Composition and Molecular Weight Determination of Aqueous Two-phase System by Quantitative Size Exclusion Chromatography†

doi:10.7503/cjcu20150567

Abstract

Abstract:

Quantitative size exclusion chromatography(SEC) was exploited to study the composition, molecular weight and molecular weight distribution of aqueous two-phase system of dextran and poly(ethylene glycol)(PEG) following phase separation. Tie lines constructed by SEC method were compared with the cloud point curve of the system obtained by titration. An excellent agreement was found between the tie line end points and the cloud point, except for the data points of the PEG-rich phases close to the critical point. The molecular weight and molecular weight distribution of the two polymer species in two co-existing phases obtained by SEC indicate that the mismatch is caused by the uneven distribution of macromolecular components between two phases upon phase separation. Having a broad molecular weight distribution, dextran in the two phases show prominent molecular mass difference. The molecular weight of dextran in the dextran-rich phase is significantly higher than that in the PEG-rich phase. As the initial polymer concentration of the system increases, the molecular weight difference of dextran in the two phases becomes more significant. However, we have not observed such a trend for PEG because of its narrow molecular weight distribution. Accurate data on the phase diagram and molecular weight of two polymer components in the co-existing phases can be obtained using quantitative SEC. The above results will not only help to understand the phase diagram of dextran-PEG-water ternary system, but also provide guidance to its application in extraction and separation of biological materials.

Key words: Size exclusion chromatography, Aqueous two-phase system, Phase separation, Dextran, Poly(ethylene glycol)

CLC Number:

O631.4

TrendMD:

ZHAO Ziliang, LI Qi, XUE Yanhu, JI Xiangling, BO Shuqin, LIU Yonggang. Composition and Molecular Weight Determination of Aqueous Two-phase System by Quantitative Size Exclusion Chromatography^†[J]. Chem. J. Chinese Universities, 2016, 37(1): 167.

Figures/Tables 7

Fig.1 Cloud point curve of the aqueous solution of dextran and PEG at (25±1) ℃ obtained by titration ^The homogeneous solution in the left vial has a composition in the one-phase region, the right one shows phase separation in the two-phase region. Au nanoparticles are dispersed in water and added to the mixed solution to enhance the color contrast.

Fig.2 Volume fraction(φd) of the dextran-rich phase as a function of the normalized distance from the cloud point(w/wcl-1) for polymer solutions of different mass ratios(wd/wp) between dextran and PEG(A) and dependence of the volume fraction φd on the mass ratio wd/wp at w/wcl=1.01(B)

Fig.3 Dependence of the RI peak area(ARI) as a function of polymer concentration(cinj) of the solutions injected into the SEC columns for dextran(a) and PEG(b)

Fig.4 SEC chromatograms of the coexisting dextran-rich(A) and PEG-rich phases(B) at

Fig.5 Phase diagram of the ternary dextran-PEG-water system^The cloud point curve(same data as in Fig.1) is shown as a solid curve. The compositions of the initial solutions(with mass ratio wd/wp=1.23) for which SEC measurements after phase separation are performed are indicated by black crosses. The end points of the respective tie lines(dashed lines) consist of red crosses indicating the compositions of the dextran-rich phases and green crosses indicating the compositions of the PEG-rich phases. The midpoints(blue circles) of the tie lines were extrapolated to determine the critical point.

Table 1 Molecular weight of dextran in dextran-rich and PEG-rich phases

No.	ε	Dextran-rich phase			PEG-rich phase
No.	ε	10^-3 M_w	10^-3 M_n	M_w/M_n	10^-3 M_w	10^-3 M_n	M_w/M_n
Native	______	380.0	173.7	2.19	380.0	173.7	2.19
1	0.0098	453.9	192.9	2.35	249.3	152.6	1.63
2	0.030	426.9	182.0	2.35	208.9	138.3	1.51
3	0.092	394.1	172.1	2.29	167.6	120.8	1.39
4	0.134	397.9	177.8	2.24	152.3	112.5	1.35
5	0.200	380.6	170.3	2.23	136.0	103.1	1.32
6	0.302	369.8	164.2	2.25	116.9	90.0	1.30
7	0.389	373.3	167.1	2.23	109.4	84.8	1.29
8	0.508	371.3	164.6	2.26	97.1	76.9	1.26
9	0.599	370.6	163.1	2.27	95.9	69.4	1.38
10	0.729	375.2	166.0	2.26	82.2	66.9	1.23
11	0.982	372.4	163.7	2.28
12	1.552	368.7	161.8	2.28
13	2.087	368.6	155.8	2.37
14	2.825	374.7	160.5	2.34
15	3.605	368.7	161.8	2.28

Table 2 Molecular weight of PEG in dextran-rich and PEG-rich phases

No.	ε	Dextran-rich phase			PEG-rich phase
No.	ε	10^-3 M_w	10^-3 M_n	M_w/M_n	10^-3 M_w	10^-3 M_n	M_w/M_n
Native	_____	8.45	7.64	1.11	8.45	7.64	1.11
1	0.0098	8.38	7.57	1.11	8.35	7.58	1.10
2	0.030	8.12	7.33	1.11	8.35	7.61	1.10
3	0.092	8.08	7.25	1.11	8.30	7.52	1.10
4	0.134	8.18	7.31	1.12	8.41	7.65	1.10
5	0.200	8.02	7.06	1.14	8.39	7.63	1.10
6	0.302	8.02	7.00	1.15	8.28	7.53	1.10
7	0.389	7.98	6.89	1.16	8.40	7.63	1.10
8	0.508	7.93	6.69	1.19	8.38	7.60	1.10
9	0.599	7.88	6.55	1.20	8.50	7.70	1.10
10	0.729	7.78	6.22	1.25	8.29	7.49	1.11
11	0.982				8.44	7.65	1.10
12	1.552				8.63	7.82	1.10
13	2.087				8.43	7.64	1.10
14	2.825				8.40	7.57	1.11
15	3.605				8.56	7.76	1.10

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