V. UZINA, K a u c h u k i rezina, No. 2, 8, 1941 B. A. DOGADKIN, Kolloid. zh. 21: 762, 1959 O. N. GRIGOROV, I. F. KARPOVA, Z. P. KOZ'MINA and D. A. FRIDRIKHSBERG, Rukovodstvo k prakticheskim zanyatiyam po kolloidnoi khimii (Practical Work on Colloid Chemistry). p. 196, Izd. LGU, 1955 A. LOTTERMOSER and H. RADESTOCK, Z. angew. Chem. 40: 1506, 1927 I. I. ZHUKOV, S. L. TALMUD and V. A. ZIL'BERMAN, Sintetiche~kii ka~olat~k, N$. 6, 21, 1935 I. I. ZHUKOV and F. M. SIMKHOVICH, Kolloid. zh. 1: 11, 1935 T. P. TYAZHELOVA, Kolloid. zh. 1: 90, 1935 G. P. YAMPOL'SKAYA, V. N. IZMAILOVA, V. A. PCHELIN and A. V. ¥OLYNSKAYA, Vysokomol. soyed. 7: 1956, 1965 (Translated in Polymer Sei. U.S.S.R. 7: 11, 2147,1965 Ftor i ego soyedineniya (Fluorine and its Compounds). Edited by G. Simons, vol. 1, p. 315, Izd. inostr, lit., 1953 F t o r i ego soyedineniya, Edited by G. Simons, eel. 2, p. 328, Izd. inostr, lit., 1956 G. A. YAKOVKIN, Freony. Svois~va i primenenie (Refrigerants. Properties and Applioation), p. 25, Goskhimizdat, 1956
54t
REFERENCES 1. B. GROTEN, Analyt. Chem. 36: 1206, 1964 2. J. MASHIKO, A. FOSHIZEN and A. NAKAO, J. Chem. Soc. Japan, Industr. Chem. Sec. 68: 7, 1206, 1965 3. C. E. R. JONES and G. E. J. REYNOLDS, J. Gas. Chromatogr. 5: 251, 1967 4. M. DRESSLER and M. KREJI~I, Chelnicke list s~ 61: 1571, 1967 5. B. G. BELEN'KII, Yu. V. ZHURAVLEV, T. V. PETROVA ct al,. Gazuvaya khrom~ttograliya (Gas Chromatography). VII, p. 97, N I I T E K h I M , 1967 6. T. S. WEST, Information Bull. IUPAC 26: 39, 1966 7. L. A. WALL, Sbornik: AnMiticheskaya khimiya polimerov (A~Laly~icalChemistry of Polymers). Ed. by G. Klein, Pyrolysis, vol. I[, "Mir", p. 152, 1965
METHODS OF INVESTIGATION VOLUMETRIC METHOD FOR THE STUDY OF SWELLING AND SYNERESIS AND ITS APPLICATION TO PROTEIN GELS* ~rE. YE. I~I~AUI)O ~md V. B. TOLSTOGUZOV Institute of tt(,I.ero-organie Compound~, U.S.S.R. Aca(b~my of Sciences
(Received 12 February 1969) THJ,: problem of dev,qopit~g a simple and. accurate method for det.crminmg t h,' velum,'trie d.,grce of swelling of subsl.ane~,s with diffewnt mechanical propcrt.ics up Io now r~,mains tll~ urgent problmn. A critical revi~,w is given of s¢,vera] method~ [1, 2]. Yermolellke and. Maz(,l' Ill dev,qepcd, two m~t.hods for determining vohnnetrie swelling. A shortcoming of the first, m,,l~hod, as a,lso of eth,~r well-l¢nowlx methods [3-6], is the distortion of rosult~s of measur(,nu,l~|s owing to ent.rapping of solvent, drops by the sample. In add, itiom tlmse methods are mtsuitable for |he investigation o1" swelling of soft, uustaMe or, (ut the eoutrary, fairly hard specimens. W}wn determinilxg swelling accord.ing to She sceoi~d method [1], the swelling specimen is under the pressure of the mereuIT colmrm, which obviously affects the results of the measuremetits. This shorteomixtg is also observed, with other methods based on l,he principle of measurements proposed, by Logtermoser and. l~adestoek [7-10]. The metho4 proposed b y Przheborovskii and Tile [2] is free from these drawbacks and involves weighing samples before and after swelling in two liquids in which they do not swell. To obtain fairly accurate results it is essential tba~t the liquid.s are of very different densities and consequently of d.ifferent chemical natures. However, it will be pointed out that this conflition is not always feasible. We developed a simple method, for determitfing swelling of substances with different, mechanical properties, which is based on the d.ireet determination of the specimen volume before and after swelling b y immersion in a liquid which is inert in relation to the specimen. The specimen is placed in a perforated, dish (Fig. 1) provided, with a stopper; the dish is made of a materiM which does not swell in the liquids used. * A thin steel wire passes • Vysokomol. soyed. A12: No. 2, 474-476, 1970. • For fluorocarbon and fluoroehloroearbou liquids it is recommended to use organie glass.
542
Y~.. YE. BRAUDO and V. B. TOLSTOGUZOV
through the openings in the walls of the dish t~ form a loop. The dimensions and shape of the specimen are selected so t h a t during swelling the specimen is not in contact with the walls of the dish. To determine the volume of the specimens, a modified apparatus is used [5, 6]
i ¢ IlO
:l,-I
FIG. 1. The wails and base of the dish are perforated (not shown in the Figure). The diameter of the openings is 1 ram. (Fig. 2). On the central tube of 3"5 cm a a scale is used with divisions corresponding to 0.05 cm s (1). The dimensions shown in Figs. 1 and 2 correspond to a n initial specimen volume of 0.2-0.5 cm s a n d a m a x i m u m degree of swelling of I0. The cross section of the device is sealed with graphite lubricant. The device is placed in a n air oven for the time of measurement. The zero reading of the device is previously determined; therefore, it is arranged vertically so that the stopper is in the upper position, the liquid allowed to flow in a large container, the device turned through 180 ° and the position of the meniscus recorded at certain intervaIs. The time required for achieving a constant level is determined empirically for each t 2
20
-j
~ m . 2, Device for the measurement of specimen volmne: I-- scale (one division =0.05 cma); 2 - - N S h 14.5.
liquid. Thus, for the fluorocarbon liquid we used. it was 15 min; for octane, 5 min. The liquid volume in the device should be such that the meniscus with zero reading is in the lower part of the scale. At least two measurements are made for determining the position of the meniscus. After establishing the zero reading the dish containing the specimen is placed in the apparatus, the dish is fastened by a loop to the hook on the stopper of the apparatus and the
V o h n n e t r i e metb.od for s t u d y of swellir~g and syneresis
543
m e a s u r e m e n t r e p e a t e d as described. Tlte v o h u u e of the e m p t y dish wittl the lid and loop is p r , viously d,'t,ermit~ed by a similar method. Swelling of the sample is effeeged in tim dish used. for d e t e r m i n i n g the volume, or separately, if t.he specimen is fairly strong. A f t e r swelling, the '.~peeimen or dish w i t h the specimen al'~ freed from free solvent residues b y a. filter paper or b y light, agitation. The v o l u m e of the swollm~ spectral,it is t h e n determiIled as described. "Whei~ select, ixtsa tt~e inert liquid to d e t e r m i n e the specimen v o l u m e air o p t i m u m eombinaLieu of volatility rout. viscosity should be a i m e d at. The use of tfighly velar, lie liquids involw,s errors of lnoasul',?lnt'nt; witlt highly viscous liquids a e o n s t a n t position of the Ineniseus is slowly ~mhiow'd, Wll,,n w a t e r or m m t h e r liquid ~aqt]t a leith surface tension is used for tire vohun,' nleasurem~'nt, to facilitate overflow from one eontainer into another, addition of surt'~m,~ act, ire substmmes is r e c o m m e n d e d . V~hel~. working wittt protein ~els t h e seh:el.,ion of llte iluq't liquid involves certain dittieulties sil~.ee these, st~})slane('s ttavt~ a solubilizing ability in relation to tmn-polar compounds, ineluding parafl3nie h y d r o c a r b o n s [11]. I t is kImwn glint m i x t u r e s of fluorocarbons and hydrocarbons show considerable posit|w> doviation~ from the R a o u l t law [12, 13]; fluorocarbons are v e r y sparingly soluble in w a t e r [12 I. W e tltorefore assumed t h a t fluorocarbons and possibly fluoroeltloroearbons are n o t sohttfilized, by proteins in praetiee. Tim a s s u m p t i o n was eol~firmt'd ext)oriment.ally b y th,, absence of ~lny ~Vei~llt v a r i a t i o u of dishes containing the gelatin gel after r e t e n t i o n for a wt~ek in a thtorocarbon liqui(l and in F r e o n - l l 3 . Therelbre, to determi~w (.lu! d(,gt'e(~ of swelling of protei~ gt.is w~' used a fluorocarbon liquid of tim following properties: r~0: : 10.6 eS; p~0 := 1"98; the iniiial |)oiling 1)oint aa 10 m m H g was tfigher t h a n 70 °, solid|fleet, ion 1)oint below --52:'. W e not(, tllai, waL(,r solubility its. tltese liquids is also \'t,ry low: 0-004 [~'4 for I~'t'('on - 1 [3 aL 20 ° [14], of the order of 0.001 }{ for fluoroearlion liquids [121. As aI~ e x a m p l e of the m e t h o d we deseribe resttlts of doi.erminillg the degree of swelling in w a t e r of p h o t o g e l a t i n e gels purified b y L~q)us. Prior to doLernfination the gel specimens we're l'e~airted at 10 :[_ 1.5 ~for 5 days. Swellil~g in 200-fold \vat.or excess was carried, out at, the siuno t e m p e r a t u r e for 6 da.ys. In|tin,1 eo~eentration, '~{; ])egree of swelling,
2"5 5'11 7"5 10"0 0.57:! 0.03 0.86 ! 0.02 0 . 9 8 ± 0 . 0 2 1 ' 1 1 ~ 0 - 0 1
3~he t].~wiee described can also be ust.d to invest,|gab; ~he syuer~,sis kitmt, ies of gels. The liquid in wlfieh syirert~sis is carried out s}}.otll~l not dissolve the sytieretie liquid a n d should be h e a v i e r ttmlt t]fis liquid. The speeilnen v o l u m e is d~termined as described, in a separat, e device, or in a d,~vice in which syneresis is efl'eeted if the liquid contained, is fairly inert in relatimi l,o the gel. The. liquid in the device should be sat, u r a t e d wit.It tim solvent c o n t a i n e d by the g~d. Syueresis is o b s e r v e d b y measuring tt~e v o l u m e of the synoretie liquid s e p a r a t e d and (if the liquid in wtfieh the lneasuremel}.t is earn'led out is not volatile) the gel volume. I t is only essential to eI~sttre t h a t the drops of the synoretie liquid do not adtmre to the specimen, dish, or t h e wails of the device. W e used F r e o n - i i 3 to investig~tte syn,r~sis oI( prot, eiu gels. It.s a d v a n t a g e over fire ttuorocarbon liquid is it,s lower viscosity; however, owing to l.tt, increased volatility of F r e o n (b.p. 48 °) the resull, s o[' d e t e r m i n i n g the v o h u n e of g,,1 samples are less reliable,
CONCLUSIONS (1) A mel~hod was d~velep~,d 1,o del,ermine tht~ volumel,rie dogree of swollilxg of substanee~ wit,h differm~t nleehal~iea.i prol)erties. (2) I t is p o i n t e d o u t ttl~t.l~ ftuoroeal'boltS or tluoroehloroear|mns should be used as i~ort liquids for t h e s t u d y of prot, ebz gels. (3) A met,hod is proposed for the invcst.igatiou of sy~ercsis of gels.
Translated by E . SEMERE
544
Y z . YE. BHAUDO and V. B. TOLSTOGVZOV
REFERENCES 1. 2. 3. 4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14.
I. N. YERMOLENKO and M. I. MAZEL', Kolloid. zh. 14: 390, 1952 Ya. S. PRZHEBOROVSKII and V. :K. TILE, Kolloid. zh. 16: 304, 1954 B. DOGADKIN, K. PECHKOVSKAYA ~nd L. CHERNIKINA, Kolloid. zh. 8: 31, 1946 R. V. UZINA, K a u c h u k i rezina, No. 2, 8, 1941 B. A. DOGADKIN, Kolloid. zh. 21: 762, 1959 O. N. GRIGOROV, I. F. KARPOVA, Z. P. KOZ'MINA and D. A. FRIDRIKHSBERG, Rukovodstvo k prakticheskim zanyatiyam po kolloidnoi khimii (Practical Work on Colloid Chemistry). p. 196, Izd. LGU, 1955 A. LOTTERMOSER and H. RADESTOCK, Z. angew. Chem. 40: 1506, 1927 I. I. ZHUKOV, S. L. TALMUD and V. A. ZIL'BERMAN, Sintetiche~kii ka~olat~k, N$. 6, 21, 1935 I. I. ZHUKOV and F. M. SIMKHOVICH, Kolloid. zh. 1: 11, 1935 T. P. TYAZHELOVA, Kolloid. zh. 1: 90, 1935 G. P. YAMPOL'SKAYA, V. N. IZMAILOVA, V. A. PCHELIN and A. V. ¥OLYNSKAYA, Vysokomol. soyed. 7: 1956, 1965 (Translated in Polymer Sei. U.S.S.R. 7: 11, 2147,1965 Ftor i ego soyedineniya (Fluorine and its Compounds). Edited by G. Simons, vol. 1, p. 315, Izd. inostr, lit., 1953 F t o r i ego soyedineniya, Edited by G. Simons, eel. 2, p. 328, Izd. inostr, lit., 1956 G. A. YAKOVKIN, Freony. Svois~va i primenenie (Refrigerants. Properties and Applioation), p. 25, Goskhimizdat, 1956