Food Hydrocolloids Yol.7 no.5 pp.397-405, 1993

Osmotic pressure measurements for gellan gum aqueous solutions

E.Ogawa

Showagakuin Junior College, Ichikawa, Chiba 272, Japan

Abstract. Osmotic pressure measurements for aqueous solutions of a gellan gum and standardsamples, such as three kinds of pullulans and a dextran, were made to determine number averagemolecular weights Mn and osmotic second viral coefficients A 2 using a Hewlett-Packard high speedmembrane osmometer. From the measurements of the standard samples, some technical problemsfor the osmometry of aqueous solutions were discussed. It was found that the values of Mn obtainedfor the standard samples were close to but somewhat smaller than the Mw values determined bysedimentation equilibrium measurements, Osmometry of the polyelectrolyte of gellan gum wascarried out at 40°C in tetramethyl ammonium nitrate solution using the sample converted intotetramethyl ammonium salt. The Mn values of the sample obtained were 5,4 x 10~ and 5.5 x 10~ in0.05 and 0.075 rnol/drn tetramethyl ammonium nitrate solutions respectively. The A 2 valuesobtained were rather large due to the Oonnan effect.

Introduction

Deacetylated gellan gum is a linear anionic heteropolysaccharide composed oftetrasaccharide (f3-o-glucose, f3-o-glucuronic acid, f3-D-glucose, and a-L­

rhamnose) repeat units, and a carboxyl side group (1,2). This is a veryinteresting polymer of wide potential application in the food industry (3). Thedilute solution properties of this polymer, however, seem to remain ambiguous.So far a few studies have been done on measurements by light scattering,intrinsic viscosity and optical rotation (4-10).

In this work the osmotic pressures for gellan gum aqueous solutions werestudied to obtain number average molecular weights and osmotic second viralcoefficients. Osmometry is one of the basic methods of measuring the molecularweight of polymers. Nevertheless, reports on the osmometry for polysaccharideaqueous solutions are very few because of the technical difficulties of the method(11-13). To discuss this technical problem, osmotic pressure measurements forthe aqueous solutions of standard polysaccharides, such as pulullan and dextran,were carried out prior to the measurement of gellan gum solutions.

Materials and methods

Preparation of solutions of pullulan and dextran

Three kinds of puIIulan samples of Shodex STANDARD p-82 and a dextransample fractionated and purified at the Faculty of Pharmaceutical Science,Nagasaki University (14), were used for the measurements. The puIIulan anddextran samples were dried in vacuo at 90°C for 6 h and at 105°C for 4 h,respectively. These samples were dissolved in deionized water which was leftovernight in a refrigerator.

Preparation of solutions of gellan gum

A geIIan gum sample (deacetylated geIIan gum; San-ei Kagaku Co.; lot 62058)

397

E.Ogawa

Table I. Metals in the gellan gum samples

Sample

Gellan gumTMA-gellan

1350420

290004200

3870420

Mg(f-lg/g)

1550840

Metal contents were measured by flame spectra photometry (Perkin Elmer Model 3100).

was swelled with deionized water at 45°C for 5 h and the solution stirred at 105°Cfor 10 h to dissolve the sample completely. The metals contained in the sampleare shown in Table I.

Preparation of solutions of tetramethyl ammonium gellan gum

The tetramethyl ammonium gellan gum (TMA-gellan) was prepared by thefollowing method. As mentioned above, the gellan gum was solubilized indeionized water (0.5-1.0 gil). The solutions were filtered through a 0.45 urnMillipore filter at 105°C, and then passed through a column of cation-exchangeresin (Amberlite 1R 120B) at room temperature. The effluent was concentratedby evaporation under reduced pressure. After neutralization of the concentratedsolution with TMA-hydroxide solution, it was poured into isopropanol/water(90:10) mixture to precipitate the TMA-gellans. Finally, the precipitate wasdried for 48 h at 40°C under a vacuum. The conversion to TMA salt was checkedby ionic contents of the TMA-gellan sample (Table I).

TMA-gellan was dissolved in TMA-nitrate (N03) solution and stirred for 1 hat 60°C. This solution was dialyzed against the TMA-N03 solution at 45°C.Concentrations of the solutions were determined by using a Union-GikenDifferential Refractometer Model RM-102. The value of (dn/dc) at 633 nm was0.140 (cmVg) at 40°C for 0.075 mol/drrr' TMA-N03 solution without dialysis.

Osmotic pressure measurements

A Hewlett-Packard High Speed Membrane Osmometer Model 503 (Figure 1)having a special type of glass capillary tube was employed for the osmoticpressure measurements. Schleicher and Schuell membranes (AC62) wereconditioned to the solvent system in use and degassed under reduced pressure atambient temperature immediately prior to installation in the instrument. Solventrequired for setting up the instrument was degassed in a similar fashion.Measurements were made at 25 or 40°C.

Results and discussion

Osmotic pressure measurements for the aqueous solutions ofpullulan and dextran

Osmotic pressure measurements were carried out at 25°C. Four or five solutionsin the concentration range from 0.3 to 1.0 g/dl were employed for each polymersample. Accuracy of the reading was ±0.02 em for the solvent (both sides of themembrane were filled with water), which was lower than the respective values of

398

Osmotic pressure of gellan aqueous solutions

g

h

b

Fig. I. Diagram of the osmotic pressure instrument. a Sample cell. b membrane. c solvent ce ll. c airbubbl e . d glass capillary. e light source . f optical detector, g solvent reservoir . h elevator. iservo mo to r. j amplifie r.

common organic solvents (e .g. toluene , ±O.OI ern) . In the case of the solution(the upper side was filled with aqueous solution) the accuracy was ±O.04 em ,which was also lower than the case of the organic one (toluene solution ,±O.02 ern) . It is considered that the low accuracy of these readings is mainlycaused by the low mobility of the air bubble in the capillary tube filled withwater. Moreover, it seems that the low fluidity of aqueous solvent or solutionsand the difficulty of degassing them also contribute to this low accuracy ofreadings . The equilibration time of a measurement of the aqueous solvent(solut ion) was typically s 15 min , which was approximately equal to that of theorganic solvent (solution). Adsorption of the polysaccharide samples onto thecellulose membrane was not observed under this experimental condition.Therefore for each solution at least three to five measurements were made andthe average values were recorded as the reading for each solution.

Average molecular weights, Mn, and osmotic second virial coefficients, A z, ofthe samples were determined according to the following equations (15).

'IT/c = RT(l/Mn + Azc)

('IT/C)'I: = (RT/Mn) ';' (1 + MnA 2c12)

(1)

(2)

Here 'IT is the osmotic pressure , C is the concentration of the solution in g/dl, R isthe gas constant , and T is the absolute temperature . Plots of -nk: versus c and ('ITIct versus c for the aqueous solutions of the standard samples , such as pullulans

399

E.Ogaw3

o. 4r---------------,

0.3

TIle

( em)

0.2

o. 1

P:Pullul an

K: Dextran

1.2)

0.8(g/ dc

0.4o.aL..--_L------JL--J_----L_---L_-----J

0.0

Fig. 2. Plots of ,,/(' versus (' for the aqueous solutions of pull ulan and dcxt an . p pullulan, k dextran.

and a dextr an, are show n in Figures 2 and 3, resp ectively. It is noted that th eplots of each sample solution fit a single line. The number average molecularweight s and the second virial coe fficients obtained res pe ctively from theint ercepts and slopes of the straight lines are shown in Ta ble II. The weightaverage molecular weights Mw and the values of MwlMn of these samplesdetermin ed respectively fro m the measurements of sedimentation equilibriumand ge l filtratio n were also show n in Ta ble II. No significant differe nce wasfou nd in the va lues of Mn of these samples determ ined from equations (1) and(2) , while the A 2 va lues o f these sa mples (except for K-13 sample) obta ined fromeq ua tio n (2) containing the third virial coefficient term were sma ller th an thoseobta ined fro m eq uation (1). It was found that the Mn values of these sampleswere close to but somew hat smaller th an th e Mw values. These result s impl y th at

400

Osmotic pressure of gellan aqueous solutions

0.6K - 1 3

1/2( TIle)

0.5

1/2( em)

0.4

0.3

P: pullulan

1.2)

K: Dextran

0.8( g / dc

0.40.2 '--__-'--__...L-__..L...__-'-__-'-__--'

0.0

Fig. 3. Plots of (TI/et versus c for the aqueous solutions of pullulan and dextran. p pullulan. kdextran.

Table II. Average molecular weights and second virial coefficients for the standard samples ofpullulan and dextran

Sample Mn x 10·

cq. (I) eq. (2)

A, x 10 (em'mol/g)

eq. (I) eq. (2)

M",/MIl"'"

Pullulanp-IOOp-200p-400

Dextrank-13

9.6 10.2 3.46 3.15 10.016.7 16.6 2.31 1.99 18.635.2 33.9 2.46 1.72 38.0

8.9 11.8 1.38 2.30 10.6

1.101.131.12

1.14

Sedimentation equilibrium. H gel filtration.

401

E.O!(awa

the osmotic pressure measurements for the aqueous solutions of the standardpolysaccharide samples in this study were carried out satisfactorily.

Osmotic pressure measurements for gellan Rum aqueous solutions

The plots of TIle versus c for the gellan gum aqueous solutions at 25°C are shownin Figure ~. Since the osmotic pressure of the gellan gum solutions was higherthan the limit of the measurement (39 em water height), the measurements werecarried out in the concentration range from IUl2 to 0.05 g/dl, far below the rangeused for the pullulan and dextran. During the measurements. the solutions ofthe gellan gum were stable without gelation. It was noted that the values ofosmotic pressure of the gellan gum solutions were very high and increased withthe decrease in the concentration. This implies that the gcllan gum ionized in theaqueous solutions and the dissociated counter ions such as sodium andpotassium (Table I) also contributed to the osmotic pressures. which werecharacteristic features for the osmometry of polyelectrolyte aqueous solutions(16). It is desirable to have neutral salts present in osmotic pressuredeterminations of the molecular weights of polyelcctrolytes to reduce the effectsof the diffusible counter ions upon osmotic pressure or to reduce the Donnaneffect (17). However gellan gum is an extremely effective gelling agent. and theaddition of the neutral salts to its solutions results in gel formation. Thereforethe measurements were made in tetramethyl ammonium nitrate (TMA-NO,)solutions at 40°C using tctrarnethyl ammonium Cr\1A) gellan; the bulky countercations such as TMA cations can inhibit gelation. In spite of the presence ofbulky TMA cations, gel formation occurred depending on the concentration of

20 GELLAN GUM

15 •njc

•10

( C ill) • •5

0.02(g/d 1)

0.0 1c

Ol.---_----L__-..l-__..L--_--"__-L..-J

0.0

Flg. 4. Plots of stl« versus r: for the gcllan gum aqueous solutions.

402

Osmotic pressure of gellan aqueous solutions

the salts and gcllan gum; the onset of gelation was observed in a samplecontaining ~ 1.2% (w/v) polymer in 0.075 mol/drrr' TMA-N03 solution. Themeasurements were carried out in the concentration range from 0.1 to 0.6%(w/v) and, to obtain better estimates of the molecular weight, two different ionicstrengths (0.05 and 0.075 mol/drn' TMA-N03) were used. The measurementswere repeated 2-3 times in 0.05 and 0.075 mol/drn' TMA-NO, solutions,respectively, and the results are shown in Figure 5. The plots of TIle versus e forboth 0.05 and 0.075 mol/dm' TMA-NO., solutions of the gellan gum were

2.5...---------------------,

TMA-GELLAN GUM

2.0

TIleo o• 0 5 M

( em)

1.5

o • •• 0.075 M

••o

1.0•

0.5

0.80.60.40.2o.0 L-_L------l_-----.!_------L_-----l._-.L_----L_--L_--'

0.0C (g/dl)

Flg, 5. Plots of Tile versus c for the D.05 and 0.075 rnol/drn' TMA-NO" solutions of TMA-gellan.

403

E.Ogawa

Table III. Average molecular weights and second virial coefficients for the TMA-gellan gum

TMA-NO,(mol/dm')

0.050.075

Mil X IW' /1_ x 10'(c~\lllol/g)

X26.2

somewhat scattered, but in each case the value of TIle could be extrapolated toinfinite dilution. Using the least square method, the number average molecularweights and the osmotic second virial coefficients were obtained from equation(1) (Table 1Il). It was found that the A 2 values in both solutions were very largebecause of the Donnan effect; the A) value in 0.05 rnol/drrr' TMA-NO, solutionwas larger than that in 0.075 mol/dm' TMA-NO, solution. It was note~i that theMil values determined for the two different ionic strengths were almost equal.The molecular weight of a tetrasaccharide repeating sequence in the T\1A­gellan gum is ~700. Accordingly, it can be said from a simple calculation. that agellan gum molecule contains ~80 tetrasaccharide repeating units.

The Mn value of the gellan gum molecule has not been obtained yet.Grasdalen et at. (7) reported the osmometry of gellan gum solution. using thedegraded sample. Milas et at. (10) and Crescenzi et at. (5.6) suggested theexistence of an order (helix)-disorder (coil) conformational transition of theTMA-gellan gum in TMA-chloride solutions from the study of temperaturedependence of intrinsic viscosity, optical rotation and light scattering. Milaset at. (10) reported that at 36°C the gellan gum molecule took the disorderedconformation, and its weight average molecular weight (Mw) was 2.5 x 105

,

much higher than the Mn value obtained in this study. It is considered that theintermolecular aggregation of the gellan gum molecules may occur to someextent in the solution, and in that case the difference between the values of Mwand Mil becomes large.

Acknowledgement

The author wishes to express sincere thanks to Professor K. Ogino of ChibaInstitute of Technology for his discussions and valuable advice for this study.

References

1. Jansson.P.; Linbcrg.B. and Sandford,!'.A. (I'.IX3) Carbohydr. Res .. 124, U5-13'.12. O''\eiII.M.A. Selvendran.R.R. and Morris.V.J. (1'.183) Carbohvdr. Res., 124. 123--1:'3.3. Sanderson.G.R. and C1ark,R.C. (1983) Food Tech II 01.. 63-70.4. Brownsey.G.J .. Chilvers.G.R.. l'Anson.K. and Morris.Vr.I. (1984) 1111. J. Biol. Macromol .. 6.

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10. Milas.M.; Shi.X. and Rinaudo.M. (1990) Biopolymers, 30, 451-464.

404

Osmotic pressure of gellan aqueous solutions

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and 14.17. Richards,E.G. (1980) An Introduction to Physical Properties of Large Molecules in Solution,

Cambridge University Press, Cambridge, Chs 3 and 9.

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