Polysaccharide Applications - American Chemical Society

Chapter 21

Applications of Gel Permeation Chromatography with Multi-Angle Light Scattering to the Characterization of Polysaccharides

Downloaded by UNIV OF ALBERTA on August 29, 2013 | http://pubs.acs.org Publication Date: August 20, 1999 | doi: 10.1021/bk-1999-0737.ch021

D. Richard White, Jr. Food and Beverage Analytical Department, The Procter & Gamble Company, Winton Hill Technical Center, 6210 Center Hill Road, Cincinnati, OH 46224

Gel permeation chromatography with multi-angle light scattering (GPC-MALS) is a useful technique for the characterization of polysaccharides. Polysaccharides are typically polydisperse, exhibiting a wide range in molecular weight, sequence, and structure. Industrial polysaccharides find use in pharmaceutics and cosmetics, as well as in foods, beverages, detergents, textiles, paper and paint, to name a few. Industrial polysaccharides include, but are not limited to, such materials as native and modified starches, dextrins, dextrans, glucans, pullulans, modified celluloses, pectins, carrageenans, and gums from microbial and plant seed sources. Information as to size, structure and conformation is useful in order to better understand solution behavior, intra- and intermolecular interactions, rheology, and function. GPC -MALS permits the determination of molar mass and size from the measurement of scattered light as a function of angle. This chapter illustrates the use of the technique for the characterization of some important industrial polysaccharides.

The primary advantage of combining light-scattering with size-exclusion chromatography is that it allows for the absolute determination of molar mass and mean square radius, without the need to calibrate the column with standards, which oftentimes are not available. The first of two key principles is that the amount of light scattered is proportional to both the molecular weight (M ) and concentration (c) of polymer (1), w

R(0) = K*M c P(0) [1 - 2A M cP(0)] w

2

w

© 1999 American Chemical Society

In Polysaccharide Applications; El-Nokaly, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

(1)

299

300 where R(0) is termed the "excess Rayleigh Ratio" and is the excess light scattered above that of the pure solvent. A is the second-order virial coefficient which accounts for non-ideal scattering due to solute-solute and solute-solvent interactions. A can often be neglected in dilute solutions and/or solutions of lower molar mass polymers. The proportionality constant or optical constant, K * = 47i (dn/dc) n N ~ A, ~ , includes several known and constant terms: n is the refractive index of the of solvent, N is Avagadro's number, X is the vacuum wavelength of incident light. The refractive index increment (dn/dc) is the change in the refractive index of the solution with respect to the change in concentration of polymer in the limit of zero concentration. For co-polymers and for polymers with molar masses below about 10 g mol" , the value of dn/dc may vary significantly with molecular weight. However, for most homopolymers of higher molar mass, the value of dn/dc is essentially constant over the range of masses measured. Thus, it may be determined by making ancillary refractive index measurements of solutions of the bulk polymer at varying concentration. It can also be computed "on the fly" during a chromatographic run if the mass injected is accurately known and there is 100 percent recovery from the column. The second key principle is that the variation in the scattered light with angle depends on the mean square radius of the molecule. This is reflected in the "scattering function", P(0), 2

2

2

2

2

0

1

4

A

0

0

A

Q


3

2

1

2

P(0)=l-(2ksin0/2) (r )/3

(2)

g

where k = 27in /A, . It is important to note that P(0) -> 1 as 0 -> 0. The mean square radius, (= Zr m/M ), is the sum of the square of the equilibrium distances between scattering centers and the center of mass of the polymer. Hence, is related to the distribution of mass within the molecule and is not generally a measure of the molecule's external geometry. The square-root is termed the root-mean-square radius, r , although it is often erroneously called the "radius of gyration." It is also important to note that r is not the hydrodynamic radius, but has the distinct advantage of revealing information about a molecule's internal structure so that one can make inferences as to conformational shape, degree of branching, etc. Combining equations 1 and 2 in the fashion of Zimm (2) yields what is referred to as the Zimm formalism of the Rayleigh-Debye-Gans approximation. In vertically polarized light, 0

0

2

g

2

i

i

2

g

g

g

K*c/R(0) = 1/M P(0) + 2A c W

2

(3)

The second term on the right of Equation 3, A c , is retained to account for nonideality at higher concentration and/or in the case of higher molar mass polymers. As previously mentioned, A c can often be neglected. At the limit of zero angle and zero concentration where P(0) is unity, the function K*c/R is exactly equivalent to the reciprocal molecular weight. In Gel Permeation Chromatography (GPC) it is assumed that each chromatographic slice contains molecules of a single, or at least very narrow band, molecular weight, Mj. Therefore, once data have been processed, the effective mass moments can be calculated over the entire peak from the following relations: 2

2


301 weight-average, number average, " Z " average,

M = Zc M /Zci M^Ic/CEcM) M, = Zc M /(2c M ). W

I

i

2

I

I

i

I

The weight-average molecular weight (M ) is the parameter directly measured by light-scattering. It is the sum of the products of the concentration and molecular weight at each slice, divided by the total concentration, c (= Ecj). The number average molecular weight (M ) tends to weigh more heavily the lower molecular weight species and is more closely related to that which would be determined by measurement of colligative properties such as osmometry. On the other hand, the Zaverage molecular weight ( M J , tends to weigh more heavily the higher molecular weights and is more closely related to that determined by sedimentation equilibrium and ultracentrifugation experiments, for example. The ratio of the M to M or M is often used as an estimate of relative polydispersity of a polymer. The corresponding quantities for the mean square radii are written in an analogous fashion as: w


n

w

n

z

2

= E /I = ( E ^ V c / M i V d c / M O ^ d ^ V ^ M ^ S c M ) . w

g

iCi

Ci

2

g

2

g

In GPC, it can usually be assumed that the concentration in each slice of the chromatogram is so small that the second virial coefficient is negligible. Thus, for each slice of the chromatogram, one can plot K*c/R(0) vs. sin (6/2) to yield an intercept which is the reciprocal molecular weight for that slice. Correspondingly, the slope of the plot at the intercept permits the determination of the mean square radius for that slice. Figure 1 is a Debye Plot using the Zimm formalism (K*c/R) for a representative slice of a narrow band pullulan polysaccharide. The nominal M of this material from the manufacturer (Polymer Laboratories, Amherst, M A ) is 404,000 g mol" . Taking the slice at maximum concentration (maximum RI signal) and plotting K*c/R(9) vs sin (9/2) for 3 angles (approximately 45°, 90°, and 135°) yields M = 449,000 g mol" and r = 13 nm. 2

w

1

2

w

1

g

Experimental The experimental setup consisted of a Waters (Milford, M A ) 2690 solvent delivery system with built-in degasser, a thermostatted sample compartment, injector, and column oven. A 0.1 um in-line filter was added between the pump and injector. The columns were Waters ultrahydrogel 2000 and 250 in series, thermostatted to 45 °C. Mobile phase was either 0.025 M phosphate buffer with added 0.05 M NaCl, or 0.1 M L i C l for charged polysaccharides (such as the carrageenans), shown to inhibit gel formation. Samples were prepared in the mobile phase to contain from 1 - 3 mg mL" . Typically, the polysaccharides were dissolved by stirring at 50 °C. For the starch, samples (~5 mg mL ) were microwave solubilized at 130 °C for 10 minutes. Samples were coarsely filtered through 5um syringe-tip filters prior to analysis. The pump flow rate was 0.5 mL/min and the injection volume was 100 uL. The detectors 1

-1)



0.4 2

sin(theta/2)

0.6

90° & AUX detectors

1.0

Figure 1. Debye Plot (Zimm Formalism) for a Representative Slice of a Narrow Band Pullulan Polysaccharide.

Peak, Slice : 1,3479 Volume : 14.496 mL Fit degree : 1 Cone. : (1.289 ± 0.001 )e-4 g/mL Mw : (4.471 ± 0.025)e+5 g/mol Radius 13.6 ± 1.6 nm

2.320x10



303 consisted of a MiniDAWN (Wyatt Technology, Inc., Santa Barbara, CA) which is a 3angle laser light scattering detector capable of making measurements at fixed angles approximately 45, 90, and 135 degrees, and an Optilab interferometric refractive index detector (Wyatt Technology, Inc.) which operates at the same wavelength as the MiniDAWN, in this case 690 nm. The software was A S T R A version 4.5 (Wyatt Technology, Inc.). Calibration of the RI detector and measurements of dn/dc were accomplished with DNDC software version 5.2 (Wyatt Technology, Inc.). The first step before making measurements is to calibrate the detectors. As with all transducers, it is necessary to relate the signal output, in this case Volts, to the physical quantity of interest. In the case of the RI detector it is the refractive index change, dn, that must be measured as a function of the voltage output. This is accomplished by making RI measurements on accurately prepared solutions for which the quantity dn/dc is already known. The quantity dn can then be calculated for each solution by multiplying the concentration, c, by dn/dc. The slope of a plot of dn vs. the measured voltage output yields the calibration constant for the detector. Once the calibration constant is determined, then with a knowledge of dn/dc for a sample polymer, it is possible to calculate the concentration at any point in the run as the polymer elutes through the detectors. To calibrate the RI detector, solutions of NaCl (0.1 to 0.6 mg/mL) were passed through the RI detector via syringe pump. Voltage readings were taken when the signal had reached a stable plateau. For the light-scattering detector, one would like to relate the signal output (Volts) to the Rayleigh ratio, R(0). This is accomplished by measuring the signal at the 90° detector while passing solvent with a well-characterized R(0) through the light scattering flow cell. Toluene is a good choice because it has a high Rayleigh factor relative to most common solvents. Once the 90° diode is calibrated, the final step is to normalize the diodes at the other angles to that of the 90° signal. This is necessary because of differences in the light scattered due to the geometry of the cell, the refractive index of the cell material, and the refractive index of the solvent in which measurements will be made. For this reason, normalization must be carried out in the solvent that will be used to make the final measurements. Normalization coefficients for the low angle and the high angle are determined by measuring the signal produced by a monodisperse, isotropic scatterer in the solvent to be used. In our case, the solvent was water and bovine serum albumin (BSA, M ~ 69,000) was a suitable polymer for normalization. BSA's small radius (< lOnm) ensures that the angular dependence of scattering is very small, i.e., P(0) ~ 1. w

Results and Discussion The polymers studied here were limited to a relatively few commercially available polysaccharides for illustrative purposes. A l l the polysaccharides chosen were rather well behaved in terms of solubility and stability in the aqueous mobile phases employed. Pullulans. Pullulans are water-soluble extracellular polysaccharides synthesized by Aureobasidium pullulans (3, 4), a common and widespread fungus; they are prepared by fermentation. The chemical structure varies according to the substrate and strain of



304 microorganism used, as well as the chemical fractionation and purification methods employed. Nevertheless, pullulan is currently accepted to be a water-soluble, neutral glucan, which consists of linear chains of malto-triosyl units, linked together by l->6 alpha-D-linkages. It is a flexible, odorless, tasteless material and its film forming properties make it an ideal coating for foods and drugs. Pullulan is non-nutritive in that it is not degraded by in vivo digestive enzymes. It is commercially available in narrow molecular weight ranges and has been recommended for column calibration in size-exclusion chromatography by Kato (3) andFishman (5). Seven narrow molecular weight pullulans (Polymer Laboratories, Amherst, M A ) were analyzed separately. Three concentration levels were used so that the mass on column ranged from 0.1 to 0.3 mg for a 100 uL injection. Figure 2 is an overlay of the GPC chromatograms for the seven polymers. Both refractive index and light-scattering signals vs. elution volume are illustrated. The three concentrations are apparent from the relative magnitude of the RI signals. Comparison of the relative intensity between the LS and RI signals highlights the dependence of scattering on molar mass as well as concentration. Note that the LS signal is much higher for the larger, early eluting polymers in spite of their lower concentration. Superimposed on the chromatograms are the calculated point-by-point M values across each polymer band. If each polymer were truly monodisperse these would be horizontal lines and one would expect to see a stair-step like pattern. As observed here, there is a slight slope downward within each band indicating some polydispersity. Table I is a summary of the results obtained. Calculated results for the average M agree reasonably well with the nominal values from the manufacturer, although about 10% higher. It is noteworthy that the range of polydispersity values is essentially the same as that obtained by Fishman (5), who used differential RI detection, a different set of chromatographic columns, sodium chloride mobile phase, and a smaller injection volume (20 uL). The dn/dc values for the pullulan standards were obtained from the known mass injected on column and by assuming 100 percent recovery. Although there was quite a bit of uncertainty in the r values obtained, the range is close to values given in the literature (3, 5). The inability to calculate a reasonable r for the smaller M polymers probably reflects the lower limit of molecular radius for which one can realize a measurable angular dependence, P(0). That limit appears to be about 10 nm for X = 690 nm. w

w

gz

w

Table I. Summary of M and r Results for Seven Commercial Pullulan Standards r , (nm)* M (nominal) M (calc) dn/dc (mL/g) r , (nm) M /M 11,800 3 13,230 0.133 1.10 22,800 5 24,760 0.136 1.15 47,300 7 54,850 0.137 1.04 12 112,000 115,200 1.04 0.138 212,000 17 0.133 230,200 1.06 10^21 25 404,000 18 455,700 0.133 1.04 37 788,000 30 817,400 0.136 1.08 *data interpolated from (3, 5) w

w

w

gz

w

n

e z

g z



Figure 2. GPC-LS-RI Overlay of Seven Pullulan Standards with Molar Mass Superimposed (—) LS Signal, (—) RI Signal.



306 Dextrans. Dextrans are contiguous alpha l->6 glucosidic linkages in the main chains, with variable amounts of alpha l->2, l->3, or l->4 branch linkages (6). Dextrans are found anywhere sucrose is found and, as such, are often unwanted contaminants in sucrose refineries, sugary foods, and other sucrose preparations. When synthesized in the mouth by Streptococcus mutans, dextrans form dental plaque. Purified dextrans and their derivatives have found valuable uses as blood plasma substitutes, anticoagulants, antilipemics (sulfate esters) , and anti-ulcer therapeutics, all of which require very specific molecular weight ranges to be effective. Figure 3 shows GPC-RI chromatograms obtained from a series of dextrans available commercially (Sigma, St. Louis, MO). It is readily apparent how much more broadly these polymers are distributed relative to the pullulans. Whereas the average peak elution volume for the pullulans was ~2 mL, the dextrans elute in -4-5 mL volume. Table II gives a comparison of calculated molecular weights with the nominal M given by the manufacturer; all agree to within 10%. Again, dn/dc values were obtained by assuming 100 percent column recovery. The polydispersity numbers agree well with Fishman (5) and the calculated radii range for the highest M dextrans encompasses that in the literature. Note that the calculated molecular weights superimposed on the chromatogram exhibit a smooth trend downward, with overlap between adjacent peaks. In contrast with the narrower band pullulans, this is characteristic of broad, polydisperse polymers. w

w

Table II. Summary of M and r Results for Five Commercial Dextran Standards M (nominal) M (calc) M / M dn/dc (mL/g) ^ (nm) r , (nm)* 4 19,500 0.134 21,470 1.31 41,000 0.134 6 43,290 1.34 74,400 8 68,210 1.69 0.134 12 167,000 169,800 2.20 0.146 22 580,000 0.134 19 539,300 2.15 *Data interpolated from (3, 5) w

w

w

gz

w

n

E z

Dextrins. Unlike pullulan and dextran, which are synthesized from smaller molecules by bacterial enzymes, dextrins are hydrolysis products of larger polysaccharide material, specifically starch (7). The result is a range of D-glucose polymers, linked by alpha l->4 bonds, typically referred to as dextrin, or maltodextrin when the "dextrose equivalent" is less than 20, corresponding to a degree of polymerization (DP) greater than about 5 glucose units. Dextrins have a variety of uses as absorbents, binders, bulking agents, adhesives, films, conditioners, thickeners, or as foundation for make-up, and face powders. Dextrins are also used as an aid to spray-drying, or encapsulation to provide new dosage forms, tasteless or controlled-release drugs, and food flavors. Figure 4 is a plot of differential molar mass calculated from GPCM A L S of a commercial maltodextrin which nominally contained oligosaccharides in the range 5 to 30 DP, corresponding to a maximum molecular weight of about 5000 g



4

1.0x10

12.0

14.0

Elution Volume (mL)

16.0

18.0

20.0

* t + a X

22.0

DEX_19K DEX_41K DEX_74K DEX167K DEX580K

Figure 3. GPC-RI Overlay of Five Dextran Standards with Molar Mass Superimposed.

1.0x10 10.0

3

f

6

1.0x10

1.0x10

7

(

1.0x10

1.0x10

9

10

1.0x10

1.0x10

Molar M a s s vs. Volume



0.8

Figure 4. Differential Molar Mass Plot for a Commercial Maltodextrin (—) Run !,(---) Run 2.

Molar Mass (g/mol)


Run 2

MALTODEXTRIN

MALTODEXTRIN Run 1

309 1


mol" . It can be seen that in addition to the material in the molecular weight range of a few thousands, there is a significant amount of material in the higher range of 100,000 to roughly half a million, unexpected for a DP 30 dextrin. This is likely an indication of "left-over" high molecular weight material from incomplete hydrolysis. This information would likely be missed by conventional detection methods but could be important in terms of the functional properties of this material. Gums. Gums are hydrophyllic polysaccharides that are particularly known for their water thickening and gelling properties; also termed hydrocolloids. Hydrocolloid gums are usually classified in terms of their origin. For example, seed gums arise from the endosperm of seeds or other ground plant products. Plant gums fall into the general category of extracts isolated from land plant sources, such as pectin. These should be distinguished from other extract gums derived from seaweed sources such as alginates or carrageenans. Bacterial gums are fermentation products such as xanthan and gellan. Animal gums are also extract gums but derived from animal sources and protein based, an example being gelatin. Xanthan. Xanthan gum is one of the most versatile gums, used extensively in foods, pharmaceuticals, and personal care products. It is a bacterial gum derived from Xanthomonas campestris (8). Its main chain is 1->4-beta-D-glucopyranosyl units, identical to cellulose. However, every other unit in the main chain is substituted with a trisaccharide unit of beta-D-mannopyranosyl linked l->4 to a beta-Dglucopyranosyluronic acid, and linked l->2 to a 6-acetyl, alpha-D-mannopyranosyl unit. The molecular weight is probably about 2 million, although much higher molecular weights have been reported (9). Although xanthan is anionic, pH has almost no effect on its viscosity. Xanthan is a thickener that is stable to heat and shear. It is a high molecular weight, rod-like molecule which forms aggregates through hydrogen bonding and polymer entanglement. These networks dissociate when stress is applied giving xanthan its pseudoplastic properties. There is much controversy as to changes in the structure with changing solution conditions. Single, double and even triple helical structures have been proposed. Xanthan's pseudoplastic property improves the flow properties of many viscous liquid products. It provides lubricity to heavy creams, is compatible with other gums and starches, and works synergistically to increase viscosity. Guaran. Guaran is the purified polysaccharide from guar gum, which in turn is derived from the endosperm of guar seeds, and is therefore a seed gum (10). It is a polymer of D-galactose and D-mannose with a mannan backbone, that is a linear chain of 1 ->4 beta-D-mannopyranosyl units with every other unit being substituted with a l->6 alpha-D-galactopyranosyl unit. The polymer is neutral, and forms very high viscosity, pseudoplastic (shear thinning) solutions at low concentrations. It is often used in combination with other gums.


310


Carrageenans. Carrageenans are sulfated algal polysaccharides and are heteropolymers with alternating alpha l->3 D- and beta l->4 D-galactans (11). There are conformational differences between the non-gelling lambda form and the gelforming kappa and iota forms. Carrageenan has diverse industrial applications including its use in creams, toothpaste, shampoos, pharmaceutical and industrial suspensions, etc. There is also some anticoagulant activity and some evidence of inducing growth of new connective tissue. Gellan. Gellan is an anionic polysaccharide from Pseudomonas elodea (10). It finds use mostly in food applications. Its gelling properties are highly dependent on cation concentration. It is used in synergy with other gums, mono- and oligosaccharides, often in sugary foods. Figure 5 is an overlay of GPC-LS chromatograms of the four gums investigated. Following the recommendation by Lecacheux (12), we used 0.1 M L i C l as the mobile phase to avoid potential gel formation. Each of the gums were made up at roughly the same concentrations, so one can readily see from this plot the relative molecular size as indicated by the intensity of the light scattering signals and the order of elution. Xanthan is obviously the largest polymer, followed by guar, then K carrageenan, and finally gellan. Some peak deformity in the xanthan and significant tailing of the xanthan and guar may indicate less than optimum chromatographic conditions. Table III summarizes the results obtained. The dn/dc value for xanthan

Table III. Summary of M and r Results for Some Commercial Gums w

:

Gum xanthan guar gellan

gz

_ni=

_—6

1

M (xl0 gmol )(lit.)* M / M (lit.)* dn/dc (mL/g) r > m ) (lit)* 0.141 4.87 ± 2.8 (2.2, 7) 228 ± 1 5 1.06 113 + 3 1.98 + 2 0.14 1.16 70 ± 5 0.298 ± 0.02 0.133 1.58 (0.15-0.56) 0.115 73 ± 3 (60) K-carrageenan 0.495 ± 0.03 (0.468), 1.53 (0.34-0.58), (0.42) *datafrom (9, 12, 13) w

w

n

was taken from the literature (9). The dn/dc value of 0.133 for gellan was obtained by assuming 100 percent column recovery and was considered reasonable. However, the value obtained for guar in this manner was less than 0.1, an unreasonable value for polysaccharides in aqueous media, and probably a result of poor column recovery. Because a dn/dc value for guar could not be found in the literature, a value close to that of xanthan was chosen. The M obtained for xanthan was within the range of those reported in the literature (9). The apparent molecular weight may be solution dependent due to changes in the solution conformation of xanthan. The radius of xanthan was estimated to be over 0.2 micron, which is quite large and may necessitate the inclusion of a second virial coefficient in Equation 3, although this was not investigated. Thus, the absolute accuracy of these calculated results may be questionable. The M for guar was higher than expected, as the literature would seem w

w



311

1.2

Elution Volume (mL)

Figure 5. GPC-LS Overlay of Four Commercial Gums.


312 to suggest a M in the 200,000 range. Both the M and the radius were about half that of xanthan. The polydispersity numbers for xanthan and guar were relatively low. The results for gellan and K-carrageenan were very much in line with expected results.


w

w

Starch. Starch is the principle food-reserve polysaccharide in plants and serves as the main carbohydrate in the diet of man and animals. It is a mixture of two polysaccharides, both being D-glucans. Amylose, which normally comprises 20-30% of starch, is a linear alpha-D-l->4 glucan with a DP of several thousand and a molecular weight probably under 1 million (14-16). Amylose is thought to be a random coil in neutral solution. Amylopectin is 70-80% of starch and is also an alphaD-l->4 glucan, but with alpha-D-l->6 branch points. Most uses of starch capitalize on its high viscosity and gel-forming properties. Physical and chemical modifications, such as partial hydrolysis to dextrins, conversion to ester, ether derivatives, or amino derivatives can have profound effects on starch behavior. Figure 6 shows GPC-LS chromatograms for a purified potato amylopectin and a soluble potato starch (Sigma, St. Louis, MO). Note the relative monodispersity of the amylopectin relative to the starch. Examination of the calculated molecular weight vs. elution volume for the two materials shows a region early in the elution where the slopes overlap; here presumably only amylopectin is eluting from the column. For the intact starch, there is a point at which the slope departs from that of the pure amylopectin, implying coelution of lower molecular weight material. The slope decreases more rapidly with elution volume, probably as the linear amylose makes up more of the eluting material. It should be kept in mind that molecules are separated in size-exclusion based on their hydrodynamic volume. Thus, one would expect the molecular weight of linear molecules to drop off more rapidly with elution volume than that of compact, highly branched molecules. It is also useful to examine a log-log plot of radius vs. molecular weight (Figure 7). For a highly branched molecule approximating a sphere, one would expect that the radius would increase as a cube root of the molecular weight. Thus, the slope of log r vs. log M would approach 0.33. On the other hand, for a rigid rod the radius should increase linearly with molecular weight and the slope would be ~ 1. For a random coil the radius would increase as approximately the square-root of M yielding a slope in the log-log plot of -0.5. It is noteworthy that the average slope for the purified amylopectin is 0.35, close to expected for the branched polymer. For the starch, the slope is nearer to 0.5, reflecting perhaps the random coil behavior contributed by the amylose portion. Results are summarized in Table IV. Using a dn/dc value of 0.146 mL/g (17), an average M of 2.4xl0 g mol" with a corresponding radius of 39 nm was obtained for the potato starch. These values agree quite well with the 2.0xl0 g mol" and 39 nm, respectively, reported by Fishman (16) who took a similar approach. For the amylopectin, the M was estimated to be about 10 g mol" with a radius of 60 nm. Although the high M and r for the amylopectin are undoubtedly pushing the capability limits of the 3-angle Mini-DAWN detector, the results of the replicate analyses shown here give an appreciation for the repeatability that can be obtained. g

w

w

6

w

6

1

w

7

1

w

gz


1


Figure 6. GPC-LS Overlay of Commercial Potato Amylopectin (X) and Soluble Potato Starch ( • )with Molar Mass Superimposed .



R.M.S. Radius (nm)


315 Table IV. Summary of M and r Results for Replicate Analysis of Potato Amylopectin and Soluble Potato Starch M / M dn/dc (mL/g) r ^(nm) sample M (xl0 gmol ) 58 amylopectin (run 1) 2.24 0.146 10.48 57 amylopectin (run 2) 0.146 10.04 2.45 39 0.146 starch (run 1) 3.46 2.30 39 starch (run 2) 0.146 2.47 3.25 w

g>z

6

w

1

w

n

E


Conclusion We have shown here some basic light-scattering principles and application of the technique to a variety of polysaccharides. To summarize, GPC-MALS is a technique that permits the estimation of absolute molecular weight and radius of polysaccharides without the need for column calibration methods or standards. The technique is suitable for characterizing a wide variety of industrial polysaccharides. The results obtained are of comparable accuracy to those obtained by other methods. G P C - M A L S is a useful tool for the characterization and quality control of polysaccharides of industrial importance, particularly where functional properties are dependent on molecular size or conformation. Acknowledgements The author thanks his colleagues, Ms. Pat Hudson of the Procter & Gamble Co. for her assistance in the laboratory, and Ms. Helena Soini of the Procter & Gamble Co. for providing the microwave-solubilized starch samples. Appreciation is also expressed to Dr. Ron Myers of Wyatt Technology, Inc. for helpful comments. Literature Cited 1. 2. 3. 4.

Wyatt, P. J. Analytica Chimica Acta, 1993, 272, 1. Zimm, B. H. J. Chem. Phys. 1948, 16, 1093. Kato, T.; Tokrya, T.; Takohoski, A. J. J. Chromatogr. 1983, 256, 61. Tsujisaka, Y.; Mitsuhashi, M . In Industrial Gums: Polysaccharides and Their Derivatives,3 ed.;Whistler, R. L.; BeMiller, J. N . Eds.; Academic Press, San Diego, CA, 1993, pp 447-460. Fishman, M . L.; Damert, W. C.; Phillips, J. G.; Barford, R. A. Carbohydrate Res. 1987, 160, 215. DeBelder, A.N. In Industrial Gums: Polysaccharides and Their Derivatives, 3 ed.; Whistler, R. L. and BeMiller, J. N . Eds.; Academic Press, San Diego, CA, 1993, pp 400-425. Chronakis, I. S., Crit. Rev. Food Sci., 1998, 38, 599. Kang, K. S.; Pettitt, D. J. In Industrial Gums: Polysaccharides and Their Derivatives,3 ed.;Whistler, R. L.; BeMiller, J. N . Eds.; Academic Press, San Diego, CA, 1993, pp 341-397. rd

5. 6.

rd

7. 8.

rd


316

9. 10.

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Polysaccharide Applications - American Chemical Society

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