Chapter 18
Comparison of Secondary Effects in Aqueous Size Exclusion Chromatography Between Sodium Poly(styrenesulfonate) Compounds of Different Sulfonations Strategies in Size Exclusion Chromatography Downloaded from pubs.acs.org by MACQUARIE UNIV on 02/19/19. For personal use only.
Preliminary Study Sadao Mori and Toshitaka Oosaki Department of Industrial Chemistry, Faculty of Engineering, Mie University, Tsu, Mie 514, Japan
Hydrophobic interactions of sodium poly(styrenesulfonate) compounds (NaPSS) of different sulfonation were investigated. One was commercially available and was considered to be less than 85% degree of sulfonation. The other was prepared in our laboratory from sodium styrenesulfonate monomer and was considered to be 100% sulfonation. Column was Shodex PROTEIN KW-804 packed with glycerylpropylgroup bonded s i l i c a gel. Mobile phase was sodium phosphate buffer at pH 7.0 at different ionic strengths. Calibration plots for these two types of NaPSS pullulan showed that retention volumes of NaPSS increased with increasing the ionic strength, but the extent was different between two types of NaPSS. Hydrophobicity of low sulfonated NaPSS was originated from both unreacted phenyl groups and the backbone C-C linkage. Highly sulfonated NaPSS still exhibited some hydrophobicity which may be originated from the backbone C-C linkage. Aqueous size exclusion chromatography (ASEC) i s the technique that permits the separation and the measurement of molecular weight (MW) averages of water-soluble polymers which include i o n i c or nonionic synthetic polymers and proteins. However, since both the stationary phase and the polyer solute i n ASEC of i o n i c polymers possess numerous polar groups, mutual interactions between them w i l l lead to nonideal SEC. These mutual interactions are divided into two secondary e f f e c t s : ion exclusion and hydrophobic interactions. In the previous paper (I), elution behavior of sodium poly(styrenesulfonate) (NaPSS) compounds compared with nonionic linear polysaccharide (pullulan) on several types of ASEC columns was reported at varing ionic strengths of the mobile phase. The divergence of the hydrodynamic volume c a l i b r a t i o n curves of NaPSS from that of the pullulan was observed and the c a l i b r a t i o n curves of NaPSS changed with changing the ionic strength. These phenomena were ascribed to the combination of three separation e f f e c t s : size exclusion, i o n 0097^6156/96/0635-0347$15.00/0 © 1996 American Chemical Society
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exclusion, and hydrophobic interactions. There are a number of similar studies concerning the secondary effects i n ASEC of sodium poly(styrenesulfonate) compounds (2-6). NaPSS samples used i n the previous study were purchased from the commercial source and were prepared from polystyrene samples of narrow MW d i s t r i b u t i o n s by sulfonation. Therefore, the degree of sulfonation of the NaPSS samples used i n the work was supposed to be less than 85% and the unreacted phenyl groups may be considered to be the main s i t e s of hydrophobic interactions. In the present work, the preparation of NaPSS of 100% degree of sulfonation has been attempted and the e l u t i o n behavior of both com mercial NaPSS samples (designated as low sulfonated NaPSS) and NaPSS samples prepared i n our laboratory (designated as highly sulfonated NaPSS) has been compared. This i s the preliminary report on this matter. The detailed study on this subject w i l l be published e l s e where .
Experimental P r e p a r a t i o n o f NaPSS o f 100% degree o f s u l f o n a t i o n . Sodium styrene sulfonate (monomer)(reagent grade, Tokyo Kasei, Japan) was p u r i f i e d by r e c r y s t a l i z a t i o n two times with the water-acetone system. 5.3 g of the monomer, 0.3 g of sodium s u l f i t e , and 0.14 g of potassium peroxydisulfate (these two reagent were used as polymerization i n i t i a t o r s ) were dissolved i n 15 mL of degassed d i s t i l l e d water and the solution was reacted for 1 h at 45 C under reduced pressure. The reaction solution was then poured into excess acetone with s t i r r i n g . The precipitated material was dissolved i n water and the solution was poured into excess acetone. This process was repeated two times and then the obtained p r e c i p i t a t e was dried at room temperatures under vacuum. The polymer sample was then fractionated into eight fractions by f r a c t i o n a l p r e c i p i t a t i o n , dissolving the polymer sample in 4 Ν sodium iodide solution i n the concentration of 1% and kepting the solution at 20 C, followed by dropping 9.1 Ν sodium iodide solution into the polymer solution. Precipitated material was f i l t e r e d and each f r a c t i o n was p u r i f i e d by d i s s o l u t i o n of the f i l t e r a t e d f r a c t i o n into water, followed by r e p r e c i p i t a t i o n of the material into acetone. ASEC. Measurements were performed on a Jasco high-performance l i q u i d chromatograph Model TRIROTAR-V (Jasco, Tokyo, Japan) with a r e f r a c t i v e index detector Model SE-11 (Showa Denko, Tokyo, Japan). A column was Shodex PROTEIN KW-804 (300-mm χ 8-mm id.d) packed with glycerylpropyl group-bonded s i l i c a gel (a glycophase-bonded support). Pullulan standards which are nonionic l i n e a r polysaccharides were purchased from Showa Denko and NaPSS standards were from Pressure Chemical (Pittsburgh, PA). The mobile phase was made up from sodium monohydrogen phosphate, Na2HP04, and sodium dihydrogen phosphate, NaH2P04, to the disired i o n i c strength at pH 7.0. Ionic strength was changed from 0.005M to 0.3M. The flow rate was 1.0 mL/min. The sample were dissolved i n the solvent used as the mobile phase i n the concentration of 0.05% and i n j e c t i o n volume of these sample solutions was 0.1 mL.
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Results Calibration plots f o r low sulfonated NaPSS, highly sulfonated NaPSS and pullulan standards are shown i n Figure 1 at various ionic strengths. The effect of the ionic strength on retention volume f o r pullulan was almost n e g l i g i b l e . In contrast to p u l l u l a n , the s i g n i f i c a n t difference i n retention volume for NaPSS with ionic strength was observed. The i n t e r s i t i a l volume was estimated to be at 6.0 mL with low sulfonated NaPSS having MW 10 . Retention volume of both types of NaPSS increased with increas ing the ionic strength. At lower ionic strengths (0.005 M and 0.01 Μ ) , both types of NaPSS eluted from the column e a r l i e r than pullulan and they possessed the almost s i m i l a r c a l i b r a t i o n plots at each ion i c strength. At higher i o n i c strengths (0.2 M and 0.3 Μ ) , they 6
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8 10 12 14 Retention V o l u m e , mL
16
Figure 1. C a l i b r a t i o n plots for pullulan ( © ) , low sulfonated NaPSS ( f i l l e d marks), and highly sulfonated NaPSS (open marks). A s o l i d l i n e i s a p u l l u l a n c a l i b r a t i o n curve and broken l i n e s are c a l i b r a t i o n curves f o r low sulfonated NaPSS. Ionic strength; # Ο , 0.005 M; H • , 0.01 Μ; γ ν > · ? • Ο > ° · · 0
2
Μ
3
Μ
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eluted l a t e r than pullulan and highly sulfonated NaPSS eluted e a r l i er than low sulfonated NaPSS having the s i m i l a r MW. This difference i n retention volume became larger with increasing the ionic strength.
Discussion Shodex PROTEIN KW-804 column s t i l l has some amount of unreacted s i lanol groups on the surface of packings. Early e l u t i o n of NaPSS r e l a t i v e to pullulan on the column i s governed by the ion exclusion e f f e c t as reported i n the previous paper. The extent of the electro s t a t i c interactions between NaPSS and the packings was almost the same, implying that the electrorepulsive force of low sulfonated NaPSS i s s i m i l a r to that of highly sulfonated NaPSS. Glycerylpropyl(l,2-dihydroxy-3-propoxypropyl) groups which are bonded on the surface of the packings are hydrophilic and therefore, hydrophobic interactions between the support and NaPSS solutes are supposed to be small. However, as reported i n the previous paper, l a t e elution of NaPSS r e l a t i v e to pullulan i s governed by hydropho bic interactions between NaPSS and the packings. The extent of the hydrophobic interactions of low sulfonated NaPSS i s larger than highly sulfonated NaPSS, implying that the hydrophobic interactions of low sulfonated NaPSS are governed by both unreacted phenyl groups and the backboneC-C linkage of propoxypropyl groups on the packings. Hydrophobic parts of highly sulfonated NaPSS are only the backbone C-C linkage, therefore, hydrophobic interactions of highly sulfonat ed NaPSS are smaller, r e s u l t i n g early e l u t i o n than low sulfonated NaPSS. In conclusion, hydrophobicity of low sulfonated NaPSS i s o r i g inated from both unreacted phenyl groups and the backbone C-C l i n k age. Highly sulfonated NaPSS s t i l l e x i b i t s some hydrophobicity in the ASEC system examined i n this work and the main s i t e of the hy drophobicity i s originated from the backbone C-C linkage. Low s u l fonated NaPSS i s supposed to have unreacted ohenyl groups (Dubin,P., Indiana University Purdue University Indianapolis, personal communi cation, 1994).
Literature Cited (1) Mori, S. Anal. Chem. 1989, 61, pp 530-534. (2) Spatorico, A. L. Beyer, G. L. J. Appl. Polym. Sci. 1975, 19, pp 2933-2945. (3) Rochas, C.; Domard, Α.; Rinaudo, M. Eur. Polym. J. 1980, 16, pp 135-140. (4) Callec, G.; Anderson, A. W.; Tsao, G. T.; Rollings, J. E. J. Polym. Sci. Polym. Chem. Ed. 1984, 22, pp 287-293. (5) Dubin, P. L.; Tecklenburg, M. M. Anal. Chem. 1985, 57, pp 275-279. (6) Soria, V.; Campos, Α.; Garcia, R.; Parets, M. J. J. L i q . Chromatogr. 1990, 13, pp 1785-1808.
