Interaction of Anionic Compounds with Gelatin I: Binding Studies

RESEARCH ARTICLES

Interaction of Anionic Compounds with Gelatin. I: Binding Studies JAVA GAUTAM'AND HANSSCHOTT~ Accepted for Received March 17, 1992, from the School of Pharmacy, Temple University, Philadelphia, PA 19140. * Present address: Alcon Laboratories, Inc., Fort Worth, TX 76134. publication January 26, 1994". Abstract 0 Even though gelatin is the most widely used polymeric excipient in pharmaceutical products, scant attention has been paid

to its interaction with small organic molecules. The present work deals with the interaction of gelatin and four monosulfonated or monocarboxylated azo dyes having hydrocarbon moieties of different sizes. These dyes were used as models for anionic drugs, which make up a significant percentage of all new drugs. Most binding studies used ultrafiltration to separate free from bound dye, followed by spectrophotometricdye assays. One binding study was based on the shift in pH when a dye was added to gelatin solutions. All binding isotherms consisted of two linear segments. The initial segments, which start at the origin, represent the partitioning of the dyes between the dissolved gelatin and the aqueous buffer solution. They changed abruptly to horizontal plateaus, which represent the binding limit. Increases in pH from 5.00 to 7.00 reduced the binding of the sulfonated dyes but increased the binding of the carboxylated dye. At pH I 7.00, where even the carboxylic acid groups are fully ionized,the carboxylated dye and its sulfonated analog were bound to gelatin to the same extent. The binding of all dyes decreased with increasing temperature ( i a , the standard enthalpy of binding was negative), with a change of the solvent medium from water to 0.15 M ammonium acetate, and with decreasing size of the hydrocarbon moieties of the dyes. The binding of the dyes to gelatin was always reversible and the standard entropy changeassociated with it was negative. At the experimental condfins chosen, particularly pH values at least 1.9 units below the isoionic point of the gelatin of 8.9, electrostatic attraction between the dye anions and the basic sites of gelatin was the major binding force. Hydrophobic effects played a secondary but perceptible role, causing the dyes with the largest hydrocarbon moieties to be bound the most strongly and extensively to the gelatin.

Introduction The binding of small organic molecules to polymers plays an important role in a number of biological and technological processes, such as the transport of drugs bound to plasma proteins, enzyme catalysis (Michaelis-Menten complexes), protein denaturation, carcinogenesis and antineoplastic drug action when nucleic acids are the substrate, bacteriostatic effects and lysis when cell membrane proteins are the substrate, and dyeing and waterproofing of fibers and fabrics. Despite the numerous pharmaceutical,photographic, and foodrelated uses of gelatin, scant attention has been devoted to its interaction withsmall molecules.' The exception is its interaction with surfactants, especially sodium dodecyl sulfate, which has been investigated in some detail, and with dyes. For instance, complexeswith sodium dodecyl sulfate were used to fractionate gelatin.2 Among the gelatin properties affected by the interaction with sodium dodecyl sulfate and other surfactants were viscosity3 and surface tensi0n.~85 The effect of anionic: cationic? and Abstract published in Advance ACS Abstracts, March 15, 1994. 922 / Journal of Pharmaceutical Sciences Vol. 83, No. 7, July 1994

nonionics surfactants on the helical structure of gelatin was investigated by circular dichroism. The effect of dyes on the consistency of gelatin solutionsgand gels10 and on the swelling of gelatin films'' was investigated for the photographic industry. The effect of dyes on gelatin c a p ~ u l e s ' ~and J ~ films14was studied by pharmaceutical scientists. The remaining studies of the interaction of dyes with gelatin and other proteins were fundamental in nature.15-19 Many of the newer drugs, such as antibiotics, nonsteroidal antiinflammatory agents, and prostaglandins, are acidic or anionic. Some of them are dispensed in capsules or microcapsules where gelatin is part of the wall material. The interaction of such drugs with gelatin may affect their release and bioavailability. The purpose of the present work was to study the binding of model anionic compounds to gelatin. Acid azo dyes are convenient because they are commercially available in a variety of homologous series of different structures and they can be readily assayed spectrophotometrically. While most azo dyes with a single carboxylate group have low water solubility, monosulfonated dyes are more soluble. Therefore, only one fairly soluble monocarboxylated dye was used in the present binding studies with gelatin, along with three monosulfonated dyes. None of the publications referenced above shed much light on the binding mechanism(s), i.e., the thermodynamics of binding, the types of bonds involved, and their relative contribution. To obtain this kind of information, our specificobjectives were (i) to characterize a specially prepared gelatin sample with respect to its content of basic and acid functional groups and of oligomers that might pass through ultrafiltration membranes, (ii)to investigate quantitatively the binding of acid dyes to gelatin by determining the binding isotherms at various temperatures and pH values, by employing a wider concentration range than had been done previously, and (iii) to derive the pertinent thermodynamic binding parameters. The structure of the dyes and the pH values were selected to assess the relative importance of the strength of their acid groups and the size of their aromatic hydrocarbon moiety on the extent of binding. As an additional goal, in order to be useful, the foregoing information must provide insight into the mechanism(s) and the types of forces involved in the dye-gelatin interaction.

Experimental Section Materials-The free dye solution was to be separated from the dissolved gelatin and dye bound to it by ultrafiltration. Therefore, it was important for the gelatin to be practically free of fractions of low enough molecular weight to pass through the ultrafiltration membrane. The sample employed, Type A gelatin (lot no. 1, Type K & K T-5542) was a gift of Kind & Knox, Division of Knox Gelatine, Inc., Sioux City, IA. It was manufacturedby acid treatment of pork skinsunder conditions designed to minimize the amount of low molecular weight fractions. The average molecular weight of the gelatine was determined by sizeexclusion chromatography. The column used was a TSK type SW column (TosoHaas, Montgomeryville, PA) packed with a silica-based, rigid,

0022-3549/94/1200-922$04.50/0

@ 1994, American Chemical Society and American Pharmaceutical Association

Table 1-Acld

Dyes and Thelr Structure Dye

Structure

2-[(4-Hydroxyphenyl)zo] benzoic acid, sodium salt

Molecular Weight

Symbol

264.24

HABA

4-[(4-Hydroxyphenyl)zo] benzenesulfonicacid, sodium salt 4-[(2-Hydroxy-l-naphthyl)azo] benzenesulfonicacid, sodium salt (C.I. 15510)

OH

350.39

0-11

4-[(2-Hydroxy-l-naphthyl)zo]-l-naphthalenesulfonic acid, sodium salt (C.I. 15620)

PH

400.39

AR-88

hydrophilic gel with spherical particles of 10-13 in diameter.20 The gelatin had a bloom strength of 301 g and a viscosity of 5 CPat 6.67% solid content and 60 0C.20 The pH of gelatin manufactured from pork skins is so high that the moisture present in solid, granular gelatin may produce hydrolytic degradation on storage. In order to limit the extent of solid-state hydrolysis, sulfuric acid is added to the gelatin solutions prior to drying. Consequently, the pH of a 1.5% solution of commercial pork skin gelatin is 5.42.20 The pH of a 1.0% (w/v) solution was found to be 5.62. The term gelatin in this paper refers to the gelatin as supplied by the manufacturer. The dyes used, their abbreviations, and structures are listed in Table 1. HABA-H (lot no. 165496 780, FlukaBioChemika, Buchs, Switzerland) was neutralized to the sodium salt, HABA, with sodium hydroxide to pH 7.00. HASA (lot no. MANO1, TCl, Tokyo, Japan) was 98% pure and used as supplied. 0-11(lot no. 237189 287, Fluka Chemika) and AR-88 (ref N13693, Atlantic Industries, Inc., Nutley, NJ) were recrystallized from anhydrous methanol and dried over phosphorus pentoxide to constant weight. All other chemicals were ACS reagent grade. Water was double distilled and maintained free of carbon dioxide. Potentiometric Titration of Gelatin-The gelatin was deionized with a mixed bed of ion-exchange resins. A cation-exchange resin (AmberliteIR-120Plus H, lot no. 6-9454 of Rohm and Haas, Philadelphia, PA) in the hydrogen form and an anion-exchange resin (Amberlite IRA402-OH, lot no. 6-9688) in the hydroxide form were mixed in the ratio of 1:2.5.21 The resin bed was formed in a jacketed glass column maintained at 35.0 "C. A 2.0% (w/w) gelatin solution in water was passed through this column at the rate of 20 g/h. The deionized solution was stored at 4O C . An aliquot of the solution was dried at 105 OC to constant weight in order to determine the solids content. Deionized gelatin solutions were titrated at 30.0 "C in a 600mL jacketed beaker. In order to exclude atmospheric carbon dioxide, the titrations were performed under a positive nitrogen pressure. The jacketed titration vessel was covered with a lid that had openings for a nitrogen inlet and outlet, for a burette, and for an electrode. Each 200-g aliquot of 1.0% (w/w) gelatin solution was titrated with 0.2 N HCl and 0.2 N NaOH. After the addition of each 0.05-mL portion of titrant, the solution was stirred with a magnetic stirrer for 3 min to assure that a steady pH value was attained. The titrations ranged from pH 1.50 to 11.50. Back-titrations were done to check for hydrolysis of amide groups: None occurred. Blank titrations were performed on 200-g aliquota of water at 30.0 "C. The same equipment and method were used to determine the ionization constant of the carboxylated dye, HABA-H. Dye Binding Studies-Most binding studies were made at constant pH by separating the gelatin and dye bound to it from the free dye by means of ultrafiltration and then determining the two dye concentrations spectrophotometrically. However, a few dye binding measurements were made by adding the dyes to deionized gelatin in water and measuring the shift in pH resulting from the binding reaction. These methods are described below.

Ultrafiltration-The ultrafilter consisted of a cylindrical nylon framework 9.5 cm long with a 2.7-cm outer diameter, over which a tubular cellophane membrane was stretched.22 The tubing (Spectra/Por 7 of Spectrum Medical, Inc., Los Angeles,CA),is reported to have a molecular weight cutoff of 1OOO. It was soaked in water and rinsed extensively. The upper end of the cellophane tube was closed with a double knot. The lower end was closed by a rubber stopper with a glass tube through which vacuum was applied to remove the ultrafiltrate. The supported membrane was mounted in the center of a jacketed cylindrical glass container which was filled with the filtrand. The lower end of thecontainer was closed by a rubber stopper directly beneath the first stopper. The glass tube which drained the ultrafiltrate ran through the second stopper as well. The ultrafiltrate ran down the inside surface ofthe membrane and the frame, thereby practicallyeliminatingdialysis.P The glass container with the tubular membrane in the center was filled with 750-mL solution aliquots containing gelatin, dye, and buffer that were maintained a t the desired constant temperature. The solutions were stirred during the ultrafiltration to prevent the buildup of highly concentrated solution at the membrane. One milliliter of ultrafiltrate was collected every 30 min and analyzed until three consecutive aliquoh had the same dye concentration, indicating that equilibrium had been attained. Rates of filtration of water a t constant reduced pressure were measured before and after each ultrafiltration run to verify the integrity of the tubular membrane. Desorption studies were carriedout by diluting the solutions in the ultrafilter with measured amounts of buffer solution and proceeding as before. When the ultrafiltrand solutions were prepared in water instead of the buffer solution, their pH was adjusted to 7.00 with a few drops of 2 N NaOH. Spectrophotometric Analyses-Dye concentrations were determined with a H P 8451diode array apectrophotometer (Hewlett Packard Corporation, Palo Alto, CA). Absorbances were measured at the wavelengths of maximum absorption listed in Table 3. Traces of gelatin were occasionally present in the ultrafiltrate. These minute amounta were determined by adding ninhydrin and measuring the absorbance at 570 nm corresponding to the ninhydrin-proline complex.26 pH Shifts-0-11 solutions were added gradually to 100-g aliquoh of solutions of 0.1 % (w/v) of deionized gelatin in the titration vessel at 40.0 "C. After the addition of each 2.0-mL portion of dye solution but before the pH was measured, the mixtures were stirred for 3 min to attain equilibrium. The reported pH shifts were corrected for the pH changes of blank solutions containing the dye and water but no gelatin.

Results and Discussion Characterization of Gelatin-The molecular weight of the gelatin sample, determined by size-exclusion chromatography (Figure l),was 114000.20 This is a number-averagevalue because Journal of Pharmaceutical Sciences / 923 Vol. 83, No. 7, July 1994

4

'.

t 60

Table 2-pH Ranges Used for Determining the Ionic Groups of Gelatin and Their Concentration

I

Total carboxylic acid groups Total basic groups a-Amino (terminal) 4- imidazole

1.5-6.5 1.5-8.93 6.5-8.5

0.876 0.925 0.050

8.5-1 1.5 >11.5

0.460 0.415

(histidine)

+Amino (lysine + hydroxylysine) Guanidino (arginine)

p

0 103

Concentration, mmol/g gelatin

pH Range

Group

,

, ,

..__, .

104

':.;*La

105

Table 3-Wavelength of Maxlmum Absorptlon (Lax) and Molar Absorptlvity ( 6 ) Used for Assaying Free and Bound Dyes

c,

106

t,

Limo1 cm

A,, Dyea

Molecular weight

Additional Peak of Bound Dye

Single Peak of Free Dye

nm

X

,A,, n, f l

L/molcm

nm

X lo-*

n, fl

~~

Flgure 1-Molecular weight distribution of gelatin.20Key: (-) Molecular weight versus % area; (- - -)molecular weight versus cumulative % area.

348 (3503 HASA 354 (3539 0-11 486 (4853 AR-88 508 (5 103 HABA

a

1.39

6, 0.9992

544

5.31

4, 0.9927

2.24

6, 0.9999

568

5.16

4, 0.9946

2.07

5, 1.0000

584

3.98

4, 0.9978

1.90

5, 0.9988

526

7.65

4, 0.9988

Names and structures are shown in Table 1 . For linear Beer's

law plot. Reference 24. Reference 18. Reference23. Reference

16.

solutions on addition of the base, respectively. By using these relationships, MAand MBwere computed and the amounts of combined acid or alkali per gram of gelatin were calculated by -1 dividing Mx - MA and Mz - MB by the gelatin concentration Q 4 8 12 expressed in grams per liter.26 PH Figure 2 is a plot of bound acid or alkali (expressed as millimoles Figure 2-Titration curve of type A gelatin in water at 40.0 O C . Key: (0) of combined H+ or OH- per gram of gelatin) versus pH. Milliequivalents of H+ consumed; (+) milliequivalents of OH- consumed. Thepoints represent individual measurements. Each segment the chromatographiccolumn was calibrated with gelatin fractions was checked by back-titration. The amount of H+ or OHwhose molecular weights had been determined by osmometry, reacted at a given pH represents the net charge of the gelatin. which measures number averages. Table 2 lists the pH ranges used for determining the various The pH of a 1.5% (w/v) solution of deionized gelatin in water ionic groups of gelatin from the titration curve and their at 25.0 "C was 8.93. This value is the isoionic point, which is concentration^.^^ assumed to be equal to the isoelectric point (IEP). It agrees pKa of HABA-H-This measurement involved titrating a with that reported by the manufacturer (8.9-9.2 for 1.5% 100-mL volume of a 2.64 X 104 M HABA-H solution with 0.2 solutions).20 N NaOH solution under a positive nitrogen pressure at room Figure 2 showsthe titration curve of deionized gelatin in water. temperature. The pH at which 50 % of the carboxylicacid groups When the titration was carried out in 0.15 M NaCl instead of of HABA-H were neutralized, namely, 4.8, is the pK, of HABAwater, the curve was indistinguishable from Figure 2. MeasureH. ments of pH upon the addition of acid or base to the isoionic Assay of Free and Bound Dyes-The four near-ultraviolet/ gelatin were translated into titration curves as follows.26 Asvisible absorption spectra of the free dyes contained a major, suming that the activity coefficients of the acid and the base very broad and asymmetric peak. Table 3 lists its wavelengths were the same in the presence and absence of gelatin, the equation of maximum absorption. The four plots of absorbance versus for the addition of acid is as follows: free dye concentration were straight lines whose intercepts did not differ significantlyfrom zero. The slopes,representing molar absorptivity, the number of points on which they were based, and the linear regression coefficients are also listed in Table 3. where M is the molar concentration of acid (or base), and the The main peaks of the four spectra of the bound dyes were subscripts X and A refer to the blank and gelatin solutions on indistinguishable from the respective peaks in the spectra of the addition of the acid, respectively. Similarly, for the addition of free dyes: Their shapes, the wavelengths of maximum absorption, base: and the molar absorptivities were identical. However, the spectra log MB = pHB - pHz log Mz of the bound dyes exhibited a weak new peak at a higher (2) wavelength (Table 3). A similar weak new peak in the absorption where the subscripts Z and B refer to the blank and gelatin spectrum of HABA-H was produced by binding to albumin.24

:= I . , , ,

+

924 /Journal of Pharmaceutical Sciences Vol. 83, No. 7, July 1994

~

P

10

s

rA

A

X

.-c

-

8

a

0

-

, " 6

r

4

0

0 a

-n! n

Jd

4

2

P

0

2

0

6

4

8

Mols free dyelL x lo4 Figure 3-Effect of pH on the binding of 0-11 by gelatin at 40.0 OC.Key: (A) (m) Adsorption at pH 5.00 in 0.15 M ammonium acetate; (0)desorption at pH 5.00 in 0.15 M ammonium acetate; (B) (0)adsorption at pH 7.00 in water; (0)desorption at pH 7.00 in water; (C) (X) adsorption at pH 8.93 10.41 in water; (D) (A)adsorption at pH 7.00 in 0.15 M ammonium acetate; (A)desorption at pH 7.00 in 0.15 M ammonium acetate.

-

6

4

2

id

Figure 5-Binding of various dyes by gelatin at pH 7.00 and 40.0 OC in 0.15 M ammonium acetate. Key: (A) (*) Adsorption of AR-88; (0) desorption of AR-88; (B) (W) adsorption of 0-11; (0)desorption of 0-11; (C) (A)adsorption of HASA; (A)desorption of HASA; (D) ( 0 )adsorption of HABA; (0)desorption of HABA.

G-NH3+C1- + D-SOCNa' 0

ciyetL x

Such abrupt transitions have also been observed in pharmaceutical systems.29 The characteristics of the binding isotherms are listed in Table 4. In some instances the horizontal segments, which represent the binding limits, were reached well before 100% of the basic groups of the gelatin (G) had reacted with the sulfonate or carboxylate groups of the dyes (D). Binding occurred via ionexchange reactions and resulted in the attachment of the dye anions to the gelatin via electrovalent bonds according to the scheme:

8l

0

MOIS free

2

4

6

8

1

0

1

2

Mols free dyeL x 1d Figure 4-Effect of temperature and presence of buffer on the binding of HABA by gelatin at pH 7.00. Key: (A) (A)Adsorption at 40.0 OC in water; (A)desorption at 40.0 OC in water; (B) (0)adsorption at 33.0 OC in 0.15 M ammonium acetate; (0)desorption at 33.0 OC in 0.15 M ammonium acetate; (C) (W) adsorption at 40.0 OC in 0.15 M ammonium acetate; (El)desorption at 40.0 OC in 0.15 M ammonium acetate. Small amounts of gelatin, ranging in concentration from 0 to 0.002% (w/v), were present in the ultrafiltrates. They are

ascribed to an oligomer fraction with a molecular weight
+

G-NH3+-03S-D

+ NaCl

(3)

Hydrophobic bonding and hydrogen bonds are also involved in the binding of the dyes to gelatin (see next section). The fact that about one-half of the binding limits (columns 7 and 8 of Table 4) fell considerably short of the complete neutralization of the basic groups of the gelatin (Table 2) by the dye anions is attributed to competition of the carboxylate ions of the gelatin with the sulfonate or carboxylate ions of the dyes. The possibility that steric hindrance between dye anions binding to adjacent basic groups along a gelatin chain blocks some of these basic sites and prevents their complete neutralization is examined in the Appendix. All dyes reached binding equilibrium within 15 min after mixing their solutions with the gelatin solutions. Desorption equilibria were reached between 12 and 30 min after dilution. The desorption was faster for the smaller dye molecules than for the larger ones. Points for both the binding or adsorption and the desorption of each dye by gelatin fell on the same curve. The absence of hysteresis indicates reversible binding. Thus, the binding of dyes to gelatin is a t,rue complexation reaction.30 The binding was affected by pH, temperature, and the presence of a buffer. It was also affected by the nature of the dyes, especially the pK, of their acid groups and the size of their hydrophobic moieties. The gelatin concentration did not affect the binding. pH-In the case of the three sulfonated dyes, there was an increase in binding when the pH of the buffer solution was reduced from 7.00 to 5.00 at constant ionic strength (Figure 3, curves A and D): The slopes of the initial segments of the binding isotherms increased by a factor of 4.3-5.6 and the plateaus, representing the binding limits, increased by a factor of 1.0-2.6 (Table 4). A t pH 5.00, the sulfonated dyes neutralized the basic groups of the gelatin almost completely. This increased binding Journal of Pharmaceutical Sciences / 925 Vol. 83, No. 7, July 1994

Table 4-Slopes

Dyea

(Partklon Coefflclents) and Plateaus (Blndlng Limns) of the Blndlng Isotherms of Acid Dyes to Gelatln

Slope of Initial Segment, Ammonium Acetate Temperature, mol bound dyelg gelatin Concentration, M OC mol free dye/L (n, fib

PH

HABA HABA HABA HABA HABA HABA

7.00 7.00 7.00 7.00 5.00 5.00 HASA 7.00 HASA 7.00

0.15 0.15 0.00 0.00 0.15 0.15 0.15 0.15

HASA 7.00 HASA 7.00 HASA 5.00

0.00

0-11

0-11 0-11 0-11 0-11 0-11 0-11

0-11 O-IId AR-88 AR-88 AR-88 AR-88 AR-88 AR-88

7.00 7.00 7.00 7.00 7.00 7.00 5.00 5.00 8.93 7.00 7.00 7.00 7.00 5.00 5.00

+

10.41

0.00 0.15 0.15 0.15 0.15 0.15 0.00 0.00 0.15 0.15 0.00 0.15 0.15 0.00 0.00 0.15 0.15

33.0 40.0 33.0 40.0 33.0 40.0 33.0 40.0 33.0 40.0 40.0 33.0 40.0 42.0 45.0 33.0 40.0 33.0 40.0 40.0 33.0 40.0 33.0 40.0 33.0 40.0

0.57 0.43 1.33 1.08 0.21 0.18 0.59 0.48 1.66 1.33 2.39 1.83 1.48 1.40 1.28 4.85 3.85 8.96 7.09 3.89 4.99 3.96 14.17 11.09 27.69 19.41

(6, 0.9687) (8, 0.9987) (4,0.9999) (4, 0.9987) (4, 0.9978) (4, 0.9989) (6, 0.9957) (8, 0.9987) (4, 0.9969) (4, 0.9987) (7, 0.9997) (6, 0.9954) (6, 0.9989) (4, 0.9986) (4, 0.9987) (4, 0.9843) (4, 0.9997) (4, 0.9989) (4, 0.9997) (5, 0.9987) (8, 0.9989) (6, 1.0000) (4, 0.9997) (4, 0.9997) (4, 0.9989) (6, 0.9997)

Binding Limit,

mol bound dye/g gelatin Binding Limit as x 104 % of Saturation Average f SD Binding, 1008c 3.46 f 0.10 2.94 f 0.02 6.14 f 0.01 6.67 f 0.17 2.23 f 0.13 1.76 f 0.08 3.60 f 0.12 3.21 f 0.01 7.64 f 0.07 7.22 f 0.01 8.45 f 0.01 5.00 f 0.01 4.52 f 0.50 4.34 f 0.08 4.13 f 0.13 8.63 f 0.09 8.21 f 0.05 8.95 f 0.07 8.68 f 0.05 7.25 f 0.00 8.75 f 0.02 7.52 f 0.01 8.86 f 0.10 8.75 f 0.25 9.05 f 0.03 8.98 f 0.00

37 32 66 72 24 19 39 35 83 78 91 54 49 47 45 93 89 97 94 78 95 81 96 95 98 97

a Names and structures are shown in Table 1. Number of ultrafiltration runs on which the slope is based and linear regression coefficient. Moles of bound dye, expressed as percent of the number of moles of basic functional groups present in the gelatin substrate; 8 is defined by eq 5. dFrom pH shifts; all other values were determined by ultrafiltration.

at the lower pH was due to decreased ionization of the carboxylate groups of the gelatin, which reduced their competition for the basic groups of the gelatin. In the case of the carboxylated dye HABA, there was a decrease in binding when the pH of the buffer solution was reduced from 7.00 to 5.00 The slopes of the initial segments decreased by a factor of 2.4-2.7 and the binding limits decreased by a factor of 1.5-1.7 (Table 4). The decreased binding at the lower pH was due to reduced ionization of the carboxylate groups of HABA. With a pKa of 4.8, they are only 61% ionized at pH 5.0 compared to 99% at pH 7.0. Temperature-The slopes of the initial segments and the plateaus representing the binding limits decreased with increasing temperature for all four dyes regardless of pH and electrolyte concentration (Figure 4, curves B and C, and Table 4). The decrease in binding of the dyes to gelatin with increasing temperature indicates that the reaction is exothermic. Gelatin Concentration-The amounts of bound dye per gram of gelatin at a constant free dye concentration were not affected by varying the gelatin concentration between 0.1 and 12.5 g/L. Most binding studies were performed at 5.0 g/L of gelatin. Buffer-The concentration of the ammonium acetate buffer, 0.15 M, was chosen to make it isotonic with blood. The presence of this buffer lowered the binding of all four dyes (Figure 4, curves A and C, and Table 4): The slopes of the initial segments of the binding isotherms were reduced by a factor of 2.3-2.8 and the binding limits were reduced by a factor of 1.0-2.3 when the medium was changed from water to 0.15 M ammonium acetate while maintaining the pH constant a t 7.00. This decrease in binding in the presence of ammonium acetate is attributed to the screening effect of the acetate ions: The sulfonate or carboxylate ions of the dyes bind to the basic groups of gelatin 926 /Journal of Pharmaceutical Sciences Vol. 83, No. 7, July 1994

by electrovalences. The acetate ions surrounding the basic groups of gelatin in high concentration screen the electrostatic attraction of these groups for the dye anions, thereby reducing the binding. An alternate but related explanation to account for the reduced dye binding in the presence of ammonium acetate is the competition of the acetate ions with the sulfonate and carboxylate ions of the dyes for the basic groups of gelatin. Because of the relatively high concentration of ammonium acetate, the reaction:

G-NH3+-03S-D

+ CH,-COO-NH,+ .= G-NH,+-OOC-CH~ + D-SO;NH,+

(4)

proceeds to some extent in the forward direction despite the lack of hydrophobic and hydrogen bonding between the acetate ions and gelatin. Size of the Dye Molecule-Increases in the number of carbon atoms of the sulfonated dyes increased their binding (Figure 5). When one benzene ring of HASA was replaced by a naphthalene ring, resulting in 0-11, the slope of the initial segment of the binding isotherms increased %fold and the plateau, representing the binding limit, increased by a factor of 1.0-1.4. When the benzene ring of 0-11was replaced by a naphthalene ring, resulting in AR-88, the slope increased again 3-fold and the binding limit increased by a factor of 1.0-1.8 (Table 4). Acid Strength of Dye-The dyes HABA and HASA have similar structures, except that the former is carboxylated (pK, = 4.8) while the latter is sulfonated (pK, < 1). At pH 7.00, where both dyes are completely ionized, there was no significant difference in the binding of HABA and HASA (Figure 5, curves C and D).

At pH 5.00, the binding of HASA was greater than that of HABA The slopes of the initial segments of the binding isotherms of HASA were 12-13 times greater than those of HABA and their plateaus, representing the binding limits, were 4-5 times greater (Table 4). The lesser binding of HABA at pH 5.00 compared to HASA is due to reduced ionization of its carboxylate groups. Solubility of Dyes in Presence of Gelatin-The solubility of the free dyes in 0.15 M ammonium acetate was not affected by the presence of gelatin. For instance, the solubility of HABA at 25 "Cwas 0.379 and 0.384 mol/L in 0.15 M ammonium acetate and 0.15 M ammonium acetate containing 1.0 g/L of gelatin, respectively. The concentration of bound dye in the latter solution was 3.46 X 10-4 mol/L. Gelatin increased the overall molar dye solubility by an amount approximately equal to its percent (w/v) concentration. As is seen in Table 4 , 8 sometimes fell considerably short of unity at level-off Df values, i.e., the plateaus of the binding isotherms were reached before all of the basic sites of gelatin chemisorbed dye anions. The possibility that the dye anions bound to some of the basic gelatin groups by ion pairing blocked the access of additional dye anions to adjoining basic groups is examined and discarded in the Appendix. Thermodynamic Treatment of Binding Isotherms-The binding parameters were calculated from the slopes of the initial segments of the binding isotherms by the following equation:

(5) where K is the binding constant, r is the number of millimoles of bound dye per gram of dry gelatin, n is the number of millimoles of basic groups per gram of dry gelatin, namely, 0.925 (Table 21, Df is the concentration of free dye (in moles per liter), and 8 = r / n is the fraction of the basic gelatin groups neutralized by bound dye anions. The standard free energy of binding per mole of dye, AGO, was calculated by the equation:

AGO = -RT In K

(6)

where R is the gas constant and T the absolute temperature. The standard enthalpy of binding, AHo, was calculated by the van't Hoff equation, which takes the following form:

(7) when two binding constants, Kl and Kz, were determined at T I and Tz, respectively. In the one set of experiments with 0-11 where the binding was studied at four different temperatures between 33.0 and 45.0 "C, the van't Hoff equation was used in the following form:

A straight line was obtained when In K was plotted against the reciprocal of the absolute temperature (r = 0.9998), indicating that AHo was constant in this temperature range. The slope, -AH"/R, was 2922 K, which corresponds to AH" = -5800 cal/mol of dye (see Table 6). The binding of 0-11to gelatin between 33.0 and 45.0 OC obeyed the van't Hoff equation (eq 8) with unusually high precision. Therefore, we assumed that the binding of the other three dyes also obeys the van't Hoff equation in that temperature range, conducted their binding experiments at only two temperatures, namely, 33.0 and 40.0 OC, and calculated the standard enthalpies of binding by means of eq 7. Because of the transitions that

occur in aqueous gelatin systems, it is unlikely that AHo remains constant and that eq 8 is obeyed at temperatures as little as -5 to 10 "C below that range. Because the binding experiments were carried out at constant temperatures, the standard entropy of binding per mole of dye, AS", was calculated by the usual equation:

AGO = AH" - TAS"

(9)

In the one set of experiments where the binding of 0-11was determined at four temperatures, the standard entropy of binding was checked by the following form of the Gibbs-Helmholtz equation:

which results from differentiating eq 9 with respect to temperature, provided AS" and AHo are independent of temperature. The plot of AGO versus T between 33 "C (306 K) and 45 OC (318 K) was linear ( n = 4, r = 0.995), indicating that ASo as well as AH" is independent of temperature in that range. The slope of that line equals the standard entropy of binding. Shifts i n pH-In addition to studying the binding of 0-11 to gelatin in water a t constant pH by ultrafiltration, a binding isotherm was also determined without any separation by measuring the gradual shift in pH when a deionized gelatin solution was titrated potentiometrically with the dye solution. According to the scheme:

D-SO
+ GNH,+OH- * D-S0;+H3N-G

+ Na+ + OH-

(11)

each dye anion (D-S03-) that binds to a basic group of isoionic gelatin (G-NHa+OH-) releases one hydroxide ion. Thus, the increase in pH gauges the extent of dye binding. A 0.1 M solution of 0-11 (pH = 7.00) was added in 2.0-mL increments to 100 mL of a 0.1 % (w/v) solution of isoionic gelatin in water maintained at 40 "C (initial pH = 8.93). The pH increased continuously until it reached 10.41 and remained constant on further addition of dye solution. The maximum pH increase of 1.48 units was produced by 1.00 X 103 moVL of 0-11. Taking the pK of water at 40 OC as 13.535, an increase in pH from 8.93 to 10.41 corresponds to the release of 7.25 X lo"mol/L of hydroxide ions and the binding of the same number of moles of dye anion. This is equivalent to the neutralization of 78.4% of the basic groups of gelatin by the bound dye. The 40 "C binding isotherms of 0-11obtained by ultrafiltration in water at pH 7.00 and by the pH-shift method (pH 8.93 10.41) have similar shapes (Figure 3, curves B and C). Their initial segmentsare almost superimposable,making their binding constants nearly identical (see Table 5). This agreement is expected because, at pH 2 7.00, the carboxylic acid groups of gelatin are fully ionized. Therefore, their tendency to bind to the basic groups of gelatin, in competition with the sulfonate ions of 0-11,is the same during ultrafiltration at pH = 7.00 and during the pH shift from 8.93 to 10.41. The plateau, representing the binding limit, determined by ultrafiltration is somewhat higher than that determined by pH shifts. This is attributed to the lesser ionization of the primary amino groups of gelatin in the higher pH range met during the pH shifts compared to the constant pH of 7.00 employed during ultrafiltration. Thermodynamic Binding Functions-The values are listed in Table 6. The three sulfonated dyes constitute a homologous serieswith increments of four aromatic CH groups. Their binding constants and the absolute values of their negative standard free energies of binding in each of the three media (water and

-

Journal of Pharmaceutical Sciences / 927 Vol. 83. No. 7, JuEy 1994

Table B-Comparlson of 40.0 OC Binding Isotherms of 0-11 In Water Obtained by UltraflHratlon and pH Shlft

Method

pH Range 8.93’ 7.00

pH shift

Ultrafiltration a

10.41

Form of Gelatin

Binding Limit,

K,

mol bound dye/g gelatin

x

104

en

7.25 8.21

Isoionic As supplied

L/mol dye 0.78 0.89

x

10-3

4.21 4.16

Defined by eq 5.

Table 6-Thermodynarnlc

Dyea HABA

PH

Parameters for the Blndlng of Acid Dyes by Gelatln K,

Temperature,

0.15

33.0

0.62

3.91

0.00

40.0 33.0

0.47 1.44

3.83 4.42

0.15

40.0 33.0

1.17 0.23

4.39 3.31

0.15

40.0 33.0

0.20 0.64

3.30 3.93

0.00

40.0 33.0

0.52 1.79

3.89 4.56

0.15

40.0 33.0

1.44 3.43

4.52 4.95

2.75 1.98 1.59 1.51 1.38 5.24

4.89 4.62 4.59 4.58 4.57 5.21

7.00

OC

L/mol dye

-A@, kcal/mol dye

Ammonium Acetate Concentration, M

X

-A@, kcal/mol dye 7.53

HABA

HABA HASA

HASA

HASA

7.00 5.00 7.00

7.00

5.00

0-11

7.00

0.15

0-11

7.00

0.00

40.0 33.0 40.0 42.0 45.0 33.0

0.15

40.0 33.0

4.16 9.69

5.18 5.58

0.00 0.15

40.0 40.0 33.0

7.66 4.218 5.39

5.56 5.19 5.23

0.00

40.0 33.0

4.28 15.32

5.20 5.86

0.15

40.0 33.0

11.99 26.93

5.84 6.20

40.0

20.98

6.19

0-11

5.00

0-11 AR-88 AR-88

AR-88

8.93 7.00

-

10.41

7.00

5.00

-A9, cal/mol dye K 11.8

5.65

4.00

3.80

1.61

5.65

5.61

5.92

4.45

6.01

3.52

5.8OOb

3.873O 3.892d

6.28

3.49

6.39

2.65

-

-

6.27

3.42

6.66

2.63

6.79

1.91

a Names and structures are shown in Table 1. From eq 8. Mean of four values calculated with eq 9. From eq 10. From shift in pH. All other values were determined at constant pH by ultrafiltration.

0.15 M ammonium acetate at pH 7.00 and 0.15 M ammonium acetate at pH 5.00) increase with their number of carbon atoms: The larger dyes have a higher affinity for gelatin and are more extensively bound to it. The absolute values of their negative binding enthalpies also increase with the number of carbon atoms per dye molecule, C: Even on a molar basis, the larger dyes are bound more exothermically. Two of the three plots of AHo versus C in the three media (Figure 6) are linear. Their regression equations are, in 0.15 M ammonium acetate at pH 5.00

AHo = -4.84

- 0.0975C

( n = 3, r = 0.9999) (12)

and in water at pH 7.00: 928 / Journal of pharmaceutical Sciences Vol. 83, No. 7, Ju& 1994

AHo = -4.81

- 0.0925C

( n = 3, r = 0.9999) (13)

Extrapolation to C = 0 provides a rough estimate of the electrostatic component of the binding enthalpy, namely, -4.8 kcal/mol of bound dye, because the dispersion forces between gelatin and the dye molecules stripped of their hydrocarbon moieties are only a small fraction of the dispersion forces involving the complete dye molecules. Obviously, the linear extrapolation from C = 12 to C = 0 is speculative. The slopes of 100 cal per mol of aromatic CH are of the right order of magnitude for dispersion forces. The standard entropies of binding are negative (unfavorable for binding), and their absolute values decrease with increasing

-

slopes (Figure 3) and practically identical binding constants (Table 5), which validates eq 11. (ii) The binding of the carboxylated dye was less extensive at pH 5.00 than at pH 7.00 while the binding of the sulfonated dyes was more extensive at pH 5.00, approaching stoichiometric neutralization of the basic groups of gelatin (e 1.00). (iii) The binding constants for each dye in water were 2.66 f 0.17 times greater than the corresponding values in 0.15 M ammonium acetate at comparable temperatures, due to competition between the dye anion and the acetate anion for the basic gelatin groups (eq 4). These facts are incompatible with hydrophobic interaction, which is a rival mechanism for the binding of small organic molecules to polymers in water. Hydrophobic Effect-Hydrophobic interaction or bonding occurs during the binding of a relatively nonpolar organic solute (or one having a substantial hydrocarbon moiety) dissolved in water by a water-soluble polymer, when the binding forces involved are primarily dispersion forces, without substantial contribution from electrostatic attraction. This binding process is either endothermic, which is unfavorable, or only slightly exothermic. The driving force that makes AGO negative and thereby promotes binding is the release of water associated with the small solute molecules and with the binding sites of the polymer chains. When the small molecules are bound to chain segments, the hydrocarbon moieties of both are pushed together in order to reduce the energetically unfavorable water-hydrocarbon interface. Much of the highly structured water that had surrounded both the small organic molecules and the binding sites of the polymer chains is thereby released and becomes disordered. This decrease in order represents a substantial entropy gain, one that is considerably larger than the entropy loss suffered by the small organic moleculeswhen they become attached to the polymer. The TAS" term containing the total entropy change is positive and exceeds the positive AHa value, making AGO negative according to eq 9 and thereby causing the binding to take p l a ~ e . ~ O . ~ ~ The present process of anionic dyes binding to basic sites of gelatin is exothermic. It is driven by the negative AH" and occurs despite the negative AS". This is characteristic of Coulombic interactions.30 The entropy change is negative because the loss of randomness suffered by the dye anions being affixed to the gelatin chains plus the reduction in conformational freedom suffered by the gelatin chains rendered stiffer by the attached dye anions is larger than the gain in randomness as water previously structured around the hydrocarbon moieties of the dye molecules and the binding sites of the gelatin chains I released when the two come into contact. However, the following two observations indicate that hydrophobic bonding makes a contribution to the dye binding by gelatin, albeit one that is smaller than the Coulombic attraction: (i) Each increase of four aromatic CH groups in the size of the dye molecule at a given temperature and ionic strength increased the value of K (in units of liters per mole of dye) by a factor of 2.90 f 0.13. (ii) All binding entropies were negative, but they were small and decreased in absolute value with increasing C, i.e., they approached zero and positive values as the size of the dye molecules increased. The entropies of binding of large and/or amphoteric dye molecules by gelatin at low pH were reported to be positive, which was attributed to hydrophobic bonding.18 Hydrogen Bonding-Our choice of dyes was not designed to evaluate the contribution of hydrogen bonds to dye binding by gelatin because all four dyes had one phenolic hydroxyl group. In view of the high concentration of amide groups in gelatin and by analogy to the interaction of phenols with povidone32 (which has pendant lactam rather than main-chain amide groups), hydrogen bonding is likely to occur. However, it is unlikely to affect the standard enthalpy of binding because each time the phenolic hydroxyl group of a dye molecule undergoing electro-

-

-4.51 ,

,

0 4

, , , , , , , 8 r- 12 16 20 b

Figure 6-Effect of C, the number of carbon atoms per molecule of sulfonated dye, on the enthalpy of binding. Key: (A)at pH 5.00 in 0.15 M ammonium acetate; (0)at pH 7.00 in water; (0)at pH 7.00 in 0.15 M ammonium acetate.

C . Negative entropy changes indicate a reduction in randomness or an increase in order: During the binding process, the dye anions lose their freedom of independent motion. The gelatin chains, already relatively rigid because of the partial doublebond character of the C-N backbone bonds, are rendered even more rigid by the attached dye anions and can assume still fewer conformations in solution. The dye anions themselves are coplanar and rigid. The absolute values of the negative binding entropy become smaller as the size of the dye molecules increases (see Table 6). This observation is ascribed to an increase in the number of water molecules released upon binding, water molecules that were previously hydrating the dye anions and the gelatin chains at the binding sites (see below). Published binding studies of sulfonated azo dyes to gelatin, based on polarography and equilibrium dialysis,ls reported somewhat more highly negative AG" and AH" values. The AS" values were negative for small dye molecules but positive for larger ones. These studies were conducted in more acidic solutions (pH 3-5) and the maximum free dye concentrations were an order of magnitude smaller than the present values: The binding isotherms never reached the plateaus.

Conclusions One of the two purposes of this work was to determine the mechanisms by which acid dyes are bound to basic gelatin. This section contains an analysis of the data of Table 6 and presents conclusions about the binding mechanisms and the types of forces that operate between the dyes and gelatin. A t physiological pH, the carboxylated dye (which is representative of anionic drugs) is completely ionized, and its results are comparable to those of the sulfonated dyes. Electrostatic Interaction-Since the amphoteric gelatin was used at pH values at least 1.9 units below its isoelectric point, it was in its cationic form, and electrovalences or Coulombic forces (represented by eq 3) would be expected to play the major role in the binding of small organic anions. The following experimental facts corroborate the prevalence of Coulombic forces in dye binding: (i) When 0-11was added to deionized gelatin, the pH increased as each bound dye anion released one hydroxide ion (eq 11). Moreover, the binding isotherms of 0-11in water determined by ultrafiltration at a constant pH of 7.00 and by the pH shift at pH values above the isoionic point of 8.93 had the same initial

Journal of pharmaceutical sciences / 828 Vol. 83, No. 7, July 1994

static binding forms a hydrogen bond with an amide group of gelatin, two hydrogen bonds are broken: one between a water molecule that had been hydrogen bonded to the amide group and another between a water molecule that had been hydrogen bonded to the dye's phenolic hydroxyl group. The two released water molecules are free to hydrogen bond to each other. Thus, the net number of hydrogen bonds is not changed by the dye binding. Furthermore, the hydrogen bonds between water and amide group, water and phenolic hydroxyl, phenolic hydroxyl and amide group, and between two water molecules have nearly the same bond energy.

Appendix Complex Coacervation-The compositions listed in Table 6 were single-phase solutions. However, phase separation occurred when concentrated solutions of 0-11or AR-88 were added to gelatin solutions in water. The coacervate phase, containing most of the gelatin and dye, separated as droplets. The other phase was a very dilute solution of gelatin and dye. The coacervatedroplets measured between 20 and 100pm within 15 min of mixing. They settled slowly and coalesced to a clear layer in approximately 24 h. No phase separation was observed with HABA and HASA in water, nor with 0-11or AR-88 in 0.15 M ammonium acetate. As an example of coacervation,119.48gof asolution containing 0.205 g of gelatin and 2.8 X 10-3 mol of 0-11 in water at 33.0 "C and pH 7.00 separated into 4.42 g or 3.7 % (w/w) of a coacervate phase and 115.06 g or 96.3% (w/w)of aqueous serum. The former contained 0.043 g of gelatin and 5.84 X lo4 mol of 0-11while mol of the latter contained 0.162 g of gelatin and 2.22 X 0-11. The ratio of dye to gelatin was 1.4 X mol of 0-II/g of gelatin in both phases. The number of equivalents of 0-11present in either phase exceeded the number of equivalents of basic groups pertaining to the gelatin present in the same phase by a factor of 15. The phase separation was sensitive to changes in pH, temperature, dye concentration, and the presence of the buffer, ammonium acetate: Coacervation occurred between pH 5 and 8 and at temperatures below 40.0 "C. For a 5 g/L gelatin solution in water at pH 7 and 33.0 "C, coacervationwith 0-11was observed in the concentration range of 5 X lo3 to 8 X mol/L of 0-11. Below and above this 0-11concentration, the solution remained homogeneous at pH 7 and 33.0 "C. When the medium was changed from water to 0.15 M ammonium acetate, no coacervation was observed at any of the above conditions. Geometrical Considerations-Many dye binding limits fell considerably short of the neutralization of all basic groups of gelatin by dye anions (see above). The possibility that some of these basic groups were stericallyblocked by dye moleculesbound to adjacent basic groups and were, therefore, inaccessible is examined below. Pork skin gelatin has 84 basic amino groups per 1000amino acid repeat units. This number is obtained from Table 2 and from the number-averagemolecular weight per amino acid repeat unit, namely, 91.035.33 Moreover, the basic amino acid repeat units along the gelatin chain are not clustered together.34 On average, there is one basic group in 11.9 amino acid repeat units. The dye molecules measure 12.2 A in length compared to 3.6 A for an extended amino acid repeat unit.35 Assuming that the bound dye anions lie flat on and parallel to the backbone of the gelatin chains and that these chains are in an extended, planar zigzag conformation, each dye molecule covers 12.2/3.6 = 3.4 amino acid repeat units. Therefore, it is unlikely that a dye molecule bound to a basic group blocks an adjacent basic group

930 / Journal of Pharmaceutical Sciences Vol. 83. No. 7, July 1994

along the polypeptide chain. Even on complete neutralization of all basic gelatin groups by bound dye anions, only 28% of the length of the gelatin backbone is covered and obstructed by bound dye. The conclusion that bound dye anions do not sterically block adjoining basic groups of gelatin is corroborated by the fact that AR-88, the largest dye molecule, also had the largest 8 values.

References and Notes

1. Ofner, C. M., III; Schott, H. J. Pharm. Sci. 1987, 76, 715-723. 2. Stainsby, G.; Saunders, P. R.; Ward, A. G. J. Polym. Sci. 1954,12, 325-334. 3. Greener, J.; Contestable, B. A.; Bale, M. D. Macromolecules 1987, 20, 2490-2498. 4. Wuestneck, R.; Mueller, H. J. Colloid Polym. Sci. 1986,264,971-_I. n3 5. Music, S.; Suveljak-Zuljevic;Wolf, R. H. H. Colloid Polym. Sci. 1980.258.1299-1302. 6. Wuestneck, R.; Wetzel, R.; Buder, E.; Hermel, H. Colloid Polym. Sci. 1988,266, 1061-1067. 7. Wuestneck, R.; Buder, E.; Wetzel, R.; Hermel, H. Colloid Polym. Sci. 1989, 267, 429-433. 8. Wuestneck. R.: Buder. E.: Wetzel., R.:. Hermel. H. Colloid Polvm. Sci. 1989, 267,'516-519. ' 9. Brand, B. P.; Roche, M.; Winstanley, D. In Photographic Gelatin; Cox, R. J., Ed.; Academic: New York, 1972; pp 135-156. 10. Trapeznikov, A. A.; Fedotov, G. V.; Korotina, T. I. Kolloidn. Zh. 1979, 41, 103-109. 11. Sheppard, S. E.; Houck, R. C.; Dittmar, C. J. Phys. Chem. 1942, 46, 158-176. 12. Cooper, J. W.; Ansel, H. C.; Cadwallader,D. E. J. Pharm. Sci. 1973, 62, 1156-1164. 13. Smith, H. L. J. Pharm. Sci. 1974,63,639-641. 14. Kellawav, I. W.; Marriott,. C.;. Robinson, J. A. J. Can. J. Pharm. Sci. 1978,.13, 87-90. 15. Malik, W. U.; Ahmad, S. Electroanal. Chem. Interfacial Electrochem. 1973. -, 47. -155-160. -- --16. Shimizu, Y.; Kimura, M. Sen'i Gakkaishi 1981,37, T27-T31. 17. Shimizu, Y.; Kimura, M. Sen? Gakkaishi 1981,37, T200-T204. 18. Shimizu, Y.; Kimura. M. Sen'i Gakkaishi 1981.37. T236-T240. 19. Shimizu, Y.;Kimura, M. Shiga-Kenritsu Tanki Daigaku Gakujutsu Zasshi 1989,35, 1-5. 20. Keenan, T. R., Kind and Knox, Division of Knox Gelatine, Inc., Sioux City, IA, personal communication, Oct 11, 1988. 21. Janus, J. W.; Kenchington, A. W.; Ward, A. G. Research (London) 1951,4, 247-248. 22. Schott, H. J. Phys. Chem. 1964,68,3612-3619. 23. Holmes, W. C. J. Am. Chem. SOC.1924,46,631-635. 24. Rutstein, D. D.; Ingenito, E. F.; Reynolds, W. E.; Burke, J. M. J. Clin. Inuest. 1954, 33, 211-221. 25. Troll, W.; Cannan, R. K. J. Biol. Chem. 1953,200,803-811. 26. Veis, A. In The Macromolecular Chemistry of Gelatin; Academic: New York, 1964; p 101. 27. Kenchington, A. W.; Ward, A. G. Biochem. J. 1954,58,202-207. 28. Giles, C. H.; Smith, D.; Huitson, A. J. Colloid Interface Sci. 1974, 47,755-765. 29. Schott, H.; Royce, A. E. J. Pharm. Sci. 1985, 74,957-962. 30. Keipert, S.; Becker, J.; Schultze, H.; Voigt, R. Pharmazie 1973,28, 145-183. 31. Scholtan, W. Arzneimittel-Forsch. 1964,14,469-473; 1139-1146. 32. Molyneux, P.; Vekavakayanondha,S.J. Chem. Soc., Faraday Trans. 1 1986, 82, 291-317, and four earlier publications in this series. 33. Eastoe, J. E.; Leach, A. A. In Recent Advances in Gelatin and Glue Research; Stainsby, G., Ed.; Pergamon: New York, 1958; 34. Hulmes, D. J. S.; Miller, A.; Parry, D. A. D.; Piez, K. A.; W o o B h ~ ~ ~ ~ Galloway, J. J. Mol. Biol. 1973, 79, 137-148. 35. Pauling, L.; Corey, R. B.; Branson, H. R. Proc. Natl. Acad. Sci. U.S.A. 1951, 37, 205-211.

.

Acknowledgments The gift of the gelatin sample and its characterization by Kind & Knox is gratefully acknowledged. This paper was adapted in part from a thesis submitted by J. Gautam to Temple University in partial fulfillment of the Doctor of Philosophy Degree requirements and was presented at the American Association of Pharmaceutical Scientists' sixth annual meeting, Washington DC, Nov 17-22, 1991.