.
`
`,
`
`The thermotropic phase behavior of ascorbyl palmitate: an infrared spectroscopic study1
`
`HELMUT SAP PER,^ DAVID G. CAMERON, A N D HENRY H. MANTSCH
`Division of Chemistry, National Research Council of Canada, Ottawa, Ont., Canada KIA OR6
`Received March 27, 1981
`
`I
`
`I
`
`1
`
`I
`
`HELMUT SAPPER, DAVID G. CAMERON, and HENRY H. MANTSCH. Can. J. Chem. 59,2543 (1981)
`The infrared spectra of aqueous potassium ascorbyl palmitate were studied as a function of temperature using Fourier transform
`infrared techniques. From a light scattering experiment the Krafft point of 0.1 M potassium ascorbyl palmitate was determined to be
`48°C. The temperature-induced changes in infrared spectral parameters such as frequency and bandwidth characterize this Krafft
`point as a phase transition from a conformationally ordered, poorly hydrated solid phase, to an isotropic micellar phase. The phase
`transition of this "pseudosoap" occurs over a temperature range of about 1O0C, reflecting the progressive hydration of the solid upon
`micellization, a behavior typical of surfactants such as soaps.
`
`HELMUT SAPPER, DAVID G. CAMERON et HENRY H. MANTSCH. Can. J. Chem. 59,2543 (1981).
`En utilisant les techniques infrarouges de la transformation de Fourier, on a etudie les spectres infrarouges des solutions aqueuses
`de palmitateascorbyle de potassium en fonction de la temperature. A partir d'un experience de dispersion de la lumiere, on a
`determine que le point Krafft pour une solution 0,l M de palmitateascorbyle de potassium est de 48°C. La temperature provoque des
`changements dans les parametres spectraux tels la frkquence et la largeur des bandes qui caracterisent ce point Krafft en tant que
`transition de phase i partir d'une conformation ordonnee, ma1 hydratee dans la phase solide, jusqu'a une phase micellaire
`isotropique. La transition de phase de ce "pseudo" savon s e produit au dessus d'un intervalle de temperature de 10°C environ
`refletant ainsi I'hydratation progressive du solide lors de la formation des micelles, c'est un comportement typique des savons.
`[Traduit par le journal]
`
`Introduction
`Ascorbyl palmitate, the C-6 monoester of vita-
`; min C with palmitic acid, was first synthesized by
`Swern et al. in 1943 (1) with the intention in mind
`to combine the reductive properties of ascorbic
`I acid with the lipid solubility of a fatty acid. This
`' derivative of ascorbic acid is actually a lipophilic
`I vitamin C and has been shown to be as biologically
`1
`active as its original hydrophilic counterpart (2, 3).
`j Since both vitamin C and palmitate esters are
`natural food ingredients, ascorbyl palmitate is now
`widely used in the food industry as a natural
`1 preserver of oils and fats (4, 5).
`1
`The effectiveness of ascorbyl palmitate in pro-
`'
`tecting against oxidation is similar to that of other
`commercial lipid antioxidants, such as a-tocopherol
`(vitamin E). In fact, it has been shown that toco-
`pherols become more powerful antioxidants when
`used in combination with ascorbyl palmitate (5, 6)
`and this effect can be further enhanced by addition
`of lecithin (3,6). The synergism between vitamin C
`and vitamin E when used as antioxidants in bio-
`membranes has been recently confirmed by pulse
`radiolysis measurements (7). Thus, the chemical
`behavior of ascorbyl palmitate as a lipophilic
`analogue of vitamin C is of interest not only in view
`of its value as a food additive, but also with respect
`
`'
`
`'NRCC No. 19405.
`2NRCC Visiting Scientist. On leave of absence from the
`Institute of Biophysics, Justus Liebig Univenitit, D 6 3 W
`Giessen, West Germany.
`
`1
`
`to its possible biological role as a lipophilic vehicle
`of vitamin C.
`Despite its importance to the food industry,
`there are few data in the literature regarding the
`structure and physicochemical properties of ascor-
`by1 palmitate or its salts. In principle, one would
`expect these properties to reflect both those of
`vitamin C and those of a fatty acid ester. In
`addition, the salts of ascorbyl palmitate (at neutral
`pH ascorbyl palmitate is always a salt) are am-
`phiphilic molecules and structurally similar to
`soaps such as the n-alkanoates or the n-alkyl
`sulphates. A common property of such surfactants
`is that above a certain temperature, referred to as
`the Krafft point (8-14), the solubility of the surfac-
`tant in water increases dramatically. The salts of
`ascorbyl palmitate exhibit the same behavior. Using
`a light scattering technique and Fourier transform
`infrared spectroscopy we have studied the Krafft
`point transition, and report on the data herein.
`
`Experimental
`Materials and Sample Preparation
`Ethyl palmitate was from Sigma, St. Louis, MO and sodium
`ascorbate from Merck, Darmstadt, Germany. Ascorbyl palmi-
`tate (06-palmitoyl-L-ascorbic acid) was a commercial product
`from the U.S. Biochemical Corporation, Cleveland, OH. The
`potassium (or sodium) salts were prepared from equimolar
`amounts of ascorbyl palmitate and KOH (or NaOH), each
`dissolved in the minimal amount of absolute ethanol. The salts
`which precipitated upon mixing the two ethanolic solutions
`were filtered off, washed with absolute ethanol, and dried under
`vacuum.
`
`0008-4042/81/162543-07$01.00/0
`01981 National Research Council of CanadalConseil national de recherches du Canada
`
`Can. J. Chem. Downloaded from www.nrcresearchpress.com by 38.117.177.233 on 10/21/15
`
`For personal use only.
`
`Par Pharm., Inc.
`Exhibit 1011
`Page 001
`
`

`
`CAN. J. CHEM. VOL. 59, 1981
`
`Samples of aqueous 0.1 M (4.5 wt.%) potassium or sodium
`ascorbyl palmitate were obtained by warming the solid salt in
`the corresponding amount of D 2 0 to about 50°C in a closed flask;
`above 40°C the opaque suspension becomes a clear solution
`which upon cooling turns into a uniform curd at room tempera-
`ture. The pD (meter reading) of the clear solution was 8.1. Thin
`samples (15 pm thick in CaF, cells) of the curd were prepared
`for infrared spectroscopy according to the detailed methods re-
`ported elsewhere (15, 16).
`
`Spectra and Data Processing
`Infrared spectra were recorded with a Digilab FTS-11 Fourier
`transform infrared spectrometer equipped with a mercury
`cadmium telluride detector (Infrared Associates, New Bruns-
`wick, NJ). Typically, six hundred scans were accumulated and
`co-added using a maximal optical retardation of 0.5cm and a
`moving mirror velocity of 0.6cmIs. The resultant interfero-
`grams were triangularly apodized and Fourier transformed with
`one level of zero filling to yield a resolution of -2cm-'. The
`spectra of solid samples were recorded as KBr pellets (1 mg
`sample in 100 mg KBr) of 1 cm2 surface area. Temperature
`control was achieved by using a hollow cell mount thermostated
`by a flow of ethanol-water which gives a temperature stability of
`better than f O.l°C (17). The temperature was monitored with a
`thermocouple located against the cell window, and tempera-
`tures were continuously recorded by a Newport digital py-
`rometer equipped with a printer. The spectrometer computer
`controlled the complete operation of recording a spectrum,
`printing and incrementing the temperature, waiting for tempera-
`ture equilibration, and then repeating the sequence (18).
`Frequencies were measured by determining the center of area of
`the top five data points of a peak. Bandwidths were obtained by
`digitally subtracting a linearly interpolated baseline extending
`from 3000 to 2800cm-' and computing the widths- at % of the
`peak height. The precision (and reproducibility) of measuring
`temperature-induced changes in frequency and bandwidth is
`better than f 0.05 cm-I (19).
`The light scattering experiment was performed by measuring
`the light transmitted at 580nm through a 1 mm quartz cell
`containing the system under study. A Cary 219 uv-vis spec-
`trophotometer equipped with the automated temperature reactant
`accessory and a coupled chart drive was employed.
`Results and Discussion
`General Thermal Properties
`At room temperature potassium ascorbyl pal-
`mitate (APK) and sodium ascorbyl palmitate (APNa)
`are insoluble in water except at extremely low
`concentrations. However, on heating a water-APK
`(or water-APNa) mixture, the solubility increases
`rapidly above a certain temperature, and a clear
`solution is formed. If the solution is then cooled it
`solidifies as an opaque curd.
`This behavior is well known in ionic surfactant-
`water systems (8-15). The curd is a semicrystalline
`mesophase in which the surfactant molecules form
`lamellar structures. According to the degree of
`
`hydration and three dimensional ordering it may be
`referred to as a gel (well hydrated and unilamellar)
`or a coagel (poorly hydrated and multilamellar)
`(20). While X-ray measurements are required to
`unequivocally determine if at a given temperature a
`system forms a gel or a coagel, the extreme opacity
`observed in the aqueous ascorbyl palmitate salts is
`indicative of a coagel system.
`The temperature at which the transition occurs
`from the coagel (or gel) to the micellar phase is
`generally referred to as the Krafft point or as the
`"melting" point of the hydrated solid surfactant.
`However, the physical meaning of the solubility
`curve is different from that of ordinary solid-solute
`equilibrium curves and a stricter definition of the
`Krafft point is that of the concentration at which
`the coagel, monomers, and micelles are in equilib-
`rium (10).
`The Krafft point is dependent on the concentra-
`tion and the type of counterion. In the case of 0.1 M
`potassium and sodium palmitate soaps, Krafft
`points of 30°C and 60°C respectively have been
`reported (21). Using light scattering techniques we
`find Krafft points of 48°C and 44°C for 0.1 M APK
`and APNa respectively. That is, the influence of
`the counterion is much smaller and, contrary to
`the behavior of the corresponding palmitate soaps,
`that of sodium ascorbyl palmitate is lower than that
`of potassium ascorbyl palmitate.
`
`Infrared Spectra
`The thermotropic properties of potassium as-
`corbyl palmitate were investigated in detail by
`infrared spectroscopy. The 1800-1300 cm-' region
`of the spectra of the anhydrous solid, the ordered
`coagel, and the isotropic micellar phase of APK are
`shown in the bottom segment of Fig. 1. The top
`segments show spectra of sodium ascorbate and
`ethyl palmitate, accurate frequencies are given in
`Table 1. It can be seen that, in general, the
`spectrum of APK is comprised of the summation of
`the palmitate and ascorbate spectra, particularly in
`the micellar phase.
`Of more significance to this study are the differ-
`ences between the spectra of the solid, the coagel,
`and the micellar phases of APK. Considering first
`the spectra of the solid and the coagel (Fig. 1, C and
`B respectively), it can be seen that the C=O
`stretching bands at 1744 and 1735 cmL1 are broad-
`
`Can. J. Chem. Downloaded from www.nrcresearchpress.com by 38.117.177.233 on 10/21/15
`
`For personal use only.
`
`Par Pharm., Inc.
`Exhibit 1011
`Page 002
`
`

`
`SAPPER ET AL.
`
`2545
`
`broad band at 1660 cm-I in the spectrum of the
`solid gains intensity and has two components in the
`coagel (see Table 1). Even greater changes are
`observed in the strong C=C stretching band near
`1600cm-l. Formation of the coagel results in a
`strong narrow band at 1574cm-I, while a weak
`band near 1600cm-I is retained. The shift to lower
`frequency of the main C=C stretching band sug-
`gests a reduction of the double bond character of
`the C(2)=C(3) bond. The sensitivity of this bond to
`the state of the ascorbate moiety has already been
`demonstrated. Formation of the sodium salt shifts
`the C=O stretching band from 1753 to 1702 cm-I ,
`and the C(2)=C(3) stretching band from 1670 to
`1593 cm-I (23), and increases the C=O and C(2)=
`C(3) double bond lengths by 0.017 A and 0.035 A
`respectively (24).
`The interaction resulting in the shift of the
`C(2)=C(3) stretching band to 1574cm-I is specific
`to the coagel and must result from aparticular intra-
`or intermolecular interaction, possibly involving
`partial hydration. At this point, we are unable to
`assign the individual C=C stretching bands, other
`than to give them the general classification of
`double bond stretching vibrations associated with
`the ascorbyl moiety.
`The spectrum of the micellar phase is somewhat
`simpler than that of the coagel. The broad bands in
`this region are typical of those encountered in
`solution spectra of materials capable of engaging in
`extensive hydrogen bond formation, while the
`bandshapes indicate an enhanced mobility of the
`particular functional groups. Nonetheless, discrete
`absorptions are still present, as suggested by the
`asymmetry of the C=O stretching band contour,
`and demonstrated by the distinct frequency max-
`ima found after Fourier self-deconvolution (see
`Table 1).
`Another vibrational mode of interest is the CH,
`scissoring band around 1468 cm-I , which is used
`extensively for the characterization of the packing
`ened on formation of the coagel, suggesting some
`degree of hydrogen bond formation with water of
`of acyl chains in solid hydrocarbons, lipids, and
`hydration. Rather more dramatic changes are ev-
`surfactants (25,26). Its observation at 147 1.5 cm-I
`ident in the C=C stretching bands. The weak
`in the coagel is indicative of a triclinic packing of
`TABLE 1. Characteristic group frequencies (in cm-') of potassium ascorbyl palmitate and of selected model compoundsa
`
`FIG. 1 . Infrared spectra in the 1800-1300~m-~ region of
`0.1 M potassium ascorbyl palmitate in D,O at 56°C (A) and at
`20°C (B), polycrystalline APK at 20°C (C), sodium ascorbate in
`D,O (D) and as polycrystalline solid (E), and ethyl palmitate in
`KBr at 30°C (F) and at 8°C (G).
`
`Acyl chain vibrations
`
`Head group vibrations
`
`vas(CH2)
`2917.8
`2917.8
`2923.6
`
`v,(CH,)
`2850.3
`2850.3
`2853.2
`
`1673*
`1678
`
`v(C(ll)=O)
`v(C(l)=O)
`1725*
`1744*
`1735*
`APK, solid (KBr)
`1723*
`1745*
`1733*
`APK, coagel (D20)
`1744* -
`APK, micellar (D20)
`1724*
`- -
`1702
`ANa, solid (KBr)
`- -
`1717
`ANa, solution (D20)
`-
`1744*
`1738*
`2849.0
`2917.0
`EP solid (KBr)
`-
`1742*
`1738*
`2854.1
`2924.9
`EP melt (KBr)
`"Abbreviations: APK, potassium ascorbyl palmitate; ANa, sodium ascorbate; EP, ethyl palmitate. The asterisks indicate frequencies obtained by Fourier
`self-deconvolution (22), a technique which reduces the spectral linewidth of component bands at the expense of the line shape and the signal-to-noise ratio.
`
`v(C=C)
`-1660
`1609*
`1663*
`1599*
`
`-1660
`
`1602*
`
`1594*
`1574*
`1590
`1593*
`1591
`
`Can. J. Chem. Downloaded from www.nrcresearchpress.com by 38.117.177.233 on 10/21/15
`
`For personal use only.
`
`Par Pharm., Inc.
`Exhibit 1011
`Page 003
`
`

`
`CAN. J. CHEM. VOL. 59, 1981
`
`FIG. 2. Infrared spectra in the 1800-1500cm-' region of 0.1 M APK in D 2 0 as a function of temperature. There are 11 individual
`spectra takenin 3.6"C intervals; the top spectrum (with the largest peak height) is at 2WC, and the bottom spectrum (with the smallest
`peak height) at 56'C.
`
`WRVENUMBER, C M - I
`
`.-
`'"-
`
`LO
`E-
`
`. . . .
`
`"
`C ='-
`'"
`I
`
`II v . (D "-
`3 -
`S E
`
`Z
`W
`
`lY
`L
`
`p
`
`5-
`
`Y)
`
`5-
`
`'*
`
`.
`
`a
`
`
`
`. . . . . . . .
`'' 'li
`
`2s
`
`30
`
`' ''
`
`TEMPER~ITURE. "C
`FIG. 3. Temperature-dependence of the frequency (band
`maximum) of the C=C stretching vibration of APK in D,O.
`
`this surfactant below the Krafft point. Such pack-
`ing has previously been observed in solid n-
`alkanoates (27).
`The temperature dependence of the 1800- 1500
`cm-I region of the infrared spectrum of 0.1 M APK
`is shown in Fig. 2. The abrupt changes on transition
`from the coagel to the micellar phase are clearly
`evident. Also demonstrated are continuous spec-
`tral changes prior to micellization. As the temper-
`ature is raised from 20°C to -40°C these changes
`are evident as reductions in the heights of the bands
`at 1745, 1673, and 1574 cm-I. Other bands charac-
`teristic of the coagel (1733 and 1663 cm-l) do not
`change as rapidly. Although there will be a slight
`increase in the monomer concentration in this
`range, these spectral changes are too large to be
`simply accounted for in terms of a decrease in the
`coagel concentration. Therefore we believe that
`these changes result from progressive hydration of
`the palmitate C=O group and of the ascorbate
`moiety prior to micellication.
`In the range 40 to 51°C, the rate of change is much
`greater. This is illustrated in Fig. 3, which shows a
`
`Can. J. Chem. Downloaded from www.nrcresearchpress.com by 38.117.177.233 on 10/21/15
`
`For personal use only.
`
`Par Pharm., Inc.
`Exhibit 1011
`Page 004
`
`

`
`SAPPER ET AL.
`
`1
`1
`
`FIG. 4. Infrared spectra in the C-H
`
`WAVENUMBER, CM- 1
`stretching region of 0.1 M APK in D,O For further details, see caption to Fig. 2.
`
`I plot of the temperature dependence of the position
`: of the maximum of the C=C stretching band
`
`I
`I
`
`contour at 1574cm-l. Although the parameter
`reflects the sum of the changes in the two compo-
`nent bands, it does provide an excellent monitor of
`the rate of change of the bands. It can be seen that
`the frequency varies throughout the entire temper-
`ature range with a maximal rate of change (>90% of
`the entire change) between 45 and 51°C (48 f 3°C).
`Within this range the changes can be attributed to
`the continuously changing concentrations of the
`coagel and micellar phases.
`The C-H
`stretching region of the infrared
`;
`spectrum is shown in Fig. 4. There are four distinct
`I groups of absorption bands in this region, the
`strong antisymmetric and symmetric CH, stretch-
`ing bands around 2920 and 2850 cm-' , respectively,
`and the weaker asymmetric and symmetric CH,
`stretching bands at 2955 and 2873cm-l, respec-
`tively. Also evident is a weak Fermi resonance
`band near 2900cm-'. These bands exhibit a
`
`I
`I
`
`1
`
`thermotropic behavior similar to that of the
`vibrational modes originating in the head group.
`Two distinct types of spectra are observed in
`Fig. 4. The spectra at temperatures below 40°C are
`narrow, indicating relatively low acyl chain mobil-
`ity and the absolute frequencies are characteristic
`of fully extended all-trans acyl chains. As the
`temperature is raised, continuous changes are
`observed prior to the transition to the micellar
`phase. These changes were monitored by plotting
`the temperature dependence of the frequency and
`bandwidth of the symmetric CH2 stretching band,
`and are shown in Fig. 5. Identical plots (not shown
`here) were obtained from
`the
`temperature-
`dependence of the antisymmetric CH2 stretching
`mode.
`Below 40°C the frequency indicating all-trans
`acyl chains in the ordered coagel is almost invari-
`ant, whereas the bandwidth shows continuous
`changes,between 20 and 40°C. This demonstrates
`an increase in the acyl chain mobility within this
`
`Can. J. Chem. Downloaded from www.nrcresearchpress.com by 38.117.177.233 on 10/21/15
`
`For personal use only.
`
`Par Pharm., Inc.
`Exhibit 1011
`Page 005
`
`

`
`2548
`
`CAN. J. CHEM. VOL. 59. 1981
`
`shift to higher frequencies is due to the appearance
`of a large number of gauche conformers in the
`all-trans acyl chains, whereas the increased band-
`width is the consequence of an increase in the
`mobility of the palmitate chains. Both phenomena
`are typical of acyl chain melting phase transitions
`(19, 28, 29) and were also observed at the melting
`point of solid ethyl palmitate (Table 1).
`
`Conclusions
`Aqueous suspensions of potassium and sodium
`ascorbyl palmitate show thermotropic behavior
`typical of dilute ionic surfactant systems. In 0.1 M
`suspensions of potassium ascorbyl palmitate in
`D,O abrupt increases in the solubility are observed
`at 48 & 3"C, as are spectral changes typifying a
`transition from a conformationally ordered, poorly
`hydrated, solid state to an isotropic micellar state.
`The transition generally occupies about 10°C, and
`within this range continuously changing concentra-
`tions of both species are present. This behavior is
`typical of that observed in aqueous n-alkyl carbox-
`ylate ions (soaps). However, unlike in soaps, due to
`the different nature of the head group, the transi-
`tion temperature of the sodium salt of ascorbyl
`palmitate is lower than that of the corresponding
`potassium salt.
`
`1. D. SWERN, A. J. STIRTON, J. TURNER, and P. A. WELLS.
`Oil Soap, 20,224 (1943).
`2. A. M. ABROSE and F. DeEDs. Arch. Biochem. 12, 375
`(1947).
`3. J. DELGA and R. B o u ~ u . Ann. Pharm. Fr. 15,691 (1957).
`4. W. M. CORT. J. Am. Oil Chem. Soc. 51,321 (1974).
`5. H. KLAUI. Int. Flavour Food Addit. 7, 165 (1976).
`6. G. PONGRACZ. Int. J. Vit. Nut. Res. 43, 517 (1973).
`7. J. E. PACKER, T. F. SLATER, and R. L. WILLSON. Nature,
`278,737 (1979).
`8. F. KRAFFT and H. WIGLOW. Ber. 28,2566 (1895).
`9. N. K. ADAM and K. G. A. PANKHURST. Trans. Faraday
`SOC. 42, 523 (1946).
`10. K. OGINO and Y. ICHIKAWA. Bull. Chem. Soc. Jpn. 49,
`2683 (1976).
`1 1 . K. SHINODA, Y. MINEGISHI, and H. ARAI. J. Phys. Chem.
`80, 1987 (1976).
`12. J. DANIELSSON, J. B. ROSENHOLM, P. STENIUS, and
`S. BACKLUND. Prog. Colloid Polym. Sci. 61, l(1976).
`13. H. WENNERSTROM and B. LINDMAN. Phys. Rep. 52, 1
`(1979).
`14. K. SHINODA. Pure Appl. Chem. 52, 1195 (1980).
`15. D. G. CAMERON, H. L. CASAL, and H. H. MANTSCH.
`J. Biochem. Biophys. Meth. 1, 21 (1979).
`16. J. UMEMURA, D. G. CAMERON, and H. H. MANTSCH.
`J. Phys. Chem. 84,2272 (1980).
`17. D. G. CAMERON and R. N. JONES. Appl. Spectrosc. In
`press.
`18. D. G. CAMERON and G. M. CHARETTE. Appl. Spectrosc.
`35,224 (1981).
`19. D. G. CAMERON, H. L . CASAL, H. H. MANTSCH, Y.
`BOULANGER, and I. C. P. SMITH. Biophys. J. 35, l(1981).
`
`FIG. 5. Temperature-dependence of the frequency (@)and the
`width at $ height (A) of the symmetric CH, stretching band of
`APK in D,O.
`temperature range, behavior which has also been
`observed prior to the melting transition of phos-
`pholipids (19, 28).
`As the temperature is raised above 40°C progres-
`sive changes in both frequency and bandwidth are
`evident, the maximum rate of change being ob-
`served between 45 and 51°C. As with the head group
`absorptions this reflects the continuously changing
`concentrations of the coagel and micellar species.
`Confirmation that this is indeed the case comes
`from two sources. Firstly, the changes in frequency
`and bandwidth are not concerted. Rather, the
`maximum rates of change of frequency are ob-
`served at higher temperatures than those of the
`bandwidth. This is particularly evident in the plots
`in Fig. 5 in which the bandwidth is constant in the
`temperature range 49 to 5 1°C while the frequency is
`still changing. This behavior is typical of that
`resulting from the overlap of similar bands from
`two different species and has previously been
`observed in studies of the critical micelle concen-
`tration of n-alkanoates (29). Secondly, the con-
`tours of the CH, stretching bands at 46°C (Fig. 4)
`show flattened peak tops and are highly asymmet-
`ric compared to the spectra recorded at either
`higher or lower temperatures, indicating that.at this
`temperature the two component spectra have
`roughly equal intensities.
`The frequencies and bandwidths of the symmetric
`and antisymmetric CH, stretching vibration of an
`aqueous suspension of potassium ascorbyl palmi-
`tate increase dramatically between 45 and 51°C. The
`
`Can. J. Chem. Downloaded from www.nrcresearchpress.com by 38.117.177.233 on 10/21/15
`
`For personal use only.
`
`Par Pharm., Inc.
`Exhibit 1011
`Page 006
`
`

`
`SAPPER ET AL.
`
`2549
`
`20. J . M. VlNCENTand A. SKOULIOS. ActaCrystallogr. 20,441
`(1966).
`21. J . W. MCBAIN. J. Am. Oil Chem. Soc. 25,221 (1948).
`22. J . K. KAUPPINEN, D. J. MOFFATT, H. H. MANTSCH, and D.
`G. CAMERON. Appl. Spectrosc. 35, 271 (1981).
`23. J . HVOSLEF and P. KLAEBOE. Acta Chem. 25,3043 (1971).
`24. J . HVOSLEF. Acta Crystallogr. Sect. B, 25, 2214 (1969).
`25. Y. KOYAMA, M. YANAGISHITA, S. TODA, and T. MATSUO.
`J . Colloid Interface Sci. 61, 438 (1977).
`
`26. D. G. CAMERON, H. L. CASAL, F. E. GUDGIN, and
`H. H. MANTSCH. Biochim. Biophys. Acta, 596,463 (1980).
`27. J. H. DUMBLETON and T. R. LOMER. Acta Crystallogr. 19,
`301 (1965).
`28. D. G. CAMERON, H. L. CASAL, and H. H. MANTSCH.
`Biochemistry, 19, 3665 (1980).
`29. D. G. CAMERON, J. UMEMURA, and H. H. MANTSCH.
`J. Colloid Interface Sci. In press.
`
`Can. J. Chem. Downloaded from www.nrcresearchpress.com by 38.117.177.233 on 10/21/15
`
`For personal use only.
`
`Par Pharm., Inc.
`Exhibit 1011
`Page 007