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3 A Critical Reexamination of Molecular Weight and Dimensions for Citrus Pectins Marshall L. Fishman, L. Pepper, W. C. Damert, J. G. Phillips, and R. A. Barford

Downloaded by UNIV OF ARIZONA on January 6, 2013 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch003

Agricultural Research Service, North Atlantic Area, Eastern Regional Research Center, U.S. Department of Agriculture, Wyndmoor, PA 19118

1

E-1000 and Ε-linear μ Bondagel high performance size exclu sion chromatography (HPSEC) columns were calibrated in root -mean square (RMS) radii of gyration (R ) by using a combina tion of pullulan and dextran standards. By assuming that universal calibration applies, to pectins the number-average R in 0.05M and 0.1M NaCl for a series of pectins with varying degrees of esterification in the protonated and sodium forms were calculated from chromatograms. This procedure yields well-defined R averages that can be used readily to cal culate molecular parameters for comparison with other methods. Such comparisons were not feasible when an earlier HPSEC method was used. Furthermore, by assuming rod-like structure for the pectins, number-average lengths (l ) and degree of polymerization (DP ) were calculated from R . Importantly, DP and l values from HPSEC fell between values from end group titration and membrane osmometry which is consistent with previous findings that pectin can undergo a concentration dependent disaggregation. g

g

g

n

n

n

gn

n

Over the l a s t ten years evidence has accumulated that pectin can undergo s e l f disaggregation

(1-4).

More recently, by size exclusion

chromatography (SEC) and determination of number-average degrees of

This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Molecular Weight and Dimensions for Citrus Pectins

FISHMANETAL.

3.

23

polymerization (DP ) from end group t i t r a t i o n s (EGT) (5), we have n

demonstrated s e l f disaggregation over the pH range 3.7 - 7.3 f o r c i t r u s pectins with degree of methyl e s t e r f i c a t i o n (DM) between 37 and 73%.

Moreover, by membrane osmometry (0) and EGT we have shown

that protonated and neutralized pectins form metastable aggregates which can be dissociated by heat a c t i v a t i o n and that these a c t i vated pectins undergo concentration dependent disaggregation (6,7). Furthermore, EGT gives the D P of p e c t i n monomer whereas osmometry q

gives the D P of aggregated pectins. Downloaded by UNIV OF ARIZONA on January 6, 2013 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch003

r

A l l but protonated pectins

with low DM (35 or 37%) exhibited disaggregation with a steep con centration dependence.

Thus, neutralized pectins and protonated

pectin with medium and high DM gave van't Hoff p l o t s which exhibited a minimum at about 0.1 g/dl. Furthermore, the osmotic data could be extrapolated to 7t/c values obtained from EGT (Figure 1). S i m i l a r behavior by proteins has been interpreted as that of a nonideal d i s sociating system (8). In the case of the pectins, van't Hoff p l o t s appear l i n e a r and nonideal above 0.1 g/dl ( i . e . , they have a p o s i t i v e slope).

In the past, the l^m (τΐ/c) f o r the l i n e a r portion of the

curve has been used to obtain D P from membrane osmometry. r

obtain number-average (R (R

an(

), weight-average U*g ) * W

Here we

z-average

) RMS of r a d i i gyration from high performance s i z e exclusion

chromatography (HPSEC).

By assuming r o d - l i k e structure we obtain

DP

r

values from corresponding R^

DP

q

values from end group analysis and osmometry.

n

values.

These are compared with

Experimental

Materials.

Commercial c i t r u s pectins with degree of methyl e s t e r i -

f i c a t i o n (DM) 35, 58-60, and 70 were g i f t s from Bulmers Limited, Hereford, England.

Two other c i t r u s p e c t i n samples DM 37 and 72-73

were manufactured by Bulmers but were g i f t s from Drs. E. R. Morris and M. J . Gidley at Unilever.

The DM 57 c i t r u s p e c t i n was a g i f t


24

CHEMISTRY AND FUNCTION OF PECTINS

from Sunkist Growers, Corona, Ca. One p e c t i n sample was extracted from fresh grapefruit peels, according to standard procedure (9). I t had a degree of e s t e r i f i c a t i o n of 73 and was labeled 73G. Char a c t e r i z a t i o n and preparation of samples were as reported previously (5) with minor modification.

Samples to be neutralized with NaOH

were dissolved i n 0.01 M phosphate buffer (pH 6.1) containing 0.1 M EDTA, t i t r a t e d to pH 7 with 0.1 M NaOH, dialysed against four changes of water over 48 nr., centrifuged f o r 1 hr. at 30,000 χ g


to remove insoluble matter and then l y o p h i l i z e d .

Protonated

samples were dissolved i n deionized water (Continental Water Systems) followed by d i a l y s i s , c e n t r i f u g a t i o n , and l y o p h i l l i z a t i o n weight cut o f f of 12,000.

Dextran standards were from Pharmacia

Chemical Co., Piscataway, N.J. The M^ values of the dextran 5

stand

5

ards were as follows: T-500, 5.32 χ 10 ; T-250, 2.53 χ 10 ; T-110, 5

4

4

4

1.06 χ 10 ; T-70, 7 χ 10 ; T-40, 4.44 χ 10 ; T-20, 2.23 χ 10 ; T-10, 3

9.3 χ 10 . P u l l u l a n standards were from Polymer Laboratories, Inc., Amherst, MA. 5

5

The M values of the pullulans were 8.53 χ 10 , 3.80 χ w

5

5

4

4

4

10 , 1.86 χ 10 , 1.00 χ 10 , 4.8 χ 10 , 2.37 χ 10 , 1.22 χ 1 0 and 3

5.8 χ 10 . The pullulans had r a t i o s of M^/M^ of 1.14, 1.12, 1.13, 1.10, 1.09, 1.07, 1.06, and 1.07 respectively. HPSEC Apparatus, sample preparation and chromatographic conditions were as reported previously (5) with the following modifications.

High

performance s i z e exclusion chromatography was performed e i t h e r on a Waters E-1000 μ-Bondagel column (30 χ 0.39 cm I.D.) or an E - l i n e a r μ-Bondagel column (30 χ 0.39 cm I.D.).

Twenty μΐ of a 0.3 mg/ml

sample were i n j e c t e d . Mobile phase was e i t h e r 0.05 or 0.1M NaCl. The solvent i n the reservior was s t i r r e d with a magnetic s t i r r e r and the column was wrapped with a soft foam i n s u l a t o r . The chromatograph was kept i n a constant temperature room at 23±1°C. Flow rates were measured by an a i r bubble injected into a c a l i b r a t e d measuring pipette connected to the e x i t l i n e of the chromatograph (10). The pump was set at a nominal flow rate of 0.5 ml/min.

Long term flow


3.

25


FISHMANETAL.

rates were measured to be w i t h i n ± 2% of the nominal value. any 8 hour period, flow rates were precise to ± 0.3%.

Over

Generally,

peak maxima f o r 3 consecutive runs agreed w i t h i n 2 seconds. Peaks emerging from the size exclusion column were detected by r e f r a c t i v e index and UV absorbance at 206 nm.

Analog signals

were d i g i t i z e d at a rate of 150 points per minute and i n a remote location by a modcomp 7861 minicomputer, equipped with an analog input subsystem.


To obtain averaged root-mean-square r a d i i of gyration, p a r t i t i o n c o e f f i c i e n t s ( K ) were transformed point by point to

values

ay

as appropriate integrals were summed. Integrations were by a trape z o i d a l algorithm.

Transformations were obtained from the following

c a l i b r a t i o n curves:

K->K ln(Y) = a + a- Κ 1 av ' 0 1 av v

(1)

n

2

Κ gn

(R

z-average,

), r a d i i of gyration. These are defined by equations

R Downloaded by UNIV OF ARIZONA on January 6, 2013 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch003

a n d

gw

4-6

= Σ C. / Σ (C./R ·) · ι . ι' g i

(*)

y

gn

Ί

R gw = Σ. C.ι R g i. / Σ. C.ι R

(5)

2

= Σ C. R . / Σ C. R ι gi ι gi

gz

±

(6)

i

Where R ^ i s the radius of gyration of species i and

i s i t s con

centration. For each DM, R

< R < R , which i s the expected order, gn gw gz' I n t e r e s t i n g l y , f o r the radius of gyration at peak p o s i t i o n ( R ) r

gp

ό

(maximum concentration of p e c t i n ) , R

< R < R f o r Rgp < 135 A gn gP gw > 247 A. Typical chromatograms are 6 r

whereas R

< R

gw gp shown i n F i g . 3.

< R

gz

for R

Q

gp

y v

As indicated by Table I , pooling data without regard to column type, s a l t concentration i n the mobile phase, or p e c t i n form gave data with a standard deviation ranging from 3 - 15% of the mean f o r the number-, weight- or Z-average R^.

In case of the number average

radius of gyration, the standard deviation ranged from 3.5 - 10%. Since, i t was our i n t e n t i o n to compare molecular weight and size values from SEC with those from end group t i t r a t i o n s , and osmometry, (comparisons of log R

gn

reduced the heterogenity i n variance (16))

values were analyzed for variance at the (p^ 0.05) confidence l e v e l . Such analysis was used to determine i f R^ values were affected s i g n i f i c a n t l y by: (1) the concentration of s a l t i n the mobile phase; (2) whether the form of the carboxylate ion was hydrogen or sodium; (3) the pore size d i s t r i b u t i o n (psd) of the HPSEC columns.

No global

trends were i d e n t i f i e d because of interactions between combinations




3. FISHMANETAL.

-1.0

-0.5 0 0.5 PARTITION COEFFICIENT

29

1.0

Figure 3. Typical chromotograms f o r pectins with 0, 37 and 70% methylation e s t e r f i c a t i o n .

ο

Λ

TABLE I RADIUS OF GYRATION (R , A) FOR PECTINS

1

\ R NUMBER-AVERAGE

WEIGHT-AVERAGE

Z-AVERAGE

PEAK P0S.

0

51.7 + 2.2

65.2 + 4.1

84.5 + 11

53.7 ± 4.2

35

99.6 + 3.8

144 + 3.0

212 + 7.8

37

70.5 + 2.2

105 + 2.9

165 + 12

81.5 ± 8.2

57

131 + 9.4

207 + 17

309 + 40

258 ± 28

58-60

124 + 4.3

194 + 4.5

288 + 11

252 ± 33

70

126 + 6.0

201 + 10

301 + 12

247 ± 35

123 + 12

206 + 11

314 + 12

292 ± 35

101 + 3.3

149 + 4.0

225 + 15

135 ± 31

72-73 73G

â

123 ± 11

1

DATA AVERAGED OVER MOBILE PHASE CONCENTRATION (0.05M & 0.1M NaCl), OVER FORM (ACID & NEUTRALIZED) AND OVER COLUMN (E-1000 AND E-LINEAR) 24 DETERMINATIONS.

2

STANDARD DEVIATION OF POOLED DATA AS IN (1).

3

DATA FOR E-1000 COLUMN ONLY (12 DETERMINATIONS).


2

30

CHEMISTRY AND FUNCTION OF PECTINS

of mobile phase concentration, carboxylate counter i o n and column psd.

In another approach, a t constant degree of methylation, R

g n

values were separated at the (P^ 0.05) confidence l e v e l through differences i n t h e i r logs by the Bonferroni LSD method (17). within any horizontal row i n

Thus

T a b l e I I , means not f o l l o w e d by the

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