Dec., 1962
INTERACTIOX O F DYES .4ND POLYSACCH$RIDES
2585
ON THE INTERACTIOIV OF DYES AND POLYSACCHARIDES'V~ BY BENJAMINCARROLL AND HERBERT C. CHEUNG Chemistry Department of Rutgers, The State University, hlewark 2, brew Jersey Received September 66,1961
Quantitative studies were made on the binding of cationic methylene blue and anionic congo red by soluble polysaccharides, using a spectral technique. The interaction of methylene blue and carboxylated starches was found to be predominantly electrostatic. The dye appeared to be absorbed in the dimeric form. At infinite dilution of the dye the binding data, when extrapolated to zero ionic strength, yielded a 1-1 correspondence between the dye and the carboxylate group of the starch. The competitive effect of the buffer ions was evaluated. The binding constants for the competing cation were found to be two orders of magnitude smaller than those for methylene blue. The binding of congo red by linear polysaccharides was found to be independent of chain length of the substrate over a wide range from about 17 t o 860 glucose units. The effect of chain length was studied using corn amylose, hydrolysates of the amylose, and aniylodextrins isolated by column chromatography. Branching of the substrate was found to decrease markedly the extent of binding of congo red. Because the dye interaction was independent of chain length over a wider range than that for iodine, it appeared advantageous to use congo red for the determination of the degree of branching in starch. This technique may have general applicability for high polymers. Binding by carboxylated starches was observed for congo red at p H 8. The extent of binding was about the same as that for neutral starches, indicating the small effect of charge of the substrate on the binding affinity for this dye. A mechanism based on the concept of configurational adaptability is suggested for the binding of congo red.
Introduction Although the study of reversible binding of small ions by proteins has been reported in the literature for some time, there appears to be little work done with soluble polysaccharides. The well known interaction of iodine and starch certainly falls in.to the category of binding of small ions. This interaction, however, is restricted to larger linear molecules, and is thermodynamically irreversible. Further, in the description of starch splitting enzymes, amylocla.stic activity measurements have been restricted to the use of iodine. The use of other indicators could possibly exbend the range of substrates which presently are too small in size to sorb iodine. It appeared desirable, therefore, to search for other types of small ions which could overcome these limitations. Previous comm~nications~-~ from these Laboratories indicated the possibility of using both cationic and anionic dyes for the binding study of starch. I n the present paper some aspects of the nature of the binding of an anionic dye, congo red, and a cationic dye, methylene blue, by soluble polysaccharides are reported. Dat,a have been obtained regarding the effects of charge, chain length, and the degree of branching of the polymer on the binding affinity. I n considering the possible methods for determining adsorption, the use of equilibrium dialysis was ruled out because of the retrogradation of amylose during the time required for equilibration. The applicability of spect'ral changes has been shown for congo red. Equilibrium is achieved in a matter of seconds, and the dye follows Beer's law.4 While equilibrium values are attained instantly with methylene blue, some explanation is required regarding the adherence to Beer's law. Over a concentration range from to (1) Abstracted from the Ph.D. dissertation of Herbert C. Cheung, Rutgers, The State University of New Jersey, 1960. (2) This investigat.ion wan supported by a grant from the Corn Industries Research Foundation, Inc. (3) B. Carroll and J. R'. Van DyB, Science, 116, 168 (1952). (4) B. Carroll and J. W. Van Dyk, J . A m . Chem. S n c . , 7 6 , 2506 (1954). ( 5 ) B. Carroll and €3'. C. Cheung, J. Agr. Food Cheni., 8 , 76 (1960). (6) H. C. Cheung, El. Carroll, and (2. E. Weill, Anal. Chem.. 32, 818 (1960).
M , methylene blue goes from a solution of mainly monomers to a solution of dimers, and Beer's law is not obeyed. However, i t was found M), that over the restricted range (7 to 20 X where adsorption was studied, Beer's law was followed probably because more than 95% of the dye was in a single (dimeric) form. Beer's law also was followed by the adsorbed form of the dye. Thus, the requirements were met for using spectral changes for quantitatively determining the adsorption of a so-called metachromatic dye. Experimental Materials.-Stock solutions of methylene blue (Fisher Scientific Co ) and congo red (National Aniline Division, Allied Chemical and Dye Corp.) were prepared from histological grade materials. Both dyes were used directly without further purification; appropriate corrections for the purity of the dye were made using the spectral values reported in the literature.417 The methylene blue solution was stored in a paraffined amber bottle, and kept in the dark. All inorganic chemicals were of reagent grade. Buffers were prepared from sodium acetate and glacial acetic acid for H 5.3, and from monopotassium phosphate and sodium hyzoxide for pH 8. The ,%amylase was obtained from Wallerstein Co., Inc., New York, N. Y., and was free of the a-amylase activity. The carboxylated starches prepared by the hypochlorite oxidation process were obtained from Dr. T. J. Schoch. The carboxyl content had been determined by titration with XaOH. I n this method, the carboxyl groups were converted into the free acid form and the starch was leached with dilute acid to remove soluble materials which were titratable. Titration was then carried out in the gelatinbed sample. The degree of carboxylation was expressed on a weight basis. The high linear corn and wrinkled pea starches were obtained from Corn Products Co. The corn starch was a product of National Starch and Chemical Corp., Xew York, N. Y., and w&s defatted subsequently during the course of this work. From Dr. R. J. Dimler of the Northern Utilization Research and Development Division, U. S. Department of Agriculture, Peoria, Ill., were obtained the corn amylose 14SSP and corn amylopectin 14SSP. These substances had been fractionated in the latter laboratory using a procedure based on the Schoch method.8 With the exception of the defatted starch and the amylose, all starches were used without further purification. The amylose was treated with methanol four times and dried at 60" for 3 hr. under a reduced pressure. Moisture content was determined by heating separate portions of the substances a t 110" for 2 hr. 2. Binding - of the Dyes. Binding- was studied by . a spec. 1.
(7) A. Levine and M. Schubert, J . A m . Chem. Soc., 74, 91 (1952). ( 6 ) T. J. Schoch, Cereal Chem., 18, 121 (1941).
2586
BENJAMIN CARROLL AND HERBERT C. CHEUNQ
The average chain length of the isolated amylodextrins as well as the original amylose was obtained from their reducing values. The latter was deterniiiied from oxidation of the aldehydic groups by alkaline potassium ferricyanide, using the colorimetric procedure of Nussenbaum and Hassid.10 3. The Iodine Reaction.-The iodine sorption values were obtained from potentiometric titration using a Beckman Model G pH meter. A bright platinum electrode was used against a calomel half-cell." The colorimetric measurements were made at 615 mp. The final solutions contained 0.00370 12, 0.003% substrate, and 0.03% KI. 4. Viscosity.-A NO. 50 Cannon-Fenske viscometer was used. The efflux time for water was about 100 sec. The kinetic energy correction was assumed to be negligible. All measurements were made a t 25 f 0.1 '. 5. Analysis of Maltose Content.-A sample of starch was hydrolyzed with @-amylase. The resulting maltose upon complet,e hydrolysis was determined by an iodinethiosulfate method .12
E
5 *
Vol. 66
4
0
I=r3
2
Results
A. The Binding of Methylene Blue by Carboxylated Starches.-The effect of carboxylated starch on the spectral curve of methylene blue at M is shown in Fig. 1. Also included for comparison is the absorption spectrum of the free dye a t low concentration (10-5 M ) . These curves 550 600 650 700 were obtained a t pH 8. Addition of the substrate caused an enhancement of the p-band (615 mp) and MU a depression of the a-band (665 mp) . The spectral Fig. 1.-Effect of concentration and carboxylated starch changes may be taken as evidence of binding. on the spectral curve of methylene blue a t pH 8: dye alone a t 1 x 10-5 M ; - --dye alone a t 1 x 10-4 M ; Similar changes were observed for the free dye -.-.-. dye a t 1 X M plus 0.275 carboxylated starch when the aqueous concentration increased from (1.67% carboxyl). 10-5 to 10-4 M . No appreciable binding of methylene blue was tral technique. The extent of binding was determined detected when the Concentration of the dye was in directly in the dye-substrate mixture. Spectral measure- the vicinity of M and below. Also, no bindments were made on a Beckman DU spectrophotometer, ing was observed when the pH was below 5 . equipped with cells of 1-cm. path length. Cooling blocks were installed to permit control of temperature to within Therefore, all spectral measurements were made f0.1'. A Beckman Model G pH meter was used for pH a t pH 8 and in the neighborhood of AI. measurements. The binding data may be treated quantitatively A. Methylene Blue.-The general procedure for the bind- in terms of multiple equilibria. This procedurei3 is ing of methylene blue and carboxylated starches has been reported.6 The total dye concentration was about 10-4 M . based on the assumption of statistical binding, and The extinction coefficient of the dye was obtained by a it neglects any possible lateral interaction among differential method. For the effect of ionic strength on the the bound ions. The binding data may be repredegree of binding, a series of spectral measurements was sented by the following two equations made for each concentration of starch. Keeping the con-
l
centration of the dye fixed, the ionic strength waa varied using different amounts of buffer. A dye solution of the same ionic strength waa used as a blank. The extinction coefficient of the d e-starch mixture in the absence of salts then waa obtaineJfrom an extrapolation of the spectral data to zero ionic strength. B. Congo Red.-The present procedure for the binding of congo red by polysaccharides has been described.6 For the dependence of binding affinity on chain length, Sam lea of the substrate were prepared from corn amylose 1 4 d P which was first hydrolyzed in 6.2 N HC1. This was done b first dispersing the amylose in 1 N NaOH. The ratio oPstarch to alkaline solution was 1:20. To every 10 ml. of the amylose solution, 55 ml. of chilled concentrated HCl waa added. Then sufficient water waa added to yield a final volume of 100 ml. At given time intervals, 5 ml. of the hydrolyzed mixture was removed and neutralized with an equivalence of 1 N NaOH in a 50-ml. volumetric flask. M Five milliliters of pH 5.3 buffer and 5 ml. of 1 X congo red then were added, followed by dilution t o volume. Spectral measurements were made a t 500 mp immediately thereafter. A portion of the hydrolyzing mixture was removed after 45 min. After neutralizing with NaHCOs to methyl orange, the hydrolysate waa se arated into several amylodextrins in a charcoal-Celite cofumn. The general procedure was that of Whistler and Tu.9 (9) R. L. Whistler a n d C. C. (1952).
Tu, J . Am. Chem. Soc., 74, 3069
and
r / [ A ]= kn
- Izr
(2)
where r is the number of moles of bound dye per mole of substrate, [A] is the free dye concentration, n is the number of binding sites on the polymer, and k is the intrinsic binding constant. According to eq. 1, a plot of l/r against l/[A] should yield a straight line. This is shown in Fig. 2. The value of ra here is the ratio of moles of bound (dimeric) dye to mole of ca.rboxylate group of the starch. The intercept on the l / r , axis is l/n,the reciprocal of the number of binding sites per carboxylate group. The upper two lines (full and half-filled circles) were obtained a t an ionic strength of 0.01 for two samples of different carboxyl content. It is seen that l / n is about 2.2 (10) S. Nussenbaum a n d W.Z.Hassid, A n d . Chem., 24, 501 (1952). (11) F. L. Bates, D. French, and R. E. Rundle, J . A m . Chem. Soc., 66, 142 (1943). (12) &I. MaoLeod a n d R. Robinson. Biochem. J . , 23, 517 (1929). (13) I. M. Klotz, J . A m . Chem. Soc., 68, 1486, 2299 (1946).
Dec., 1962
IKTERACTION
2587
O F D Y E S .4ND P O L Y S A C C H A R I D E S
for both materials. The open circles represent data obtained a t zero ionic strength. Every point of this latter line was obtained by extrapolating the binding data to zero ionic strength. The value of l / n for zero ionic strength is seen to be 0.98. A 97% decre,dse in optical density was observed for the dye-substrate system when the ionic strength increased from 0.004 to 0.05. It is to be noted that in the absence of substrate the addition of electrolyte of this concentration had little effect upon the spectral properties of methylene blue. A quantitative estimate of the competitive effect of the buffer ions was evaluated by extrapolating the binding to zero ionic strength. The extrapolation was possible because the binding was not affected by the small change in pH [7-91 which was caused by the variation in concentration of electrolytes, From the binding constants of the dye a t finite and zero ionic strengths, the binding constants of the carboxylated starch for the buffer ions were calculated a t 27 and 40". This was done using a relation derived by Klotz.'3 The results are shown in Table I. The binding constants of the dye were obtained from linear plots of r/[A] us. [-4] according to eq. 2. The intercept on the r/ [A] axis is the first binding constant, ki lim [T/[A]] = 7cn = kl
TABLE I THERMODYNAMICS OF THE BINDING OF METHYLENE BLUE AT ZERO IONIC STRENGTH, A N D OF BUFFERIONS [Xa+, K+] AT 0.01 IONIC STRENGTH BY CARBOXYLATED STARCH (0.84% CARBOXYL) Dyea
+
to increase indefinitely with increase in chain length, until a chain length of about 100 to 150 glucose units is attained. The effect of chain length may be expressed quantitatively in terms of the binding constant. Using eq. 2, the values of kl,the first binding constant per glucose unit, may be calculated. The results are summarized in Table I1 for several samples ranging in chain length from 860 to about 8 glucose units. The binding constants are for the first congo red ion complexed with the linear substrate.
BINDINGO
-(O.D.), -
(O.D.)ap (O.D.)am - (O.D.)ap
where [O.D.],, [O.D.],,, and lO.D.],, are the optical densities of the sample, amylopectin, and amylose, respectively (see ref. 6). Both curves were drawn through the points obtained from the hydrolysates (open points). The filled points represent data obtained from the amylodextrins isolated by column chromatography. It is to be noted that the adsorptivity of congo red begins to level off a t about 17 glucose units in chain length. On the other hand, the binding of iodine appears
OF
TABLE I1 CONGO REDBY AMYLOSEA N D AMYLODEXTRINS AT
[ A ] -+ 0
Included in Table I are the values of the first binding constant per mole of carboxylate group, for the dye a t zero ionic strength, and 27 and 40°, and the corresponding values of ASo and AHo for both the dye and the buffer ions (Na+ and K+). These values were calculated using the standard thermodynamic relations. B. The Binding of Congo Red by Polysaccharides-The relative effect of chain length of linear polysaccharides on the binding affinity of congo red was studied using corn amylose 14SSP, and hydrolysates of the amylose. Figure 3 shows the relative adsorptivity of congo red for linear polysaccharides of various chain lengths. Included for comparison is the iodine adsorptivity obtained from colorimetric measurements. The relative adsorptjvity of an amylodextrin has been taken as the ra,tio of the spectral change for a fixed weight of substrate to that of the same weight of original (unhydrolyzed) amylose. Thus, the relative adsorptivity is given by the quantity
Buffer ions
kl a t 27" 1.74 X IO4 1.74 X 10* kl a t 40" 2.70 x 104 4.45 x 1 0 2 AHo, kcal./mole $6.33 +13.5 ASo, e.u. (cal./mole/deg.)* +40 55 5 The binding constants, kl, are for the uptake of one dye ion by one mole of carboxylate group of the substance. * These values are based on one mole of carboxyl group.
Sample
pH 5.3 A N D 23 0.1'
Average chain length
ki X 10-8
14SSP 860 glucose units 13.2 Gii 17 13.0 Giz 12 9.61 Go 8.6 8.70 G8 7.8 7.73 a The binding constants, k l , are for the uptake of one dye ion by one glucose unit of substrate.
From the data obtained a t 23 and 31°, it is possible to calculate the molar changes in entropy and enthalpy for the binding of congo red by linear polysaccharides. The values are listed in Table 111. Both AHo and ASo are positive. The degree of branching of the substrate was found to decrease markedly the binding affinity of congo red. Contained in Table I V are the first binding constants for several native starches and amylopectin. The iodine sorption values and the per cent of amylose determined from the colorimetric iodine procedure are also included in Table
IV. The effect of heating of granular amylose a t 110" in air on the binding of congo red was investigated. A t given time intervals, a portion of the heated sample was cooled to room temperature and its binding for congo red and iodine was determined. Another portion of the heated material was hydrolyzed with @-amylase, and the resulting maltose content upon complete hydrolysis was determined. Figure 4 contains the data for these heat treated samples. Here the relative adsorptivity is plotted against the maltose content, and the time of heating of the original amylose is included. The data of the dye appear to fall on a straight line, while no linear relation appears to exist for the iodine data.
BENAMIN CARROLI, AND HERBERT C. CHEUUG
2588
Tr01. 66
TABLE I11 THERMODYSAMIVS~ OF THE BINDING OF CONGO REDAND LINEARPOLYSACCHIRIDES AT pH 5.3 Chain length
0
k~ at 31'
kt at 23"
TABLE IV B I X D l S G O F POLYS4CCHARIDES FOR C O X G O 4~
REDAND I O D I N E
23"
Congo red hl x 10-3
Sample
a
-----Iodine--sorption*
% Arnrlosec
Amylopectin 1 i8 0 50 0 0" 3 io 5 20 24 5 Defatted rornstarrh 46 .5 5 25 12 0 High linear cornstarch 6 90 15 0 76 5 Wrinkled pea starch 19 4 100 Od Amylose 13 2 The binding constants, kl, were determined at pH 8 for glucose unit weight of starch. b Milligrams of iodine taken up per 100 mg. of polysaccharide. c From colorimetric measurement. Basis of calibration.
Carboxylated starches were used to study the effect of charge. Addition of carboxylated starch to congo red caused an increase in the extinction coefficient a t the spectral peak of the dye, 500 mp. This effect was similar to that caused by the addition of neutral starches. This spectral change was taken as evidence of binding. The binding values of three carboxylated starches are shown in Table
I-. BIYDIKG"O F
CONGO
T ~ B LIrE RED BY CARBOYYL4TED AT
CORNSTARCH
pH 8 ANI) 25'
lil x 10-8 0 36 1 69 0 84 4 36 OC2 oc3 1 67 3 12 The values of kl are for glucose unit weight of starch.
Sample
% carbox11
oc1
a
AH0
A
860 ghirosc units 1 32 x 104 2 09 x 104 +10 3 kea1 /mole +21 e.11. I7 I 30 x 104 2 ox x 1 0 4 +I0 0 +31 l'hr t)inding (onstants, b , , arr f o r one ghicosc unit of sirhstratr. nhctras 11w v a h r ~ ~ for s AH0 and L G ~ Oai-(' 111o1:irqiiantitirs
The effect of binding of congo red on the viscosity of corn amylose was studied a t pH 5.3 and 0.2 N NaCl. The results indicate a decrease in intrinsic viscosity from 50.0 ml./g. for the starch to 36.7 ml./g. for the dye-starch complex. Discussion The Binding of Methylene Blue. A. The Structural Form of the Adsorbed Dye.-The aband at 665 mp of the absorption curve of methylene blue (Fig. 1) is considered to be caused by the monomers of the dye The subsidiary p-band is indicative of the presence of dimers. The effect of the addition of carboxylated starch to aqueous methylene blue at lo-* M and pH 8 is similar to that caused by increasing the aqueous concentration of the free dye (Fig. 1). The same phenomenon is observedL4by adding ammonium sulfate to a very dilute solution of the dye in water (lop6&I). Since binding was detected in the present work only a t higher concentration of the dye, viz. S I , it appears that the dye was adsorbed in the dimeric form. The absence of subsidiary peaks other than the pband suggests the absence of aggregates higher than dimers. (14)
I,. Michaelis, J . Phys. Colloid Chem , 64, 1 (1950).
The ratio of monomers to dimers may be estimated for the free dye from the spectral curl-es of the dye a t X in alcohol, and a t Jl in water. This involves the assumption that, the dye exists exclusively in the monomeric form in alcohol. The extinction coefficient of the a-band is 90,400 in alcohol and is 30,000 a t LII in water. In the presence of large excess of carboxylated starch, the extinction was found to be 29,000 a t ill. It is seen that the absolute quantity of the monomeric materials is inappreciably changed upon adsorption. Thus, the correction due to change in the monomeric form of the dye may be neglected without introducing appreciable error. B. Binding at Finite and Zero Ionic Strength.Figure 2 indicates two carboxylate groups per dye ion a t 0.01 ionic strength (l/n 'V 2 . 2 ) . Since the binding was found to be dependent upon ionic strength, this value is not entirely uiiexpected. When the data are extrapolated to zero ionic strength, the value of l l n becomes 1.0 within experimental error. Figure 2 clearly shows the 1-1 correspondence between the dimeric dye ion and the carboxylate group of the starch. It is interesting to note that the significance of the value of l / n from the Klots equation (1) has been considered by other investigators in the case of protein interactions. For the case of binding of inorganic cations and albumin,lj l6 it has been assumed that the main sites of interaction are the carboxylate groups of the protein. Yet there does not appear to be any simple correlation between the number of bound cations and the number of anionic residues of the protein. I n the case of carboxylated polysaccharides at zero ionic strength, the present work indicates that it is possible to attach physical significance to the values of l/n because of the absolute method of determining carboxyl groups by titration. It appears that previous failure of other investigators to obtain reasonable agreement for the value of l / n may be due in part to the effect of buffer ions. Since the binding of proteins is sensitive toward variation in pH, it would be difficult to obtain reliable data a t low ionic strength, and hence to extrapolate the data to zero ionic strength. S o such difficulty appears to be encountered in the case of starch. C. The Thermodynamic Functions for the Binding Process.-Correction has been made for any competitive effects in calculating the binding values of methylene blue listed in Table I. A decrease of two orders of magnitude is found for the first binding constants, IC,, going from methylene blue to the small monovalent cations, Na+ and Kf. The binding constants, kl, are not the stoichiometric binding constants. To obtain the (16) E. Brand, ATLVL. .-A\ Y. A c a d . S c z . , 41, 187 (1946) (16) I. M. Klotz, a n d H. C. Curme, J . A m . Chem S o c , 70, 939 (1948).
Dcc., 1962
IYTERACTION O F DYES . O i D POLYS IPCH IIITDES
latter, T must be expressed in terms of bound dye per mole of substrate. However, kl may be considered to be proportional to the stoichiometric constant, and hence taken to be a measiire of the binding affinity. It is possible to calculate the molar change in enthalpy from temperature measurements, without knowing the stoichiometric constants. The only assumption to be made here is that n is independent of temperaturi?. Upon differentiation, the proportionality constant between k, and the stoichiometric constant drops out. The thermodynamic values are listed in Table I . Unlike the value for the change in enthalpy, the entropy change will depend upon the units chosen for the substrate. The entropy values listed in Table I have been calculated on the basis of one mole of carboxyl group. The increase in entropy for both the dye and buffer ions may be ascribed to the release of water molecules from the hydrated substrate and small ions. Such an increase appears to be characteristic of many interactions between small ions and macromolecules. A change in configuration may also contribute to the observed increase in entropy. In the absence of complexing ions, the charged carboxylated starch probably assumes a more or less extended form. Upon binding with cations, the electrostatic effect is removed so that the complex assumes a more random configuration, contributing to the observed AXQ. The strong dependence of the binding affinity on ionic strength suggests that the binding is of coulombic origin. The fact that no binding was observed below pH 5 may be taken as additional evidence substantiating this idea. It would seem desirable to compare the observed energetics with those calculated for electrostatic interaction. Direct calculation of the electrostatic free energy requires certain assumptions which may not be realistic. Instead of making a direct calculation, one may follow Iilotz's approach" by assuming that the observed binding is entirely electrostatic. Using the observed binding constants a t different temperatures, one may calculate the corresponding changes in enthalpy and entropy. This procedure leads to a valiie of $2.8 kcal. for AHo and +26 e.u. for ASo. These values are to be compared with the observed quantities of t 6 . 3 2 k d . and +40 e.u. (Table I). Suvh a calculation does not depend explicitly on the radius of the substrate molecmule. It is seen that the calculated values compare favorably with the observed values. Both the entropy and enthalpy agree in sign and in order of magnitude. 'I'ktis comparison should be considered qualitatively bccause of the precision involved in the determination of the temperature coefficient. The present iresults suggest that a carboxylated polysaccharide may be taken as a substance to test an adsorption isotherm, as for example, the Klotz equation, since the number of binding sites is well defined and can he determined analytically. When the degree of carboxylation is of a few per cent or less, the substance conforms to the simple model (17) I 31 M o t / a n 3 FI (1951).
! Fleas J
Phgs CoElotd C h r m , 66, 101
2589
I /
I
I
I
I
I
I
4
5
t
-2
3 ( 1 /A ) x I 0-4
Fig. 2.-Extrapolation to determine the number of binding sites per carboxylate group for the binding of meothylene blue and carboxylated starches a t pH 8 and 27 . The upper two lines are for an ionic strength of 0.01: half-filled circle for OC3 (1.67% carboxyl); full circle for OC2 (0.84y0 carboxyl); open circle for zero ionic strength (OC2); [A], molar concentration of free dye; To, specific adsorption per carboxylate group; total dye ca. 1 x 10-4 I f .
CHAIN LENGTH,
GLUCOSE UNITS
Fig. 3. -Relative adsorptivity us. chain length for the binding of congo red and iodine by amylose and amylodextrins. Upper curve for congo red, lower curve for iodine. Both curves are drawn through data obtained from the binding of hydrolyzed amylose (unfilled points). Included are three points which were obtained from fractionated amylodextrins (filled points). Conditions for spectral analysis: congo red: dye 1 X, 10" X , substrate 0.005qc, NaCl 0.062 N , pH 5.3; iodine: IZ 0.003cc, KI O.O3a, YaC1 0.025c2, substrate 0.0027c.
used in those adsorption equations where one assumes the absence of lateral interaction of the adsorbate molecules. The Binding of Congo Red. A. Linear Polysaccharides.-The binding of congo red by linear polysaccharides is fairly independent of chain length for substrates larger than 17 glucose units in size (Fig. 3). For the iodine interaction, however, there is a steady increase in the relative adsorptivity. The latter effect is well known. The adsorptivity of congo red for the fractionated materials falls on the same curve as that for the un-
R ~ J A M ICARROLL K AND HERBERT C. CHEUNG
2590
Vol. 66
fractionated hydrolysates. The iodine data indicate a lower affinity for the fractionated amylodextrins. This is not unexpected since the hydrolysates have a random distribution of molecular fragments and iodine is quite sensitive to the larger fragments. With the fractionated materials, there is an absence of the larger molecules, resulting in the lower adsorptivity for iodine. Because the dye binding is relatively insensitive toward the larger molecules, one would expect the fractionated materials to behave similarly to the unfractionated hydrolysates. The values of kl in Table 11, as in the case of methylene blue interaction, may be taken as a measure of the relative binding affinity. Thus, the binding affinity is approximately constant for long chain materials, the chain length studied ranging from 860 to 17. Below G17 (17 glucose units), there is a steady decrease in the values of k, with decrease in the size of the substrate molecule. Although the decrease is small, the trend is apparent. No attempt is made to obtain the intrinsic binding constant k and the value of n. Unlike the interaction of methylene blue and carboxylated starch, for which l / n is expected to approach unity, the uncertainty involved in the extrapolation of eq. 1 to obtain l/n is very large. This is the case because l / n here is expected to be different from unity. In this connection, Scatchard'* has suggested a plot of r / [A] vs. T (eq. 2). In this case even if eq. 2 is used, n is still burdened with a large error since its determination involves an extrapolation in the region of large r, and hence large [A], a region in which experimental data are poor. The binding of azo dyes has been reported for polyvinylpyrrolidone and polyvinyl alcohol by Sch01tan.l~ Hydrogen bonding was believed to be responsible for the binding. However, Frank and co-workersZ0 have expressed doubt on hydrogen bonding for the polyvinylpyrrolidone complex. I n the present system of congo red and polysaccharides, hydrogen bonding appears to be unfavorable because of the positive value of AHo (Table
111). The positive entropy effect2' may be attributed to the release of hydrated water molecules from both the dye and the substrate upon binding. It is of interest to note that the observed energetics listed in Table I11 are consistent with those characteristic for the formation of a hydrophobic bond.22 However, i t is difficult to conceive that this is the driving force in the binding of polysaccharides because of the predominant hydrophilic nature of the substrate. B. Branched Polysaccharides.-Native starch contains both the linear component amylose, and the branched component amylopectin. As indicated by the iodine values (Table IV), the binding of congo red increases with increasing amylose content, or with decrease in the degree of branching. A 700% increase in the binding constant over that for amylopectin is observed for amylose. The binding constants for the defatted and high linear corn starches are in fair agreement with the weight averages of the binding constants of the individual components. The agreement for the wrinkled pea starch is somewhat poor, being about 15% higher than the estimated value. It is well known that there are two major effects in the heating of granular amylose. One is reduction of chain length and the other is branching. Consider the data in Fig. 4, where the relative change in optical density of congo red and iodine is plotted against the maltose generated from the heated corn amylose. The amylose originally had a chain length of 860 glucose units. Reduction in chain length by a factor of 2 or 3 would not appreciably affect the adsorptivity of either indicator, as can be seen from the data in Fig. 3. Thus, the changes in optical density in the early stages of the heat treatment may be ascribed largely to branching. Since the maltose content may be taken as a measure of branching (increased branching yielding a lower amount of maltose), a linear relationship would be expected if the adsorptivity of congo red were determined primarily by the degree of branching. This is indeed the case. Apparently, the size of the starch fragments a t the end of the longest heating was still sufficiently large so as to make the behavior of the dye reflect changes in branching only. Iodine was found to respond differently. In the beginning of the heating, the adsorptivity of iodine changed slightly compared to that for congo red. This would be due to the relative insensitivity of iodine toward branching. With amylose subjected to prolonged heat treatment, iodine passed into a region of greater sensitivity because the extended fragmentation of the amylose in addition to branching has caused a marked drop in the iodine adsorption. It is not certain at this stage as to how much information one may obtain from the binding data concerning the degree of branching of the substrate molecule. The interaction of the dye is not specific for starch, but is a general phenomenon with many substances. It would appear that the adsorption procedure may prove useful in determining the
(18) G. Scatchard, Ann. AT. Y. Acad. Sci.,51, 660 (1949). (19) W.Scholtsn, Makromol. Chem., 11, 131 (1953). ('20) H. P. F r a n k , S. Barkin, and F. R. Eirich, J . Phus. Chem., 61, 1375 (1967).
(21) T h e e n t r o p y values here have been calculated on t h e molar basis of the substrate. (22) W.Kauamann, American Chemical Society National Meeting, Atlantic City, N. J., September, 1959.
HEATING T I M E
20.21 (1L
t---
I
I
20
40 60 80 % THEORETICAL MALTOSE
IO0
Fig. 4.-Effect of heating of solid amylose on the binding of congo red and iodine. Relative adsorptivity vs. per cent of theoretical content of maltose obtained upon complete hydrolysis of the heat-treated solid amylose with @-amylase. The numbers immediately above the points indicate the time of heating of the solid starch. Conditions for the binding studies identical with those in Fig. 3. Open circle, congo red; half-filled circle, iodine
EFFECTS O F IRItADIATION
Dec., 1962
ON
CATALYTIC ACTIVITYO F MAGXESIUM OXIDE
degree of branching in high polymers. This has been shown to be the case for s t a r ~ h . ~ C. Carboxylated Polysaccharides.-The observation of the binding of congo red by carboxylated starch is in contrast with the staining of oxidized granular No staining has been observed with anionic starches and anionic dyes. Also little or no staining has been detected with anionic dyes and neutral starches, such as cornstarch. While no information is available on the molecular weights and chain length of the carboxylated starches, it would seem reasonable to assume that chemical modification of the starch does not reduce the molecular weight much beyond an order of magnitude. The conclusion appears to have support due to the fact that the average value of the binding constants (Table V) for the carboxylated starches is reasonably close to that for native cornstarch (Table IV). At pH 8 the carboxylate groups are expected to be completely ionized. The data, therefore, suggest that the binding is not influenced to any appreciable extent by the presence of the carboxylate groups. This is contrary to prevailing opinion that an anionic dye will not bind an anionic substrate. The data also suggest that previous failure to detect staining of negative starches by negative dyes in the solid state may not be entirely an electrostatic effect, as has been suggested.23 Perhaps a contributing factor in the binding of dyes (23)
T.J. Schoch itnd E. C. MayPrald, Anal. Chem., 28, 382
(1956).
259 1
by starches is the internal flexibility of the polysaccharide chain. The binding in solution may be considered to be a consequence of the loss of rigidity of the starch molecules. An implication of this suggestion is that the reduction of the binding of amylopectin may be due in part to the rigid frame of the substrate. This is in accordance with the concept of configurational adaptability advanced by Karush.2 4 It is conceivable that the configurational changes are favored by complexing with certain small ions. Thus there is established on the surface of the substrate a measure of complementarity with the surface of some small ions approaching it. An important feature of this concept is that complementarity need not imply specificity. As has been pointed out by Kauzmann,25there exists the probability, in some instances, that the specific complementarity structure may be developed only in the presence of certain small ions. This appears to be the case for the amylose-iodine helix. D. Interpretation of Viscosity Data.-The lowering in intrinsic viscosity (from 50.0 to 36.7) indicates a reduction in the hydrodynamic volume of the starch-dye complex over the starch. The complex is more compact than the substrate since [q]a[?]3/2, where r is the radius of gyration of the macromolecule. (24) F. Karush, J . A m . Chem. Soc.. 7 2 , 2705 (1950). (25) W.Kauzmann, Rea. M o d . Phys., 31, 546 (1959).
EFFECTS OF NEUTRON AND ULTRAVIOLET IRRADIATION ON THE CATALYTIC ACTIVITY OF MAGNESIUM OXIDE BY JACK H. LUNSFORD AND THOMAS W. LELAND,JR. Department of Chemical Engineering, Rice University, Houston 1, Texas Receiaed December 11, 1961
This work further develops a technique for changing the electronic and defect structure of magnesium oxide catalysts. Relatively small doses of ultraviolet and neutron irradiation have been found to enhance the catalytic activity of MgO for the reaction Hf Dz F? 2HD. The enhancement by radiation depends on the extent of degassing of the catalyst prior to irradiation; the less active samples are more sensitive to irradiation. For samples activated a t 291 ultraviolet-irradiated MgO reache8 a precise saturation value of catalytic activity, while neutron-irradiated samples continue to increase in activity upon further irradiation. Both types of irradiation produce a large initial increase in activity. Changes in activation energy depend on the thermal activation and type of irradiation. The activation energy either increases or remains unchanged for neutron irradiation, while ultraviolet irradiation does not cause a change. For this system, a simple model based upon Fe+s as the irradiation-induced site has been developed. Other possibilities are discussed. It has been shown that most of the activity change must be attributed to changes in the electronic structure of the crystal rather than in the number of lattice imperfections.
+
O,
Introduction The purposes of this investigation were to observe how low neutron fluxes and ultraviolet irradiation affect catalytic activity, and to relate changes in the solid state to the catalytic activity by proposing a model for irradiation-induced activity. The model proposed is not set forth as the unique mechanism for the effect of irradiation on catalytic activity, but it should serve as a proposition toward which other research efforh can be directed. The model is produced by comparing radiation cfl'ccts on the bulk solid with the effects on catalysis. Experiments involving the irradiation of catalysts have several interesting advantages. They
can be conducted on a single sample of a catalyst, with no complications from sample variability, and without the introduction of extraneous changes, as by alloying. The effects are largely local, and some of the properties suggested as important in active centers (paramagnetism, local strain, abnormal atomic spacing) can be associated with the point defects. Furthermore, by making comparisons between radiation effects on the bulk solid and effects of similar radiation on the catalytic activity, it is possible to gain some insight into catalytic mechanisms. It appears that the magnitude of the effect of irradiation on catalysts is inversely proportional to
