Water-in-oil-in-water double emulsions for food applications: yield analysis and rheological properties

Food Hydrocolloids Vol.d no.5 pp.339-353, 1991

Water-in-oil-in-water double emulsions for food applications: yield analysis and rheological properties Bruno de Cindio l , Gianni Grasso? and Domenico Cacace/ Department of Chemical Engineering, University of Naples, Piazzale Tecchio, Naples, Italy

'Also: SME Ricerche Localita 'La Fagianeria', Piana di Monte Verna (Caserta), Italy 2 Guest

researcher

Abstract. Water-in-oil-in-water (W/OfW) emulsions are systems in which a water-in-oil (W/O) emulsion is dispersed in a second aqueous phase. The W/O emulsion exists in the suspending aqueous medium as oil globules containing smaller water droplets. In Ihis work the techniques of preparation and yield analysis of W/OfW emulsions are described; microstructure and rheological behavior are also studied with a view to possible food applications. In order 10 prepare W/O/W emulsions, a two-step emulsification technique was used. During the first step, a W/O emulsion was prepared in the presence of a lipophilic emulsifier while, during the second step, Ihe W/O emulsion was mixed with an aqueous solution of a hydrophilic emulsifier to rrovide the W/O/W system. The yield of W/OfW emulsion was evaluated by measuring the amount of an ionic marker (NaCl) which migrated from the dispersed globules 10 the continuous aqueous phase, using dialysis to separate the NaCI migrated from the aqueous medium and using ionic chromatography to detect the amount of the NaCI outcome. The microstructure was investigated by making phase-contrast microscope photographs and estimating the size distribution of the oil globules. Finally, the rheological behaviour of W/O/W systems was studied by means of both steady-shear and oscillatory measurements.

Introduction

Double emulsions are systems in which the dispersed phase is itself an emulsion (1-3). Therefore, it is possible to define 0I/W/0 2 systems (an 0I/W emulsion dispersed in a continuous oil phase 02) and W 1/O/W2 systems (a W1/O emulsion dispersed in a continuous water phase W 2 ) . Focussing our attention on the latter (Figure 1), we can depict it as two aqueous phases separated by a thin oil layer, stabilized by means of a monolayer of molecules of the emulsifying agents at both the O/w interfaces; this is similar to what happens in cellular membranes or in liquid crystals (4-5). The W1/O emulsion is suspended in the W2 phase as oil globules containing one or more water droplets, hereafter called internal aqueous compartments. W/O/w emulsions have already found industrial applications in various fields and are being carefully considered for further possibilities (6). The main applications are related to the capability of encapsulating in the internal compartments some water-soluble substances that are then slowly released, in a controlled way. The use of W/O/w emulsions for controlled release in pharmaceutics (7-8) and cosmetics is well known. Another advantageous feature of these systems is that they have both an aqueous internal phase and an

© Oxford University Press

339

Bide Cindio, G.Grasso and D.Ca cace

aqueous internal compartllents

oil globules (W/O)

(W 1 )

aqueous external phase (W:z)

Fig. 1. Structural model for a W /O /W emulsion.

aqu eous external phase. Thus we can encapsulate water-soluble components in emul sion s for which an oil external pha se is not tolerated (e .g. in cosmetics). Thi s is an essential advantage for the food industr y (3 ,9), where an external water phase is more acceptable, in terms of palatability, than an oil one. On this basis, a lot of new products (10) have been patented in the form of W/OfW emul sions, as salted creams (enc apsulation of salt), aromatic mayonnai ses (enca psulation of some aromas) etc . Further food appli cations of W/OfW emulsions are possible in the dietetic field. In fact we can prepare a W/OfW system having the same volum e fraction of dispersed phase and the same texture as a simple emulsion, but a lower oil content, due to the existe nce of the aqueous compartments in the oil globules. Thus, low-calorie mayonnaises and other similar foodstuffs may be easily produced (3). In this paper the two-step prepar ation method and a new simple procedure for estim ating the percentage of the double emulsion obt ained , based on ionic chromatography, are described . The rheological beh aviour of the W/O/W emul sions is studied by means of both steady-shear and oscillatory measurements and related to the microstructural properties, estimated by means of micrographs of the prepared syste ms. Theoretical background

Emulsion preparation The final characteristics of a W/OfW emulsion depend on a lot of parameters

340

Water double emulsions

which are fixed during the preparation procedure. Apart from the operational degrees of freedom, such as stirring temperature, time and speed, the formulation parameters playa very important role. Among these, we should consider the volume fraction of water in the initial simple W/O emulsion (wo) , the volume fraction of the simple emulsion W/O in the final W/O/w system (wow), and the type and concentration of both lipophilic (Cj) and hydrophilic (e2 ) emulsifiers. In fact , in order to obtain a W/O/w emulsion , it is necessary to use two different emulsifiers. The first one has a mainly lipophilic character and is dissolved in the oil phase to form the W [/0 emulsion. The second one has a mainly hydrophilic character and is dissolved in the external aqueous phase W 2 to disperse in this latter phase the simple WI/O emulsion globules (11). W/O/w emulsions are usually prepared by three different methods: (i) Mechanical agitation, at a very high stirring speed, of a mixture of an aqueous solution of a hydrophilic emulsifier and an oil phase containing a lipophilic emulsifer (12). (ii) Phase inversion: in fact, by increasing the concentration of the oil dispersed phase in a WIG emulsion, before obtaining the inverted O/W system , it is possible to observe an unstable mesophase characterized by the formation of a W/O/W emulsion (13,14). (iii) Two-step emulsification (15) . This procedure seems to be the most reliable. It consists , essentially, of preparing a W 1/0 simple emulsion using a lipophilic emulsifier (first step) and dispersing it in a second aqueous phase W 2 with the aid of a hydrophilic emulsifier (second step) . Yield analysis

During the preparation procedure, there is formation of both simple and double emulsions. Therefore, once the dispersion has been prepared, it is necessary to estimate the percentage Y of W/O/w emulsion in the whole system. It may be evaluated by dissolving, during the preparation, an ionic marker (e.g. NaCl) in the internal aqueous phase WI and by measuring, after the preparation, its concentration in the external aqueous phase W2 . It is possible to make the following assumptions. (i) The marker may be transferred from the WI to W 2 phase only by rupture of the oil layer. Therefore we may neglect the amount of marker that crosses the untouched membranes by molecular diffusion, according to the ionic nature of the marker. (ii) A W/O/W emulsion breaks down only by rupture of the oil layer. Thus, the amount of marker found in the W2 phase is directly related to the extent of the oil layer rupture (hypothesis i) and therefore it is a measure of the yield of the W/O/w emulsion (hypothesis ii). We can assign to Y the value 100% when the concentration C of marker in the external phase is 0 (no passage of salt, no rupture ofthe oil layer, total formation of a W/O/w system) and the value 0% when we find in the external phase the concentration Cm a x corresponding to the total amount of the marker initially 341

B.de Cindio, G.Grasso and D.Cacace

dissolved in the internal phase. Assuming a linear relationship (15) between Y and C, the following equation for Y is obtained: Y = 100

(1 _ _ C_) Cm ax

(1)

Experimental

Materials The water phase W l is a solution of NaCl (Sigma, ACS grade) at the concentration indicated in the following. The water phase W2 is a solution of polyoxyethylene sorbitan monooleate (Tween 80; Sigma, technical grade; HLB = 15). As oil phase 0, a mixture of liquid paraffin (Baker Chemicals, density 0.88 g/cm') and sorbitan monooleate (Span 80; Sigma, technical grade, HLB = 4.3) was used. Double-distilled water was used throughout all the experiments. Apparatus For both emulsifying steps a mechanical stirrer (Heidolph RZR 20(0) was used. It was equipped with a rectangular shaped paddle (50 x 120 mm, thickness 2 mm) in the following named 'rotor l' or, alternatively, with a three-blade propeller (diameter 20 mm), named hereafter 'rotor 2'. To prepare the WIO emulsion, a Pyrex cylinder (50 mm height, 90 mm diameter) was used. To obtain a homogeneous system, three glass pins (35 mm height, 6 mm diameter) were fixed vertically from the bottom of the cylinder and symmetrically placed 2 mm from the walls, as suggested in ref. 15. The chromatographic measurements (16) were performed with an ionic chromatograph Dionex QIC equipped with an anionic exchange column HPICAS4A and a pre-column, with a conductivity suppression membrane AFS-l and a conductometric detector. A Dionex 4270 computing integrator was used as data acquisition system. Microphotographs were obtained by means of a phase-contrast Zeiss microscope equipped with a photocamera. A Haake-Rotovisco RV20 viscometer was used for all rheological tests. The shear flow was obtained by means of two coaxial cylinders (D; = 27.83 mm, Do = 30.00 mm), using the CV100 measurement system and an ME30 sensor; for the oscillatory flow, a parallel-plate geometry was used (D = 41.74 mm, gap = 0.5 mm), equipped with a CVl00 system and a PQ45 sensor. Emulsification procedure

°

Firstly, the phase was prepared by mixing measured volumes of liquid paraffin and Span 80 in the described container and homogenizing the suspension by means of rotor 1 for 5 min at 100 r.p.m. Then a volume of V W l ml of theaqueous solution of NaCl (the phase Wl) was slowly (1 ml/min) added from a 50 ml burette by means of Hoffman tubing clamp while stirring at 200 r.p.m. When the 342

Water double emulsions

addition was completed, the mechanical agitation was continued for a further 5 min at 300 r.p.m. to obtain the simple emulsion WI/O (P w o g). The W2 phase was prepared separately by shaking a solution of doubledistilled water and Tween 80. This latter phase was added all at once to the WII o system and the mixture was stirred by means of rotor 2 for 10 min at 300 r.p.m. to obtain the final W I /O/W2 emulsion (P wo w g). The whole procedure was found to be the optimum after several attempts, and it gives results very similar to those reported in Ref. 15. Dialysis procedure

A sample of b grams of the prepared W/O/w emulsion was placed in a cellophane tube and dialyzed against a volume of 0.5 I of double-distilled water at room temperature. It was necessary to wait 48 h to reach dialysis equilibrium; afterwards, samples of the dialysed solution were chromatographed for determining Cl" concentration. Chromatographic procedure

50 J.11 volumes of the W/O/W samples or standards were automatically injected in the column. Analytical solutions of NaCI at 0.1, 0.25, 0.5,1 and 2 mg/l were used as reference standards. The optimal conditions for the chromatographic separation of the desired ionic species (CI-) from others present as impurities in the dialyzed solution, were found by eluting with a moderate eluting solution (NaHC0 3 1.7 mmoVdm3/NazC0 3 1.8 mmol/drrr'), A flux of 1.8 mllmin for the eluting solution and a flux of 2.5 mllmin for the suppressor solution (H 2S04 0.025 mmol/drrr') were used. Yield computation

When using a volume of V W 1 ml of NaCl solution (concentration Cm gil) as WI phase, in the W/O/w emulsion (weight P wo w g) there will be Cm V W I mg of NaCI. Only Cm V W 1 blPwo w mg of the latter will remain in the b grams ofW/OI W emulsion taken for the dialysis. Referring to the V d litres of dialysis solution, this amount corresponds to a Cl" concentration Cma x of 35.47 Cm VW I b158.36 Pwo w V d mg/l. Thus, substituting in equation (1), the following expression has been found for the yield: Y

= 100 (1 _ 1.65

Pw o w C V d )

Cm V W I b

(2)

Equation (2) is valid when it is possible to assume that: (i) all the salt passing through the oil layer is able to penetrate through the dialysis membrane and therefore will be found in the dialysis solution and (ii) all the salt present in the dialysis solution comes from that dissolved in the WI phase of the W/O/W emulsion (i.e. there are not other sources of NaCI in the system). In the case of our experiments, the above assumptions were found to be only 343

B.de Cindio, G.Grasso and D.Cacace

partially true and therefore it was necessary to define two correction factors. To correct for the deviations from the first hypothesis, we ha ve introduced a factor a, defined as the fraction of CI- ions that can migrate through the dialysis membrane within 48 h. It was estimated by dialysing a simple NaCl aqueous solution and measuring the percentage of NaCI which penetrated through the dialysis membrane within 48 h. Thus , instead of C, the concentration C' = Cia should be used. To correct for the deviations from the second hypothesis, we have introduced a factor ~, defined as the amount of Cl" (expressed in mg and referred to 1 g of W/OfW emulsion) coming from impurities present in the materials or in the surroundings. It was estimated by preparing and dialysing a W/O/W system where the WI phase did not contain NaCl. Thus, in the b grams of sample, there are b ~ grams of Cl" having sources other than the WI phase; therefore they must be subtracted from the C' Vd mg of CI- ions found in the dialysis solution. Therefore, the true concentration of CI- coming from the WI phase is C' = (C' V d - b ~)lVd' Equation (2) thus becomes: Y

- a ~ b)] Cm V W 1 ex b

= 100 [1 _ 1.65 Pw o w (C Vd

(3)

During our experiments we have used the following standard amounts: Cm = ~ were found to be 0.78 and 2.94 x 10- 3 mg/g respectively. Thus, finally the following equation has been obtained:

6 gil; V d = 0.5 1; VW I = 6.6 ml . The average values of ex and

Y = 100 [1 - 0.0021 P w o w (0.195 C - 5.88 x 10b

3 )]

(4)

where Pw o w and b are expressed as g units and C as mg/1. Rh eological measurements

Shear stress and viscosity were measured, at 20cC, according to the following deformation history: Increasing shear rate from 5 to 200 S-1 Constant shear rate (200 S-I) for 1 min Decreasing shear rate from 200 to 5 s-\ The W/OfW emulsions were also tested during oscillatory shear flow at small oscillation amplitude (15 mrad) in the frequency range 0.8 -;- 30 s- \. Storage and loss moduli G' and Gil were evaluated to analyze the viscoelastic behaviour of the systems. Results and discussion Yield

Five different W/O/W systems were prepared and analysed by fixing the 344

Water double emulsions

following parameters according to literature data (15): C j = 40%; C2 = 1%: cPwo = 0.15. In Table I are shown the values of cPwow used to prepare the tested emulsions. We have focussed our attention on this latter parameter in view of its importance for food applications; in fact, it is directly linked to the total amount of oil present in the system. Four samples were prepared separately for each system to test the repeatability of the experiments. For each system in Table I the values of C and those estimated for Y for the four samples, the mean values YI11 and their absolute standard deviations s, their confidence intervals and the variation coefficients are reported. The mean value of the variation coefficient is 5 -i- 6% showing a good reproducibility of the preparation and analysis methods. In Figure 2, Y is plotted versus wow. The mean values Y111 and the confidence intervals are also shown. The results suggest that: (i) Y is an increasing function of cPwow, i.e. to obtain a high yield of double emulsion we should use a large volume fraction of simple emulsion in the final double system. (ii) Y quickly increases until cPwow = 0.3, then it assumes a constant value. (iii) At low values of cPwow, Y already assumes a satisfactory value, and for cPwow 2:: 0.3 we can say that the W/O/W emulsion is completely developed. This behaviour may be explained in the following way. At higher values of cPwow, the oil globules are more closely packed in the aqueous continuous phase (Figure 3) and there is a large reduction of the water activity at the oil-water interfaces. This phenomenon makes the two main breakdown pathways (17) of a W/O/W system very difficult (i.e. the water transfer through the oil layer and the destruction of this membrane by means of the micelles of emulsifiers dispersed in the system). These results show that it is possible to prepare W/O/W emulsions having very high yield and relatively low values of cPwow as well as low oil content «25%) because a further increase of cPwow does not influence Y. This may be interesting in the food industry for the possibility of reducing the fat content in Table I. Results and statistical analysis' System

1 2 3 4 5


0.1 0.2 0.3 0.4 0.5

Ym 2

3

v.c,

c.i.

10.3 8.9 4.0 2.1 2.2

9.4 11.6 5.9 3.0 3.3

4

C

Y

C

Y

C

Y

C

Y

0.55 0.31 0.32 0.23 0.15

55.6 88.7 92.6 92.2 94.4

0.54 0.40 0.14 0.17 0.18

65.3 87.4 97.6 95.6 94.3

0.42 0.54 0.49 0.25 0.31

57.2 77.0 88.9 93.0 91.2

0.57 0.53 0.29 0.39 0.13

51.2 74.1 90.9 91.2 96.1

57.3 81.8 92.5 93.0 94.0

5.9 7.3 3.7 1.9 2.1

For each system: Y m = Y mean value in the four samples, s = standard deviation; v.c. = 100 slY m = variation coefficient; c.i. = 10. 95 s = confidence interval (where 10. 95 is the Student's 1 value for the 95% confidence level) . • C = measured concentration of Cl ions in the dialysis solution (mg/l); Y = estimated percentage of the W/O/w emulsion obtained.

345

B.de Cindio, G.Grasso and D.Cacace

100..,...---------------------------,

90

80

-

~ 70

'-'

60

50

40 -I---..---,..---T"""-.......--.----...---...--.......--.----.--....---1 0.0 0,5 0.•1 0.2 0,3 0.4 0.6

w/o/w Fig. 2. Yield of the double emulsion (Y) versus the volume fraction of the W/O emulsion in the W/O/ W emulsion (wow).

products that nevertheless retain the typical taste and texture of high-fat food as for example mayonnaises. Structure

By analysing micrographs taken just after the emulsion preparation, the diameters of the oil globules were estimated for the five prepared systems (
In Figures 7-9 are reported the experimental data of storage and loss moduli 346

Water double emulsions

25

A

Fig. 3. Micrographs of samples of W/OfW emulsions. (a)
}.J. I

= 0.1;

(b)
versus oscillation frequency, and the viscosity values versus shear rate for the five prepared W/OfW emulsions. The reported values are averaged on three different samples of the same system, tested immediately after their preparation. 347

B.de Cindio, G.Grasso and D.Cacace

30,..----------------------,

.... C\I C\I

.- 20

-....= ~ C

.~

=

,.Q

.~

.tI CI) .~

"l:l 10 ~

....>

.~

nl

~

~

o

o

(~m)

Fig. 4. Oil globule diameter percent distribution for a W/O/W system (wow = 0.3).

20

-r-------------------------,

18

S

c

16

14

12 +--.----y0.0 0.1

-,.--r----y0.2

0.3

w/o/w Fig. 5. Oil globule mean diameter versus wow.

348

-.----,r----r-""T'""--! 0.4

0.5

0.6

Water double emulsions

25

}J I

Fig. 6. Micrograph of a sample of W/OIW emulsion: the breaking down of an oil globule allows the observation of the aqueous internal compartments.

0

0.5 0.4 0.3· 0.2 0.1

-1

ns

C\ ~

be

0

-2

-3 +------..---r----.---"""T'"-----,....-------1 -1 o 2

log

(i)

(1/s)

Fig. 7. Storage modulus (G') versus oscillation frequency w for the five prepared W/O/W systems (.pwow = 0.170.5).

349

B.de Cindio, G.Grasso and D.Cacace

O-r------------------------, 0.5 0.4 0.3

D

-

0.2 0.1

-1

CO

e

-2 -+----.-----r---...---"""T---......-----; 2 o -1

log

Q)

(lis)

Fig. 8. Loss modu lus (G") versus oscillatio n frequen cy (WQW = 0.1 70.5) .

for the five pr ep ared W /OfW syste ms

w

-1,4

• • • • • • • • • • • • • • •

-1,6

0

0

.0.5

• •

0.4

-1,8

.-. III

a

iU

C! l:'"

-

a

a

a

a

a

a

a

• • • • • • • a a • • •

-2,0

00 0

0.3

0.2

-2,2 iii

a

c

EI

l:I

C

El

l:J

l:I

EI

0.1

-2,4 0

2

log

y

3

(lis)

Fig. 9. Apparent viscosity versus shear rate for the five prepared W/OfW systems (wow 0.1 7 0.5).

350

=

Water double emulsions

In the steady shear flow measurements, at each shear rate, the viscosity values at both increasing and decreasing shear rate were not significantly different; therefore their mean value was considered. The measurements at constant shear rate gave constant viscosity values and therefore it may be assumed that the tested double emulsions did not show any time-effect on the rheological properties within the experimental time. From the oscillatory results it appears that the tested systems show similar values of G' and G" at each frequency; it means that both the elastic (G') and the viscous (G") contributions to the rheological behavior are of the same order of magnitude. Both G' and G" show an approximate linear behaviour in a log-log plot; therefore the following experimental models were assumed, and the best fitting parameters were estimated

G' = A' w b '

(5)

= A" wb"

(6)

G"

In Table II, A' , b', A" and b" estimated values with their correlation coefficients are reported for the five tested emulsions. It may be observed that the value of b is -0.5 for ali the systems. This result was interpreted according to Bohlin's theory of flow (18,19) which relates the rheological behavior of the dispersed systems to their microstructure, modelled as a network where the interactions between the rheological units are represented by the coordination number z. This latter parameter may be evaluated by the equation (5) as z = lib. Therefore for our systems a value of z = 2 was found. According to the theory, this corresponds to a monodimensionallamellar microstructure ('necklace'). The same approach has been applied to the steady-shear measurements; a slow decay of the apparent viscosity versus shear rate was found and therefore our systems exhibit a small non-Newtonian behaviour. The following model was found for the shear stress: T

=

c-r

(7)

In Table III the C and d estimated values are reported for the five tested W/O/w emulsions. An approximate value of 1 was found for the d parameter. Referring Table II. Coefficients for G' and G" moduli wow

A'

b'

r"

A"

b"

r

0.1 0.2 0.3 0.4 0.5

0.0105 0.0122 0.0218 0.0223 0.0372

0.5225 0.5564 0.5121 0.5606 0.4953

0.93 0.97 0.99 0.97 0.98

0.0103 0.0137 0.0215 0.0216 0.0348

0.5567 0.6268 0.5341 0.6438 0.5882

0.94 0.95 0.98 0.98 0.99

*,

= Correlation coefficient of the measurements.

351

B.de Cindio, G.Grasso and D.Cacace

Table III. Coefficients for the shear stress model wow

c

d

r

0.1 0.2 0.3 0.4 0.5

0.0064 0.0118 0.0160 0.0333 0.0388

0.9407 0.9540 0.9934 0.8990 0.9058

-1.00 -0.96 -0.99 -0.97 -0.97

"

= Correlation coefficient of the measurements.

400

-r---------------------___,

300

100

O+---r--...,.----r--...,..---.--~---r-___,r__-r----l

o

2

3

4

5

w/o/w Fig. 10. Relative viscosity versus wow.

to the Bohlin's theory, it allows one to estimate z also as the exponent of equation (7). The unit value of z so found implies that there are no superstructural rheological units. It is only apparently in contrast with the previous result because the oscillatory tests do not alter the structural equilibrium, while shear flow destructures the system by breaking the network. Viscosity then depends only on the amount of the single droplets. Finally, 'l'Jrel versus wow is reported (Figure 10); the relative viscosity values were extrapolated for "V-O to eliminate the shear effects. A quadratic model correlates very well the experimental data; it confirms that there is only a low interaction between the rheological units.

352

Water double emulsions

References 1. Matsumot o ,S. (1985) In Shah,D .O . (ed .) , Macro and Microemulsions: Theory and Applications . AC S Sympos ium Ser ies. Ame rican Che mical Societ y, Washington, pp . 272, 415. 2. Mat sumot o ,S. (1986) J. Texture Stud. , 17, 141. 3. Matsumoto ,S. (1985) In Schick,M.J. (ed.), Non-Ionic Surfactants: Physical Chemistry. Marcel Dekker , New York, p. 549. 4. Huang,C. and Th ompson ,T .E. (1966) J. Mol. Bioi. , IS , 539. 5. Cass ,A . and Fink elstein, A. (1967) J. Gen. Physiol. , 50,1 766. 6. Davis,S. (1981) Chemistry and Industry , Oct. 3, 683. 7. Herbert,W . (1965) Lancet, ii , 771. 8. Engel ,R. , R iggi,S. and Fahrenbach ,J. (1968) Nature, 219, 856. 9. Matsumoto,S ., Koh ,H . and Michiur a,A . (1985) J. Dispers. Sci. Techno/., 6, 507. 10. Matsumoto ,S. and Kohd a ,M. (1979) In Sherman ,P. (ed .), Food Texture and Rheology. A cadem ic Press, London , p. 437. 11. Fre nkel,M . , Shwartz,R. and Ga rti,N. (1983) J. Call. Interface Sci., 94, 174. 12. Matsumoto,S., Makino,H. and Ueno,Y . (1987) J. Japan Oil Chem, Soc. , 87, 320. 13. Dokic,P ., Sherman,P. (1980) Call. Polym . Sci., 258,1159. 14. Matsumoto ,S. (1983) J. Call. Interf ace Sci., 94, 362. 15. Matsum oto ,S., Kita,Y . and Yonezawa ,D . (1976) J. Call. Interface su.. 57, 353. 16. Gr asso ,G . and Bufalo ,G . (1988) J. Chromatogr. , 454, 411. 17. Flore nce, A .T. and Whith ehill ,D. (1981) J. Call. Interface Sci., 79, 243. 18. Bohlin ,L. (1980) J. Call. Interface Sci., 74, 423. 19. Bohl in,L. (1979) J. Call. Interface s«, 69, 194.

Received on Novem ber 13, 1989; accepted on November 5, 1990

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