Chromatographic Evaluation of Resolutionand Secondary Mechanisms of Pureand Mixed Sets of SEC Columns:TSK-Gel HHR and TSK-Gel HXL

R. Garcıa-Lopera1, C. M. Gomez1, M. Falo1, C. Abad2, A. Campos1,&

1 Institut de Ciencia dels Materials, Departament de Quımica Fısica; 46100 Burjassot, Valencia, Spain;E-Mail: agustin.campos@uv.es2 Departament de Bioquımica i Biologia Molecular. Universitat de Valencia; 46100 Burjassot, Valencia, Spain;E-Mail: agustin.campos@uv.es

Received: 5 March 2003 / Revised: 11 June and 15 September 2003 / Accepted: 11 November 2003Online publication: 6 February 2004

Abstract

Size exclusion chromatographic (SEC) evaluation of secondary mechanisms and column spe-cific resolution, of five solvent/polymer systems in four sets of pure and mixed organic columnspackings based on polystyrene/divinylbenzene copolymer, TSK-Gel HHR and TSK-Gel HXL, hasbeen carried out. The combination of columns employed has been: (i) three HHR columns (set A);(ii) two HHR and one HXL column (set B); (iii) one HHR and two HXL columns (set C) and (iv) threeHXL columns (set D). Both packings offer similar characteristics (pore size, particle size, effi-ciency) but some differences have been found when eluting the same systems in differentcombination of both of them. Values of the chromatographic partition coefficient, Kp, of thevolume fraction of the network in the swollen state, /3, and the concentration effect onthe retention volume have been related. It can be concluded that the higher the /3, the higherthe crosslinking degree and Kp, and the higher the concentration effect on the retention volumefor a given solvent/polymer system. We also observe that, in general, a decrease of Kp comesalong with an increase of specific resolution, RS, and that the sets of mixed columns show lowersecondary effects different from pure size exclusion (lower Kp) and higher RS than the two sets ofpure HHR or HXL columns.

Keywords

Size exclusion chromatographyColumn packingUniversal calibrationSpecific resolution

Introduction

Size-exclusion chromatography (SEC) is

a widely employed method for polymer

molar mass determination [1]. Recent

developments in polymer characteriza-

tion through the SEC technique involve

modern columns and combination of

different detectors to obtain a better

resolution [2–10]. Instrumentation for

on-line molecular size determinations

such as light scattering and viscosity

detectors, in addition to a typical con-

centration detector (refractive index),

allow to obtain more accurate determi-

nations of molar mass distributions

(MMDs) and average molar masses

[3, 8], and also to correct the concen-

tration effect on the retention volume [3,

4]. However, in the cases where a unique

concentration detector is available, it is

desirable that size exclusion should be

the only mechanism governing the sep-

aration process inside the column, and

that secondary mechanisms such as

partition or adsorption will be negligible

[11, 12]. The size exclusion mechanism is

solely controlled by the solute hydrody-

namic volume and the average molar

mass determined from calibration

curves. In order to obtain accurate re-

sults, some chromatographic parameters

influencing the retention profile have to

be controlled. Among them, it deserves

to be mentioned the characteristics of

the packing material (particle and pore

size), the flow rate, the molar mass of

the eluting polymers, the injected

polymer concentration and the specific

resolution, RS, which measures the sep-

aration efficiency of the columns and is

related with the number of theoretical

plates [13].

Summarizing, minima secondary ef-

fects represented by the chromato-

graphic partition coefficient, Kp, and

maxima RS values, are desirable for a

better performance of a SEC column.

Moreover, the lower the concentration

effect on the retention volume the better

accuracy in the determination of

MMDs.

DOI: 10.1365/s10337-003-0181-9

Presented at: International Symposium on Se-paration and Characterization of Natural andSynthetic Macromolecules, Amsterdam, TheNetherlands, February 5–7, 2003

2004, 59, 355–360

0009-5893/04/03 � 2004 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

Original Chromatographia 2004, 59, March (No. 5/6) 355

In this respect, SEC research in our

group has been focussed on determining

the characteristic chromatographic para-

meters of different SEC columns such as

TSK-Gel HHR [12] and TSK-Gel HXL

[14]. These works revealed that, in gen-

eral, the HXL packings posses higher

crosslinking degree and lower swelling

degree than the HHR ones, for the same

solvent/polymer systems elution. Also, we

observed that, in general, the columns

with higher crosslinking degree induce

increasing secondary effects, mainly

polymer-gel matrix attractive interactions

quantified by Kp. These effects have also

been related with the preferential solva-

tion phenomenon of the polymer sample

onto the gel matrix [15]. In order to better

clarify the applicability of these packings,

we have aimed our investigations to elu-

cidate the chromatographic response of

four sets of columns, each one formed by

three columns in series. In the four sets of

columns analysed we have gradually

changed from pure HR to pure XL

columns by mixing both packings in

series. Thus, the assayed column sets

have been: (i) HR + HR + HR (set A),

(ii) HR + XL + HR (set B), (iii)

XL + HR + XL (set C) and (iv)

XL + XL + XL (set D). For each set,

the raw data are the universal calibration

plots, log(hydrodynamic volume) against

retention volume, of five solvent/polymer

systems, from which the Kp values have

been determined. Furthermore, some

equations previously reported from ther-

modynamic studies [16] have served to

obtain the volume fraction of the network

in the swollen state, /3, for the four sets.

The specific resolution of each set of col-

umns has been evaluated from the reten-

tion volumes and polydispersity index of

the eluted samples. Finally, the concen-

tration effects have been analysed by

quantifying the change on the retention

volume with the injected concentration,

DVR/Dc. Therefore, in this paper, we

compare the parameters /3, Kp, RS and

the concentration effects for the four sets

of pure and mixed TSK-Gel HHR and

HXL columns.

Experimental

Chemicals

Narrow standard polymer samples of

polybutadiene (PBD) (polydispersity

index, I ¼ 1.03–1.15) purchased from

Polymer Source Inc. (Dorval, Canada)

and poly(dimethyl siloxane) (PDMS)

(I ¼ 1.06–1.23) from Polymer Laborato-

ries (Shropshire, UK) and Polymer

Source Inc. (Dorval, Canada) were

used as polymeric solutes. Their weight-

average molar masses are compiled in

Table 1. Tetrahydrofuran (THF), ben-

zene (Bz), toluene (Tol) and 1–4 dioxane

(Diox) of chromatographic grade from

Scharlau (Barcelona, Spain) were used as

solvents or eluents.

Chromatography

A Waters liquid chromatography equip-

ment with refractive index detector was

used for SEC experiments as previously

described [17–19]. Four sets of three col-

umns (each one of 7.8 mm ID · 300 mm)

based on a polystyrene/divinylbenzene,

(PS/DVB) crosslinked copolymer TSK-

Gel from Tosohaas, Tosoh Corp.

(Tokyo, Japan) have been compared. In

the four sets of columns analysed we have

gradually changed from pure HR to pure

XL columns by mixing both packings in

series. Thus, the assayed column sets

have been: (i) HR + HR + HR (set A),

(ii) HR + XL + HR (set B), (iii)

XL + HR + XL (set C) and (iv)

XL + XL + XL (set D). Their packing

characteristics as particle size, nominal

pore size, pore and total exclusion vol-

umes (Vp and V0) are summarised in

Table 2 for the individual columns and in

Table 3 for the column sets.

All the solvents used as eluents were

previously degassed and filtered by pass-

ing them under vacuum through a

0.45 lm regenerated cellulose filter from

Micro Filtration Systems (Dublin, CA,

USA). All chromatographic experiments

were performed at 25 �C and the columns

were equilibrated overnight prior to

starting any experiment. Chromatograms

were obtained at a flow rate of 1 mL

min)1 by injection of 100 lL of sample

solution. To avoid concentration effects

[1] on the retention volumes, VR, all sol-

ute samples were injected at four con-

centrations and extrapolated to zero

concentration. The concentration effect

on VR was evaluated for every system as

DVR/Dc (mL2 g)1). The elution behaviour

was analysed in terms of the ‘‘universal

calibration’’ curves made by plotting the

hydrodynamic volumes, Vh (as log M[g])versus VR. The five systems assayed

were: THF/PBD, Bz/PBD, Diox/PBD,

Bz/PDMS and Tol/PDMS.

Viscometry

An automatic AVS 440 Ubbehlode-type

capillary viscometer from Schott Gerate

(Hofheim, Germany) at (25.0 ± 0.1) �Cwas used to perform viscometric mea-

surements. The stock solution was made

by dissolving the polymer samples in the

corresponding solvent up to a concen-

tration of approximately 1.0 g dL)1. At

least six dilutions were obtained by add-

ing the appropriate aliquots of solvent.

Efflux time of the solvent was always

above 100 s. For each solution, a 12 mL

sample, to minimize drainage errors, was

loaded into the viscometer and placed in

the thermostated bath. Measurements

started after an equilibration time of ca.

5–10 min and were continued until sev-

eral flow time readings agreed to within

0.5%. The elution time of each solution is

then determined as the average of several

readings. The dilution and measurements

are stopped when the viscosity difference

of the sample solution and pure solvent

drops below 10%.

The intrinsic viscosity, [g], for the

different solvent-polymer pairs was eval-

uated by extrapolation to infinite dilution

(zero solute concentration) of Huggins

plots: gsp/c ¼ [g] + bc , i.e. gsp/c vs. c,

being gsp the specific viscosity, c the

Table 1. Weight-average molar masses ( �Mw) of the narrow standard polymers used for the SECmeasurements in each set of columns

PDBa PBDb PBDc PDMSa PDMSb PDMSc

5950 920 6250 8100 1140 114013400 6250 12600 41500 8100 810047000 12600 42300 76030 33500 3350067300 34000 60700 188400 123000 8050086500 60700 105700 681600 188000 12300094250 105700 323000 188000268000 323000 3600001120000 360000

a used in set A; b used in set D and c used in sets B and C

356 Chromatographia 2004, 59, March (No. 5/6) Original

concentration of the solution and b a

viscosimetric parameter. All the polymer

samples yielded good linear correlations

(r > 0.99) when plotting log[g] vs.

log M from which the Mark-Houwink-

Sakurada (MHS) equation, [g] ¼ KMa,

with K and a constants for each polymer-

solvent-temperature system, were evalu-

ated [20].

Results and Discussion

Figure 1 shows the universal calibration

curves log M[g] against VR obtained with

PBD and PDMS in different eluents for

the four sets of TSK-Gel columns anal-

ysed. In the hydrodynamic volume range

here studied all the ‘‘universal calibra-

tion’’ plots were lineal (with correlation

coefficient, R > 0.998). As can be seen,

some differences arise in the elution of the

same solvent/polymer systems in the

different sets of columns. The highest

difference in retention volumes among

the different systems is observed for the

set D formed by pure TSK-Gel HXL

columns (Fig. 1d). Surprisingly, the

mixed TSK-Gel columns, set B (Fig. 1b)

and set C (Fig. 1c) show similar universal

calibration plots. It seems, a priori, that

the set D will present more secondary

effects different from pure size exclusion.

In order to better explain the observed

elution behaviour, we have selected

three values of the hydrodynamic vol-

ume (M[g] ¼ Vh ¼ 106, 107 and 108 mL

mol)1) which are representative of the

most effective mass separation range. The

retention volumes, VR, for non-ideal SEC

(when secondary mechanisms as adsorp-

tion appear) are given by [21]:

VR ¼ V0 þ KSECKpVp ð1Þ

where KSEC is the size distribution coef-

ficient for ideal SEC and Kp the coeffi-

cient accounting for interactions between

the components of the chromatographic

system, such as solute-solvent, solvent-gel

and solute-gel. In this regard, values of

Kp ¼ 1 signify the absence of secondary

polymer-gel interactions; Kp < 1 would

imply solute-gel repulsions while Kp > 1

denotes attractive interactions. The KSEC

data compiled in Table 4 have been ob-

tained with a reference system for which

Table 2. Individual column characteristics

Column Particlesize (lm)a

Effective Mw

rangea

Number oftheoretical plates, n

Asymmetryfactor

V0 (mL)b

Vp (mL) VT (mL)c

HHR G2500 5 200–40000 22700 1.16 5.90 5.10 11.00HHR G4000 5 1000–600000 20000 0.97 4.80 5.60 10.40HHR G6000 5 10000–4 · 106 28200 1.00 5.30 5.00 10.30HXL G2500 5 200–40000 17100 1.24 5.65 5.45 11.10HXL G4000 6 1000–600000 18300 1.12 6.50 5.00 11.50HXL G6000 9 10000–4 · 106 18600 1.17 7.65 3.95 11.60

a Supplied by the manufacturer;b Determined with a PS standard of molar mass higher than the exclusion limit;c Determined with a solution of Bz in THF (1 lL mL)1)

Table 3. Column sets characteristics

Column set Set name Effective Mw

rangeaV0 (mL)b Vp (mL) VT (mL)c

HHR G2500HHR G4000 A 200–4 · 106 16.40 16.80 33.20HHR G6000HHR G2500HXL G4000 B 200–4 · 106 15.83 16.17 32.00HHR G6000HXL G2500HHR G4000 C 200–4 · 106 15.68 15.32 31.00HXL G6000HXL G2500HXL G4000 D 200–4 · 106 17.07 16.63 33.70HXL G6000

a Supplied by the manufacturer;b Determined with a PS standard of high molar mass (Mw = 3800000);c Determined with small molecules such as Tol or Bz in THF (1 lL mL)1)

Fig. 1. Universal Calibration plots for different solvent/polymer systems eluted in: (a) set A ; (b) setB; (c) set C; (d) set D

Original Chromatographia 2004, 59, March (No. 5/6) 357

Kp is assumed to be the unity. PS in THF

is generally used as reference system to

evaluate KSEC since it is supposed that

elutes according to a size-exclusion

mechanism (only driven by entropic ef-

fects). However, this assumption implies

that the PS/THF universal calibration

plot (as log M[g] vs. VR) would be located

at the left-hand side (lowest VR) of the

rest of the systems where size-exclusion

and adsorption occurs. This is not the

case in the present study. Moreover and

thermodynamically speaking, and ‘‘ideal’’

reference system should be that one with

Kp ¼ 1, obtained in an independent way.

Recently, we have proposed, for a sol-

vent(1)/polymer(2)/gel(3) ternary system,

an expression that relates Kp with ther-

modynamic interaction functions [16].

Briefly:

lnKp ¼ �V2

V1/3gchr ð2Þ

where gchr ¼ 1� g12 � g13 þ g23 þ gT and

gij (i ¼ 1, 2, 3) and gT are functions that

take into account all the binary and ter-

nary interactions between the three com-

ponents of the system. These functions,

composition dependent, for the different

systems studied were independently

obtained from phase separation experi-

ments [18]. Therefore, a system can be

considered as a reference one if all the

binary and ternary enthalpic interactions,

always present, are counterbalanced and

gchr ¼ 0, then Kp ¼ 1 and the separation

process could be denoted as ‘‘ideal’’ SEC.

In practice, is rather difficult to indepen-

dently evaluate /3 and gchr and calculate

Kp according to Eq. (2) [11]. Therefore,

for practical purposes, the system with

the lowest VR is taken as reference sys-

tem, given that when secondary adsorp-

tion occurs only attractive solute-gel

interactions take place, but not repulsive

ones. For the column sets here assayed,

the system with the lowest VR is different

in every set. For this reason and in order

to make homogeneous comparisons, we

have chosen a unique system, the Tol/

PDMS as the reference system in all col-

umn sets. In consequence, the KSEC val-

ues have been calculated by applying

Eq. (1) and by using the retention vol-

umes of the same reference system, Tol/

PDMS, and the pore volumes of each set

of columns. Since the Vp is obtained by

the difference (VT ) Vo), as VT decreases

also the Vp does and KSEC will be larger

for those sets with lower pore volumes, as

seen in Table 4.

The Kp values are determined with the

corresponding Vo, Vp and KSEC data

(Tables 3 and 4) and VR given in Fig. 1 at

the three Vh selected; and are gathered in

Table 5 for every solvent/polymer system

and column set studied. The comparison

of Kp values reveals that: the set D shows

the highest values, so the highest inter-

actions and adsorption; the set A depicts

higher Kp than the mixed columns set B

and set C; set C has higher Kp than set B,

that is more adsorption or secondary ef-

fects when the proportion of XL columns

increases; the pure HR (set A) and XL

(set D) sets depict higher Kp values than

the mixed columns, sets B and C. More-

over, it is worthwhile to point out that as

a consequence of the choice of a common

reference system (Tol/PDMS) for all the

column sets, obviously appear values of

Kp < 1 in Table 5. Concretely, for the

system THF/PBD in set A and for Diox/

PBD in sets B and C, due to the fact that

their VR are lower than those corre-

sponding to the Tol/PDMS. However, in

the present context, values of Kp < 1

do not mean the existence of repulsive

solute-gel interactions given that are val-

ues relative to that of the reference system

but not absolute values.

By comparing the Kp data in all pac-

kings it is shown that, in general (except

for the Diox/PBD system), the lower

adsorption effects occur in the set B. This

evidence could be attributed to a lower

chain density or crosslinking degree in the

TSK-Gel HHR than in the TSK-Gel HXL,

leading to a decrease of the solute-gel

interactions. Overall, the highest Kp value

is observed for the set D, pure XL col-

umns. Therefore, the set of columns can

be ordered according to the increasing Kp

value or, in other words, from lower to

higher secondary effects as: set B < set

C < set A < set D.

On the other hand, the volume frac-

tion of gel matrix, /3, involved in the

chromatographic separation process can

be evaluated from Eq. (2) in the way that

has been recently explained with detail

[11, 12, 14–16]. The values obtained are

also presented as average �/3 values in

Table 5. As can be seen, the tendency of

/3 is similar to that of Kp for every

system, i.e. both increase or decrease

simultaneously. Moreover, /3 is directly

related to the crosslinking degree of the

polymeric gel network [12]. Thus, we can

conclude that Kp, /3 and the crosslinking

degree mostly evolve similarly when

changing from a set of pure TSK-Gel

HHR columns to a set of pure TSK-

Gel HXL.

Table 5 also depicts the concentration

effect on the retention volume expressed

as the relationship DVR/Dc, where DVR

and Dc are the change of retention vol-

ume and concentration, respectively. This

concentration effect depends on the mo-

lar mass of the polymer (concretely, this

effect is evidenced for Mw > 100000

g/mol) and is caused by two contribu-

tions: hydrodynamic effects, such as the

macromolecular crowding, and thermo-

dynamic effects like the attractive solute-

gel enthalpic interactions (or preferential

solvation of the polymer by the gel

matrix). The former is independent of the

packing used but not the second effect.

From what we know, there is no an un-

ique model to quantify both effects

simultaneously; on one hand there are

some models to predict the solute

crowding on the retention volumes [22–

26], and on the other, recent analysis re-

lates the contribution of the gel packing

with the preferential solvation phenome-

non [15] or with the enthalpic polymer-

stationary phase interaction [9]. For this

reason, the present analysis compares the

values for the same solvent/analyte sys-

tems eluted in different packings. As the

macromolecular crowding experimented

by the solute is the same, the observed

differences can be mainly attributed to

the solute-gel enthalpic interactions, and

then related to the Kp values. Therefore, it

is noticed that the trend of DVR/Dc is

strongly influenced by the Kp, /3, and

crosslinking degree data, in agreement

with previous studies [15]. In general, the

major concentration effect due to the low

capacity adsorption phenomena occurs in

the gel that presents major Kp value,

as set D (pure XL), except for the

system Diox/PBD. Therefore, it could

be stated that the higher the cross-

linking degree the higher the concentra-

tion of chemical residues coming from

the gel synthesis, and so the higher

Kp values representing secondary SEC

Table 4. Values of KSEC for the four sets ofcolumns at the three hydrodynamic volumes(in mL mol)1) selected

KSEC

Set name Vh = 106 Vh = 107 Vh = 108

A 0.308 0.205 0.102B 0.298 0.173 0.048C 0.338 0.221 0.104D 0.267 0.152 0.037

358 Chromatographia 2004, 59, March (No. 5/6) Original

effects such as solute-gel attractive inter-

actions. Thus, when comparing the con-

centration effect among systems where

only the gel matrix changes, the Kp values

are directly related with the concentration

effect.

In SEC chromatography, two factors

are of equal importance in order to

achieve a better efficiency: i) minima

secondary effects, and ii) maximum res-

olution of the gel matrix. Regarding the

separation efficiency, when the injected

sample is formed by different species, it is

important that the respective elution

peaks appear at clear well different times.

In this sense, another important feature

of a SEC gel matrix is the resolution, R,

or column capacity to separate species 1

and 2 of molar masses M1 and M2, which

is defined as [13]:

R ¼ 2ðVR;2 � VR;1Þw1 þ w2

ð3Þ

where VR,1 and VR,2 are the retention

volumes of species 1 and 2, and w1 and w2

are the widths of peaks 1 and 2 at base-

line. Since R depends on the pair of

polymers to be eluted, it is convenient to

use a parameter independent of the molar

mass. To do that, the specific resolution,

RS, is defined as [13, 27]:

RS ¼2ðVR;2 � VR;1Þ

ðw1I1 þ w2I2Þ logðMw;2=Mw;1Þð4Þ

being the polydispersity index, Ii ¼(Mw,i/Mn,i) (i ¼ 1, 2), and Mw and Mn the

weight and number average molar mas-

ses, respectively. Values of RS measure

the separation efficiency of the columns

and the higher the RS value, the better the

column resolution. The RS data compiled

in Table 6 and calculated for the four sets

of columns are very high when compared

with old polystyrene gels [28] where val-

ues of RS between 0.43 and 0.60 were

found. Inspection of Table 6 reveals that:

the set B has better resolution than the

sets A, C and D; the set A has better

resolution than the sets C and D; the set

D has better resolution than the set C. In

general, these results indicate that the

columns TSK-Gel HHR have higher res-

olution than the columns TSK-Gel HXL,

and that the mixed columns with HHR as

major component show the best resolu-

tion of any other combination of col-

umns, in good accord with the universal

calibration plots (Fig. 1). Moreover and

from data of individual columns gathered

in Table 2, it is noticeable that the col-

umn HXL-G6000 posses a low number of

theoretical plates, the lowest pore volume

and the highest particle size. All theses

characteristics imply a poorer specific

resolution, specially the particle size

[13, 28]. Therefore, the specific resolution

will decrease more markedly in those

column sets where the HXL-G6000

column take part, that is, in sets C

and D as observed in Table 6. Summa-

rizing, the column sets can be ordered

according to their decreasing specific

resolution capacity as: set B > set A >

set D > set C.

Table 5. Values of Kp, /3, DVR/Dc for the different systems studied in the four sets of columns at the three hydrodynamic volumes

System Column set Vh = 106 (mL mol)1) Vh = 107 (mL mol)1) Vh = 108 (mL mol)1) DVR/Dca(mL2 g)1)

Kp 102�/3 Kp 102�/3 Kp 102�/3

THF/PBD A 0.981 3.10 0.966 3.08 0.935 2.62 50B 1.011 3.04 1.080 3.16 1.285 3.18 50C 1.015 3.05 1.080 3.16 1.513 3.15 150D 1.307 3.45 1.515 3.38 3.013 3.43 125

Bz/PBD A 1.205 7.18 1.160 4.96 1.038 4.99 85B 1.131 6.09 1.206 6.13 1.450 6.12 75C 1.163 6.07 1.249 6.13 1.785 6.11 75D 1.876 6.72 2.466 6.73 6.726 6.73 150

Diox/PBD A 1.265 3.50 1.364 3.57 1.659 3.55 125B 0.959 2.27 0.968 3.44 1.024 3.79 50C 0.890 2.21 0.855 2.30 0.738 3.40 50D 1.056 3.11 1.004 3.30 0.628 3.25 25

Bz/PDMS A 1.246 9.20 1.244 9.2 1.246 9.20 100B 0.996 9.20 0.984 7.4 0.911 9.20 50C 1.004 9.20 1.000 9.2 0.988 6.10 50D 1.557 18.6 1.886 18.6 4.257 18.60 75

Tol/PDMS A 1.097 15.86 1.095 15.86 1.113 15.86 75B 1.000 8.7·10)7 1.001 1.1·10)4 1.113 1.12·10)4 75C 1.000 1.000 1.000 75D 1.003 17.60 1.008 17.60 1.001 17.50 75

a The concentration effect was evaluated with a PBD of Mw= 360000 g mol)1 and PDMS of Mw= 188000 g mol)1

Table 6. Values of the specific resolution, RS, and of the magnitudes involved in equation 4

Column set Systema VR,1 (mL) w1 (mL) VR,2 (mL) w2 (mL) RS

Bz/PBD 22.80 1.30 20.45 1.30 2.10A Bz/PDMS 22.60 1.80 20.75 1.30 1.80

Tol/PDMS 22.75 1.90 20.70 1.30 1.94

Bz/PBD 23.35 1.20 20.85 1.30 2.32B Bz/PDMS 23.00 1.90 21.00 1.30 1.89

Tol/PDMS 23.40 2.10 20.80 1.30 2.31

Bz/PBD 23.00 1.60 20.90 1.50 1.57C Bz/PDMS 22.80 2.30 21.00 1.70 1.36

Tol/PDMS 22.80 2.30 21.00 1.60 1.40

Bz/PBD 23.10 1.70 20.80 1.30 1.78D Bz/PDMS 23.00 2.60 21.00 1.40 1.51

Tol/PDMS 23.00 2.20 21.10 1.10 1.74

a PDB samples: Mw1= 6250, Mw2 = 42300, Mn1 = 6000, Mn2 = 41000 (in g mol)1)PDMS samples: Mw1 = 8100, Mw2 = 33500, Mn1 = 7500, Mn2 = 31600 (in g mol)1)

Original Chromatographia 2004, 59, March (No. 5/6) 359

By comparing the Kp and RS values

compiled in Tables 5 and 6 for Bz/PBD,

Bz/PDMS and Tol/PDMS, we notice an

increase of RS at the same time that a

decrease of Kp values. By looking to-

gether all the systems, RS increase and Kp

decrease when changing from set D (pure

XL) to set A (pure HR) to set B (HR in

major percentage), but the set C gives an

anomalous trend specially concerning the

specific resolution. It could be attributed

to the highest KSEC value (Table 4) or the

lowest Vp value (see Table 3), since a low

pore volume also tends to diminish the

resolution.

Finally to conclude we can say that:

– the set with three TSK-Gel HHR col-

umns, set A, has better resolution and

less secondary effects than the set with

three TSK-Gel HXL columns, set D.

– The mixed set of columns with gel HR

as major component (set B) shows

better resolution and less secondary

effects than any other of the two pure

HHR or HXL sets (sets A and D).

– The behaviour of the set C (with XL

as major contribution) is unclear

since, on one hand it shows less sec-

ondary effects than the two sets of

pure HHR and HXL columns, but on

the other it has the worst specific res-

olution. This behaviour could be

attributed to their highest KSEC values

and lowest pore volume, and as a

consequence, less volume to permeate

the solute would affect the resolution.

– We have found for the five solvent/

polymer systems a good agreement

among the trend of �/3, Kp and

DVR/Dc values, inferring that the

higher the gel crosslinking degree the

higher the secondary effects, and also

the higher the concentration effect due

to preferential solvation of the poly-

mer by the gel (adsorption) when

comparing the same solvent/polymer

system (i.e. constant macromolecular

crowding effect) in the different sets of

columns. The set B depicts, in general,

the lowest adsorption influence on the

total concentration effect.

– We have found that the mixed set B

has better chromatographic charac-

teristics than the non-mixed ones (set

A or D) or the mixed C with XL as

major component. This fact encour-

ages us to continue investigating more

different combination of gel packings.

– Finally, we also think that the good

chromatographic properties evidenced

by the mixed set B are due to the

individual response of each column

that forms the set. For this reason,

measurements of different chromato-

graphic parameters (Kp, RS, DVR/Dc)in isolated columns are currently in

progress.

Acknowledgements

Financial support from Direccion Gen-

eral de Investigacion (Ministerio de

Ciencia y Tecnologıa) under Grants No.

MAT2000-1781 and PB98-1435 is grate-

fully acknowledged.

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