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`
`
`
`
`
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`

`

`
`
`\r
`
`.
`
`Structure Formation in Gelatin Films
`
`1. H. COOPES
`
`Research Laboratory, Kodak (Australasia) Pry. Ltd., Cobm-g,
`Victoria, Australia.
`
`3.
`
`_
`
`*'
`
`ABSTRACT. The kinetics of structure formation during the Setting
`and drying of gelatin films have been studied by means of optical
`rotation measurements on gelatin films prepared under widely varying
`conditions. The separate contributions of helical conformation and
`planar orientation to the optical rotation are resolved by analysis of
`the data obtained from measurements with the films tilted at various
`angles to the incident beam of polarized light.
`The studies are made with model systems consisting of comparatively
`thick films cast on glass plates and monitored during drying at con-
`trolled temperature and humidity.
`The effects of gelatin hardeners on structural changes are considered.
`The relationship between the structure of films and their physical
`properties is discussed.
`'
`
`INTRODUCTION
`
`The influence of drying conditions on the physical and sensito-
`metric properties of photographic films is well established. However,
`the effects are known mainly in terms of empirical, use—oriented
`criteria, which are only of very limited value in assisting an under-
`standing of the molecular processes involved. For studying the
`mechanism of structural change, optical rotation appears to be a
`suitable property, since gelatin possesses a high level of optical
`activity which is very sensitive to environmental change.
`The molecular conformations of gelatin and collagen in solution
`have been thoroughly studied in recent years,1s2 but the structure
`of gelatin films has received less attention. Infrared dichroism3-4 and
`X~ray diffraction” indicated collagen-fold formation but the
`absence of the long-range order of native collagen. In gelatin sols
`and gels the optical rotation reflects the level of c0ll21gen—fold form-
`
`121
`
`
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`‘
`
`l
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`»
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`122
`
`1. H. eoorss
`
`ation, but in gel-dried films the optical rotation is much highcr.6-3:9
`Hot-dried (45°) films show no structural order. 1° The optical
`rotatory dispersion curves of gelatin films are not significantly diff-
`erent from those of solutions, except in the magnitude of the
`rotation," suggesting that there is no significant difference in
`
`molecularconformation;thisconclusionissupportedbyMacsuga’s
`
`study of thermal transitions in gelatin. 12 However, it has been
`observed that in films the major axis of the gelatin chains tends to
`be coplanar with film plane,”’ '4 and this would appear to be the
`structural factor which causes the increased rotation.
`
`The degrees of helical conformation and planar orientation both
`have an important influence on the properties of a gelatin film. The
`setting of a gelatin gel invoives the formation of the triple chain
`collagen—fold structure. 15 Slow setting of the gel leads to the form-
`ation of larger helical segments. Gels formed under such circum-
`stances have greater thermal stability than have gels formed by
`rapid chilling. 16:” During the (_l1‘yi11g of a gelatin film the helical
`structure will remain intact if the temperature remains below the
`melting temperature of the helical segments. Under these conditions
`as the film contracts vertically, the major axes of the helical segments
`tend to become oriented in the plane of the film.
`
`.
`
`With gel—dried films, the closer the drying temperature is to the
`gel melting temperature, the higher the gelatin concentration at
`which the helical segments are formed. Hence the films dried at
`higher drying temperatures will swell less than those dried at lower
`temperatures, when immersed in water. The degree of orientation
`in the plane of the film should also be less. However, if the gel is
`allowed to mature before drying, it shows a much greater degree of
`swell than does a film dried immediately after coating.13- The
`proportion of the gelatin molecule involved in the triple chain
`helical structure may not he very high, even in well matured gels.
`Jolleylg estimates it is about 20%.
`In practice it is necessary to increase the thermal stability of
`photographic films by the use of hardeners. Hardening appears to
`improve the stability of the helical structure in aqueous solutions.
`However, if substantial hardening occurs prior to helix formation,
`the latter process may be significantly inhibited.” Thus the effect
`of hardening on film structure depends on the relative kinetics of the
`helix-forming and hardening processes.
`A method of distinguishing between the contributors of the
`helical structure a.nd planar orientation of collagen films has been
`proposed.” It is not self-evident that this approach is directly
`
`20
`
`i
`
`*3
`l
`
`*
`
`I
`‘
`
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`
`
`. STRUCTURE FORMATION IN GELATIN FILMS
`
`1 23
`
`-
`
`applicable to gelatin films, which have a less complete and less
`_ Stable helical structure. Consequently it was necessary to first of all
`test the theory with a model system consisting of a gelatin gel which
`was allowed to attain maximum helical structure prior to drying.
`The theory was then applied to films prepared under somewhat more
`realistic conditions, with concurrent helix formation and drying.
`These experiments were performed with model systems consisting
`of thick, slow drying films.
`
`EXPERIMENTAL
`
`Preparation of films
`
`The thick films were cast on glass plates which were converted
`into moulds by attaching waterproof adhesive tape to the edges.
`Into the mould 10 ml of a 50 g per litre gelatin solution was
`poured. A first extract, lime processed bone gelatin, pH 5-9,
`isoionic point 5-0, was used. Drying was carried out either under
`room conditions (20°C, 50% RH) or in a Kottermann Climatic
`Test Cabinet which could be adjusted to provide a wide range of
`temperature and humidity.
`
`Optical rotation measurements
`
`The optical rotation measurements were performed with a jasco
`ORD/UV5 Spectropolarimeter. The wavelength at which the
`measurements were made was 450 nm, as the rotation at lower
`wavelengths was too high to be measured by the instrument. The
`films were left on the glass plates during optical rotation measure-
`ments, as the plates showed no optical activity or absorption at the
`wavelength used.
`
`RESULTS AND DISCUSSION
`
`In order to establish the relationship of the contribution to
`optical rotation of the helical structure and its orientation it is
`desirable to obtain independent values of these separate contributions.
`Yannas et ul.22 in their studies of collagen, could do this readily,
`since the collagen molecules form stable helical structures. The
`optical rotation of dilute solutions was measured, which provided
`an indication of the helical contribution, and the degree of orienta-
`tion of the major axis of the helices in the film plane was determined
`by X-ray diffraction measurements.
`
`
`
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`

`124
`
`I. H. coomas
`
`With gelatin the problem is more difficult. At practical concen-
`trations for co_ating (e.g. 50-100 g per litre) only very incomplete
`collagen helix structures can be formed, and these tend to rearrange
`during drying. This difficulty was surmounted by maturing the gel
`in,a sealed container for 24 h, by which time the maximum degree
`of helical structure had been formed. Thus optimum conditions for
`stability of the helical content during drying were ensured. The
`film was set and dried at room temperature (20°C). Facilities for
`X-ray studies were not available, so it was necessary to deduce the
`degree of orientation entirely from optical rotation data. The
`optical rotation values obtained were as follows:
`
`
`
`'7:
`
`laiilr 2 lolzzlr 2 T1205‘)
`ll
`
`l&a3 lr
`
`+ l506°
`
`where [can ],. and [0522 1, are the rotations measured along the two
`molecular axes perpendicular to the axis of the helix, and [0333 ],.
`is the rotation measured parallel to the helical axis. The subscript
`1' indicates that the specific rotation values have been corrected for
`refractive index differences, and changes in path length and in the
`angle to the plane of polarized light which occur at different angles
`of incidence. The average angle of the helices to the plane of the
`film was found to be 18°.
`23
`Full details of this work have been published elsewhere.
`The next obvious step was to observe the differences in structure
`which arise when setting and drying occur concurrently. A film
`was cast on a glass plate and allowed to dry out under room con-
`ditions over a period of 24 11. The optical rotation of the film at
`various tilt angles was measured (Table 1]. Yannas et lfIll.22 proposed
`' the following relationship between optical rotation and angle of
`incidence:
`
`056 2051+
`
`(0533 E‘-‘til Sinz 9
`
`where 0 is the angle of incidence and 0:9 is the optical rotation of
`the film for a given angle- By means of this equation and the data
`given in Table 1, the following optical rotation values were obtained:
`
`I
`"E
`
`l.°‘11lr : lazzl,» = '* 508°
`11
`
`[053-3],
`
`+ 650°
`
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`
`' STRUCTURE FORMATION IN GELATIN FILMS
`
`Table 1
`
`Specific rotation of film at various angles to the incident beam.
`
`
`
` # ffldlr 0
`
`0
`512°
`10°
`473°
`20°
`458:
`30°
`3530
`45°
`2360
`
`64 50°
`
`Note. — [O53], is the corrected specific rotation. 19 is the angle of incidence of
`the light beam.
`
`However this treatment assumes that the major axis of the helices
`is completely coplanar with the film plane. In this case the rotation of
`the film when perpendicular to the incident beam is equal to [0511 1,.
`' "However if the helices are oriented at an average angle qb to the film
`plane, the rotation of the film is given by:
`
`lolfilmlr 2 [G511]? C052¢"+ [U533]? Sing‘?
`
`(2)
`
`Thus if the correct values of [(211], and [C233 ],. are known, a value of
`(la may be obtained from equation
`Unfortunately some approx-
`imations must be made at this stage. The contribution of [0133 1, to
`the measured rotation of the film is comparatively small, and the
`value quoted above may be accepted. In the previous work” a value
`of [call ], was obtained from the specific rotation of the set gel,
`prior to drying, by means of the expression:
`1%], 2
`
`(3,
`
`In the present work, in order to obtain the equivalent value, the
`dried film was soaked in water. Chilled water at 10°C was used for
`the soaking, in order to minimize changes in the helical content. The
`soaking was continued until the measured optical rotation stabilized.
`The corrected specific rotation obtained by this means was — 2?5°.
`Subsequently the film was dried again and the dry film was found to
`have a rotation identical with the original value, indicating that the
`level of helical content had not changed.
`In the swollen film the orientation of the helices will not be
`completely random, since the helical segments developed while the
`
`
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`

`126
`
`I. H. cooras
`
`film was drying. The gelatin concentration fell to 36 g per litre. The
`degree of orientation remaining in the swollen film is therefore quite
`low, and Eq.
`may be applied.
`If ['_o:gcl],. is m 2?5° and [£133], is + 650° then from equation (3),
`[all], is — 737°. Equation (2) then becomes
`
`—- T3? coszgiv + 650 sinzgb =- 512
`
`and hence qt: is 24°.
`Thus it is possible to obtain estimates of the helical content and
`degree of orientation solely from optical rotation measurements,
`although at the cost of some rather large approximations.
`A series of films were dried in a Kottermann cabinet at various
`temperatures over the range 2[}°—50°C (50% relative humidity in
`all cases), and the optical rotation and concentration monitored
`during the drying process. It was not feasible to measure the rotation
`over the full range of angles, so measurements were made only at D
`and 45° to the incident beam. The pattern of the variations in
`rotation may be appreciated by comparing the data obtained at 1, 5
`and 24h after the setting and drying of the film commenced (Table 2).
`At the higher temperatures the gel did not set for several hours,
`hence the first reading was made at the earliest practicable time.
`This series of experiments was repeated with gelatin containing 1%
`formaldehyde (w/w gelatin) (Table
`There are some anomalies in the data, but at the lower
`temperatures the general trend is for both the actual rotation values
`and the ratio of the rotation measured with the film perpendicular
`to the incident beam
`to that measured with the film inclined
`at 45° ([0-45 1,.) to increase. At the higher temperatures (40° and 50°C}
`
`-
`
`'
`
`-
`
`'
`
`The presence of the hardener appears to have little effect.
`
`the values of [(20], and the ratio [(10],/[£345], become much lower.
`
`CONCLUSION
`
`The resolution of optical rotation into helical and orientation
`contributions can be carried out with reasonable accuracy when the
`degree of helical content and orientation are high. However at lower
`levels of structural order the approximations required become so
`high as to render the method of Yannas et all." inapplicable. The
`ratio [O£9],-/[C|£45 ], may then be used as a function of the degree of
`orientation, although it is also dependent to some extent on the
`actual magnitude of the specific rotation values.
`
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`
`
`STRUCTURE FORMATION IN GELATIN FILMS
`
`1 27
`
`Table 2
`
`Optical rotation data for films dried at various temperatures. N o hardening.
`_._._
`
`'I_'emp.
`Time
`Comm.
`9
`— [(21,
`[o:0],.{[o145],,
`1°C)
`00
`(am
`
`
`_
`
`20
`
`25
`
`30
`
`1
`5
`
`24
`
`1
`
`24
`
`1
`
`5
`
`24
`
`50
`31
`
`390
`
`55
`
`390
`
`50
`
`140
`
`397
`
`4050
`00
`45
`00
`45
`
`00
`45
`
`00
`45
`
`00
`45
`00
`45
`
`00
`45
`
`372:
`20
`558:
`470
`332:
`455
`
`334:
`2410
`
`325:
`407
`
`335:
`350
`595°
`—.—
`
`795:
`473
`
`3115?:
`4%‘,
`512
`3
`35
`24
`914
`00
`5653:
`
`45
`322
`
`1.152
`1-137
`
`1933
`
`1-386
`
`2.120
`
`1-100
`
`__
`
`2-051
`
`1-792
`1-743
`
`40
`
`3
`
`779
`
`249:
`00
`204
`45
`24
`923
`00
`251:
`1-233
`45
`193
`
`
`1-220
`
`50
`
`5
`
`24
`
`933
`
`963
`
`00
`45
`00
`45
`
`95:
`115
`33:
`103
`
`0-335
`
`0-315
`
`One surprising result of the model experiments was the lack of
`any marked effect by the hardener, in contrast to the results obtained
`with previous studies. This does not necessarily mean that hardening
`does not significantly affect the structure of machine coated films,
`since the conditions are very different to those for the model systems.
`Studies of machine coated films will be needed if 3 more detailed
`
`picture of the structural changes occurring in the drying process is
`to be obtained.
`
`
`
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`

`128
`
`I. H. COOPES
`
`Table 3
`
`Optical rotation data for films dried at various temperatures, and
`containing 1% formaldehyde (wfw gelatin).
`
`Tgmp,
`Time
`Concn.
`0
`— [01],
`[£20],-1' [0l4s1r
`( G}
`(h)
`(311)
`
`
`20
`
`30
`
`40
`
`50
`
`2
`
`5
`
`24
`
`2
`
`5
`
`24
`
`5
`
`24
`
`6
`
`24
`
`59
`-
`73
`
`390
`
`04
`
`104
`
`390
`
`745
`
`394
`
`353
`
`923
`
`00
`45
`on
`45
`on
`45
`
`00
`45
`0 D
`45
`0 O
`45
`
`00
`45
`00
`45
`on
`45
`00
`45
`
`References
`
`397:
`259
`525°
`__
`342:
`505
`
`334:
`350
`445:
`313
`744:
`419
`
`220:
`249
`230:
`232
`112:
`133
`113:
`135
`
`1-476
`
`—
`.
`1-66?
`
`0-954
`
`1-422
`
`1-773
`
`0.333
`
`0-991
`
`0-342
`
`0-353
`
`'
`
`_
`a‘
`
`=
`
`Z
`
`1. Veis, A., “The Macromolecular Chemistry of Gelatin,” Academic Press,
`New York and London (1964}, pp. 117, 267.
`Von I-Iippel, P. H., In “Chemistry of Collagen”. (G. N. Ramachanclran, ed.],
`Academic Press, London and New York (1967), p. 253.
`Ambrose, E. G. and Elliott, A., Proc. Roy. Soc. (London), A206, 205
`(1951).
`Ambrose, E. G. and Elliott, A., Proc. Roy. Soc. (London), A208, 75 (1951).
`Katz,]. R., Rec. Tran. Ch£m., 51, 385 (1932).
`Robinson, C. and Bott, M. _I., Nature, Lond., 163, 325 (1951).
`Ramachandran, G. N ., In “Recent Advances in Gelatin and Glue Research”
`(ed.}, G. Stainsby, Pergamon Press, New York (1958), p. 32.
`Cohen, C.,f. Biopkys. Biochem. Cytol, 1, 203 (1955).
`. Coopes, I. H.,__I, Polym. Sci, A-I, 6,1991 {I968}.
`
`
`
`‘°9°?'-‘S'‘'.‘’‘:‘‘‘‘
`
`Mylan v. Qualicaps, |PR2017—00203
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`S‘1‘RUC.TURE FORMATION IN GELATIN FILMS
`
`1 29
`
`10,_]ohnson, M. F.,F::llows, W. D., Kamm, W. 1)., Miller, R. S., Otto, H. O. and
`Curme, H. G., In “Photographic Gelatin” (::d.), R. G. Cox, Academic Press,
`London and New York (1972), p. 99.
`11. Coopcs, I. H.,j_ Poly. Sal, A-1, 9, 3683 (1971).
`12_ Macsuga, D. D., Biopolymers, 11, 2521 (1972).
`13-. Bradbury, E. and Martin, C.,Na£ure, Land, 168, 837 (1951).
`14_ Bradbury, E. and Martin, C., Pmc. Roy. Soc. (Landon)., A214, 183
`.
`1952 .
`15. £7'lory,)P.J. and Weaver, E. S.,}'. Amer. Chem. Soc., 82, 4518 (1960).
`16. Boedtker, H. and Doty, P., Phys. Chem., 58, 968 (1954).
`17, Eldridgc,_I. E. and Ferry,_]. D.,j. Phys. Chem, 58, 992 (1954).
`I8._]opling, D. W., Appl. C:'1em., 6, 79 (1956).
`19.JolIcy, E.,Photog‘r. Sci. Eng.,14,169(197(}}.
`20. Sterman, M. I)., Faust, M. A., Geneva, D. J., Curme, H. G. and Johnson,
`M. F., In “Photographic Gelatin” (ed.], R. J. Cox, Academic Press, London
`and New York (1972), 13.133.
`21. Coopes, I. H.,
`Polym. S.-:i., A-1, 8,1793(1970).
`22, Yannas, I. V., Sung, N. H. and Huang, (3.,
`Phys. Chem., 76, 2935 (1972).
`23. Goopes, I. H., J. Palym. Sci, In press.
`
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