Improved Separation of Poly(Butylene Glycol) Oligomers
on a Cl8 Column by Gradient Reversed-Phase Liquid
Chromatography and Fine-Tuning of the Organic Modifier
System 1
K. Rissler
C o n s u m e r Care Division, C A 6.1, Ciba Specialty Chemicals Inc., K-402.5.03, 4002 Basle, Switzerland
1This paper is dedicated to Professor Gottfried G16ckner on the occasion of his 75th birthday.
Key Words
Column liquid chromatography
Poly(butylene glycol) oligomers
Summary
Poly(butylene glycol) (PBG) samples of widely differ-
ing molecular weight OY/), either in the native form or as
the corresponding ~, co-bis(1-naphthyl)urethane deri-
vatives, have been subjected to reversed-phase high-
performance liquid chromatography (gRPHPLC) on an
octadecylsilyl (Cas) silica gel stationary phase with
mobile phase gradients comprising binary eluent sys-
tems prepared from tetrahydrofuran (THF) and water
and from acetone and water, and with ternary systems
prepared from acetonitrile, THF and water and from
methanol, THF and water.
Because of the lack of a chromophore in the PBG they
were detected both by evaporative light-scattering de-
tection (ELSD) and, after conversion to the c~, ~o-bis(1-
naphthyl)urethane derivatives with 1-naphthylisocya-
nate, by UV monitoring at 280 nm.
In general, sufficient resolution of the oligomers oflow-
M samples, i.e. PBG 650 and PBG 1000, was achieved
with all the gradient systems used, and resolution in-
creased in the order acetone-water < THF-water < me-
thanol-THF-water < acetonitrile-THF-water. Whereas
the difference between chromatographic resolution (Rs)
was relatively moderate for PBG 650 and PBG 1000 for
all the solvent-non-solvent combinations used, values
were much larger for the high-Msamples PBG 2000 and
PBG 3000 either in the native form or as their ~, co-bis(1-
naphthyl)urethane derivatives. No separation of the
high-M oligomers of PBG 3000 was obtained with
acetone-water and THF-water gradients and a common
signal envelope was observed for these sample con-
stituents. In contrast, acetonitrile-THF-water and me-
thanol-THF-water ternary gradients effected satisfac-
tory resolution of large amounts of oligomers and more
than 70 oligomer peaks were recognizable. Chromato-
656 Chromatographia Vol. 51, No. 11/12~June 2000
0009-5893/00/06 656-13 $ 03.00/0 9 2000 Friedr. Vieweg & Sohn VerlagsgesellschaftmbH
graphic resolution (Rs) of high-M oligomer was mark-
edly better with acetonitrile-THF-water than with me-
thanol-THF-water.
Use of the c~, co-bis(1-naphthyl)urethane derivative of
PBG 2000 as model component for fluorescence detec-
tion, with excitation and emission wavelengths of 232
and 358 nm, respectively, resulted in either improved
detection sensitivity or much cleaner chromatograms
and thus better selectivity compared with measurement
of UV response at 280 nm.
The effect o f the different mobile-phase systems on the
separations of these solutes is explained in terms of
distribution of the solute molecules between the bulk
mobile phase and the hydrophobic network of C18
chains, bound to the surface of the silica gel base mate-
rial. In this respect, the eluent strength of an organic
modifier increases with increasing solubility and/or
solvation characteristics, in particular for the high-M
fractions. It had, nevertheless, to be taken into con-
sideration that solvents that were too "good" afforded
efficient sample elution on the one hand but markedly
impaired oligomer resolution on the other, as shown by
separation o f the high-M sample constituents by binary
THF-water and acetone-water gradients.
Introduction
In addition to the widespread use of poly(propylene
glycols) in technical applications, the substantially
more hydrophobic polytetrahydrofurans or poly(butyl-
ene glycols) (PBG) are increasingly used as flexibilizers
and tougheners in formulated epoxy-based systems.
These compounds also play key roles as long-chain c~,
co-dlalcohol components in the synthesis of poly-
urethane fibers, and polyester and polyurethane plas-
tics, and as starting materials in the synthesis of cross-
linked polyurethane casting elastomers. As a first step
PBG are often reacted with di-isocyanates to form the
corresponding isocyanate prepolymers; these are then
converted to commercially available polyurethanes.
Original
Because PBG used for prepolymer formation with di-
isocyanates are mixtures ofoligomers which, depending
on the starting material used, can be of markedly differ-
ent molecular weight, expressed in terms of Mn, Mw,
polydispersity index (i.e. Mw/Mn), and molecular-
weight distribution, it is to be expected that the proper-
ties of the product are essentially governed by these
factors. As a logical consequence, rapid and reliable
methods are needed for exhaustive characterization of
structural and physicochemical peculiarities, which
might not only vary to different extents from one man-
ufacturer to another but also within different batches
from the same producer. Although size-exclusion chro-
matography (SEC) after appropriate calibration with
PBG standards is excellently suited to measurement of
Mn, Mw and Mw/Mn,oligomer resolution is poor and
only the lower molecular weight (M) homologues, in
general with molecular weights well below 500, are
sufficiently separated from each other. In contrast,
however, gradient reversed-phase high-performance li-
quid chromatography (gRPHPLC), because of its ex-
cellent separation efficiency, affords highly re-
producible and characteristic fingerprint patterns of
oligomeric components enabling discrimination of
samples obtained from different manufacturers and of
different batches obtained from the same producer.
Few RPHPLC procedures for poly(butylene glycols)
which satisfy these requirements are, unfortunately,
available in the literature. Chromatographic investigation
of PBG of nominal M 1000 on a variety of reversed-phase
materials (C18, C8, C4, Cphenyl, el) with binary acetoni-
trile-water gradients [1], and on C~8 with mixtures of
acetonitrile, methanol, ethanol or 2-propanol with water
[2] were reported some time ago. PBG in the M range
from 650 to 3000 have been separated on a C4 stationary
phase by use of water-organic mobile phases with acet-
onitrile and methanol as the organic modifiers [3]. Al-
though satisfactory results in terms of signal resolution
were obtained by use of a binary acetonitrile-water gra-
dient on the Ca support, recent investigations of polye-
sters on Cls matrices, resulting in excellent separation
when tetrahydrofuran (THF) was added as a "co-modi-
fier" for chromatographic "fine-tuning" [4, 5], have
prompted re-examination of the chromatographic beha-
viour of PBG of widely differing M on a C18 stationary
phase with different binary and ternary mobile phases.
Because the weight fraction (%) of the polar oxygen in
the [(CH2)4-O] repeat unit, i.e. {[O]/[(CH2)4-O]} x
100, is more than 20%, substantial polar properties
should be assumed. PBG do, however, behave much
more hydrophobically than expected and few oligomers
were eluted from a C18 chromatographic support even
with 100% acetonitrile as mobile phase [1-3]. As a
consequence, high percentages of hydroxyl-containing
organic modifiers, for example ethanol or 2-propanol
[2] or, alternatively others with good solvent and/or
solvation properties for complete elution from strongly
hydrophobic stationary phases, for example THF and
acetone, are necessary. For this reason, the PBG chosen,
Original Chromatographia Vol. 51, No. 11/ 12, June 2000
with M ranging from 650 to 3000, and therefore re-
garded as low-M samples, are ideal targets to study both
of the elution characteristics of relatively hydrophobic
compounds with mobile phases in which the solubilities
of the compounds are likely to be markedly different and
to study the solvation characteristics and the depen-
dence of chromatographic resolution, Rs, on M.
To gain more insight into these phenomena, an opti-
mized chromatographic design consisting of binary
gradient systems prepared from THF and water and
from acetone and water, and ternary gradients prepared
from acetonitrile, THF and water and from methanol,
THF and water was chosen. With acetone as the organic
modifier signal monitoring could be achieved by use of
evaporative light-scattering detection (ELSD) only.
With all other mobile phases ELSD was used for the
native compounds and, after conversion of the analytes
to the corresponding bis(1-naphthyl)urethane deriva-
tive, UV detection at 280 nm. For comparative purposes,
PBG 2000, used as the model component, was also
measured by fluorescence detection (FD).
Experimental
Materials
The poly(butylene glycol) samples PBG 650, PBG
1000, PBG 2000 and PBG 3000 ("technical quality")
were obtained from BASF (Ludwigshafen, Germany).
RPHPLC was performed on a 125 mm x 4.6 mm i.d., 5/z
particle size, 100-A pore diameter Nucleosil 5Cls col-
umn from Macherey-Nagel (Oensingen, Switzerland).
Acetonitrile, methanol, THF and acetone (all HPLC
grade) were purchased from Fluka (Buchs, Switzer-
land). THF was used as obtained from the producer
without further purification and/or distillation. Water
for use in HPLC was purified with a Milli-Q reagent
water system from Millipore-Waters (Milford, MA,
USA). The derivatization reagent 1-naphthylisocyanate
was obtained from Carbolabs (Bethany, CT, USA).
Derivatization Reaction
The ~, co-bis(1-naphthyl)urethane derivatives of the four
PBG used in the study were prepared according to a
procedure described elsewhere [6], with slight mod-
ifications. Briefly, 1-naphthylisocyanate (500/~L) was
added to each PBG sample (50 mg) in a 10-mL screw-
capped vial and the reaction mixture was kept at 60 ~
for 2 h. Methanolwas then added to a final volume of
10 mL and the mixture was heated for another 15 min at
60 ~ for complete conversion of excess reagent to 1-
naphthylmethylurethane. For UV detection this solution
(10#L) was injected directly into the HPLC system
without sample pretreatment, whereas for fluorescence
detection the solution was diluted approximately 1 : 25-
1 : 50 with methanol and this solution (10 gL) was in-
jected.
657
Table 1. Gradient system 1. Table VI. Gradient system 6.

Time (min) THF (%) Water (%) Time (min) Methanol(%) Water (%) THF (%)

0 30 70 0 55 45 0
40 100 0 40 92.5 0 7.5
45 100 0 50 88 0 12
45.1 30 70 60 80 0 20
50 30 70 75 80 0 20
75.1 55 45 0
80 55 45 0

Table II. Gradient system 2.

Analytical Equipment
Time (min) Acetone (%) Water (%)
H P L C was p e r f o r m e d with a P 4000 quaternary H P L C
0 30 70 p u m p , a v a c u u m degassing unit, an AS 3000 auto-
40 100 0 sampler equipped with a 100-#L sample loop enabling
60 100 0 injection o f different sample volumes and used in the
60.1 30 70 " p u s h - l o o p " , mode, a Spectra Focus scanning detector,
65 30 70
a FL 2000 fluorescence detector and a PC 1000 data
acquisition unit, all obtained f r o m T h e r m o Separation
Products (Fremont, CA, USA). E L S D was p e r f o r m e d
with a Sedere (Vitry sur Seine, France) Sedex 45 appa-
Table IlL Gradient system 3. ratus equipped with a 2 0 - W iodine lamp.

Time (min) Acetonitrile(%) Water (%) THF (%) Chromatographic Separation

0 30 70 0 The different gradient systems presented in Tables I - V I

40 80 0 20 were used for separations with four combinations o f
60 70 0 30 solvents: (i) T H F - w a t e r (gradient system 1, Table I), (ii)
70 70 0 30 a c e t o n e - w a t e r (gradient system 2, Table II), (iii) acet-
70.1 30 70 0
80 30 70 0 o n i t r i l e - T H F - w a t e r (gradient systems 3 and 4, Tables
III and IV), and (iv) m e t h a n o l - T H F - w a t e r (gradient
systems 5 and 6, Tables V a n d VI). C h r o m a t o g r a p h y was
p e r f o r m e d at ambient temperature (approx. 22 ~ at a
flow-rate o f 1.5 m L m i n -1. For E L S D methanolic solu-
Table IV. Gradient system 4. tions (2 %, w/v; 1 0 # L ) o f the P B G were injected on to
the H P L C column. U V detection and FD were used with
Time (min) Acetonitrile(%) Water (%) THF (%) the solutions (10 #L) obtained f r o m derivatization (see
above). For E L S D the nebulization c h a m b e r was heated
0 40 60 0 to 40 ~ and the nitrogen flow was adjusted to 4.5 L m i n -1
30 80 0 20 corresponding to an inlet pressure o f 200 kPa. The U V
60 60 0 40
70 60 0 40 responses were measured at 280 n m and for FD the wa-
70.1 40 60 0 velengths were set at 232 n m for excitation and 358 n m
80 40 60 0 for emission.

Results
Table V. Gradient system 5. Blank gradient runs, p e r f o r m e d with methanol as sam-
ple solvent instead o f sample, revealed that baseline
Time (rain) Methanol(%) Water (%) THF (%) drift was only small w h e n E L S D and FD were used.
Although even at 280 n m the influence o f continuously
0 55 45 0 admixed T H F on baseline drift, in particular for the
40 92.5 0 7.5
50 92.5 0 7.5 h i g h - M samples P B G 2000 and P B G 3000, could not be
60 85 0 15 neglected, no substantial i m p a i r m e n t o f chromato-
70 85 0 15 graphic p e r f o r m a n c e was observed.
70.1 55 45 0
75 55 45 0 W h e n the four test analytes P B G 650, P B G 1000, P B G
2000 and P B G 3000 were subjected to the individually

658 Chromatographia Vol. 51, No. 11/12, June 2000 Original

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Figure 1
Chromatograms obtained from PBG 650 (a), PBG 1000 (b), PBG 2000 (e), and PBG 3000 (d) on a 125 m m x 4.6 m m i.d. Nucleosil 5Ca8 column
with a binary THF-water gradient and evaporative light-scattering detection (ELSD).

optimized elution conditions, substantial and some- When acetone rather than THF was used as organic
times very large differences in oligomer resolution were modifier in the binary aqueous-organic mobile phase
observed; this was more pronounced the higher the mo- (gradient system 2, Table II), resolution of the higher-M
lecular weight of the samples. This is impressively oligomers of PBG 650 and PBG 1000 was substantially
shown in Figures 1-7 both for the four native analytes lower (Figures 3a, b) whereas the chromatographic fin-
and for their corresponding e, co-bis(1-naphthyl)ur- gerprints of PBG 2000 and PBG 3000 (Figures 3c, d)
ethane derivatives. were similar to those obtained with THF (Figures 1c, d),
A THF-water gradient effected quantitative elution of although Rs for intermediate M oligomers was sig-
all sample constituents from the column; this is readily nificantly lower than when THF was used. Almost
apparent from Figures 1 and 2, which depict the HPLC- baseline separation of the components of PBG 650 and
ELSD and HPLC-UV traces, respectively. In both de- PBG 1000, both for native samples and for their e, co-
tection modes optimum chromatographic resolution bis(1-naphthyl)urethane derivatives, was accomplished
(Rs) is obtained for the low-M sample PBG 650 (Figures by use of a ternary acetonitrile-THF-water gradient
la, 2a)-separation is almost to baseline. Oligomer re- (Figures 4a, b, 5a, b) when a final content of 30 % THF is
solution is generally acceptable for PBG 1000 (Figures used as "co-modifier" (gradient system 3, Table III). In
lb, 2b), although sample constituents of higher M are addition, as verified by mass spectroscopic investiga-
less well resolved, especially for native PBG 1000 (Fig- tions, PBG 1000 was separated into the maximum
ure lb). For native PBG 2000 and PBG 3000, however, number of individual oligomers [7]. Although, as ex-
Rs is satisfactory only for the small amounts of low-M pected, chromatographic resolution of the higher-M
oligomers present in the samples; those of higher M are samples of native (underivatized) PBG 2000 and PBG
lumped into an unresolved broad signal envelope (Fig- 3000 (Figures 4c, d) was substantially lower than for
ures lc, d). Furthermore, as is particularly apparent in PBG 650 and PBG 1000 and the high-M sample con-
Figure 2d, and to a substantially lesser extent in Figure stituents increasingly merge into a poorly resolved
2c, substantial amounts of intermediate-M oligomers of broad peak, more than 70 individual oligomers are still
PBG 3000 and PBG 2000, respectively, were much bet- recognizable. To effect complete oligomer elution it was
ter resolved after derivatization with 1-naphthylisocya- necessary to increase the final amount of THF to 40 %
nate than as the native counterparts (Figures lc, d). (gradient system 4, Table IV).

Original Chromatographia Vol. 51, No. 11/12, June 2000 659

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Figure 2
ChromatogramsobtainedfromPBG 650 (a), PBG 1000 (b), PBG 2000 (e), andPBG 3000 (d), afterderivatizationwith 1-naphthylisocyanate,on
a 125 mm x 4.6 mm i.d. Nucleosil 5C18columnwith a binaryTHF-watergradientand UV-detectionat 280 nm.

Replacement of acetonitrile by methanol in the ternary chromatograms when UV detection was used. These
gradient resulted in reduced oligomer resolution for all peaks can be attributed to compounds formed by side-
four test analytes both in the HPLC-ELSD traces ob- reactions during methanolysis of excess reagent to 1-
tained from the native samples (Figures 6a-d) and in the naphthylmethylurethane and cyclo-dimerization of 1-
HPLC-UV traces obtained from their ~,o~-bis(1-naph- naphthyl isocyanate to e,~o-bis(1 -naphthyl)- 1,3-diaza-
thyl)urethane derivatives (Figures 7a-d); this was more 2,4-butanone [7] (Figure 8). Although the signal attri-
pronounced as the M of the sample increases. The final butable to the dimer, as revealed by LC-MS investiga-
THF concentrations effecting complete sample elution tions of the ~,co-bis(1-naphthyl)urethane derivative of
for the pair PBG 650 and PBG 1000 and for the pair PBG PBG 1000 as the target component [7], interrupts the
2000 and PBG 3000 were 15 % and 20 %, respectively- uniform sequence of oligomer signals in all HPLC-UV
substantially lower than when acetonitrile was used as chromatograms (Figures 2, 5 and 7), and when the tern-
the "primary" organic modifier. ary methanol-THF-water gradient is used the dimer
It is readily apparent from the chromatograms obtained obviously co-elutes with a PBG oligomer (see the in-
tense signal representing the "base peak" in Figures 7a-
from PBG 2000 and PBG 3000 that secondary distribu-
d), there is no further impairment of the overall chro-
tion maxima result from the use of ternary gradients
prepared either from acetonitrile, THF and water (Fig- matographic fingerprint pattern.
ures 4c, d) or from methanol, THF and water (Figures Figures 9a, b depict chromatograms obtained from the
6c, d) whereas analogous behaviour is not apparent from PBG 2000 ~,co-bis(1-naphthyl)urethane derivative by
the corresponding HPLC-UV traces (Figures 5c, d and use of fluorescence deteetion (HPLC-FD) and ternary
7c, d, respectively). An attempt is made to explain this gradients prepared either from acetonitrile, THF and
effect in the Discussion section, below. water (gradient system 4, Table IV, Figure 9a) or me-
Derivatization of the PBG with 1-naphthylisocyanate, a thanol, THF and water (gradient system 6, Table VI,
procedure which has previously been successfully ap- Figure 9b). In contrast with the corresponding HPLC-
plied to polyethylene glycols [6], gave rise to some UV traces (Figures 5c, 7c), the HPLC-FD chromato-
impurities in the "low-M region" of the corresponding grams are much "cleaner" which underlines the im-

660 Chromatographia Vol. 51, No. 11/12, June 2000 Original

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Figure 3
Chromatograms obtained from PBG 650 (a), PBG 1000 (b), PBG 2000 (c), and PBG 3000 (d) on a 125 mm x 4.6 mm i.d. Nucleosil 5C18column
with a binary acetone-water gradient and evaporative light-scattering detection (ELSD).

proved selectivity of FD over U V detection. In addition, tection sensitivity increases substantially at wave-
compared with U V signal monitoring approximately fif- lengths below 250 nm, the use o f 280 nm has some
tyfold better detection sensitivity was obtained by mea- advantages, in particular relatively moderate baseline
surement o f FD responses. The HPLC-FD trace o f PBG drift on gradual admixture o f THF during gradient se-
2000 separated by use o f methanol as organic modifier parations; this substantially reduces overall chromato-
did not further reveal the intense signal seen in Figures graphic performance when signal responses are mea-
7a-d. The latter is presumably attributable to co-elution sured below 250 nm. It is apparent from Figures 2, 5 and
o f the 1-naphthylisocyanate dimer with a PBG oligomer. 7 that no substantial impairment o f chromatographic
fingerprinting o f the c~,co-bis(1-naphthyl)urethane deri-
vatives was observed at 280 nm when mobile phases
containing substantial amounts o f THF were used.
Discussion
THF is rarely used as the only modifier in aqueous-
As reported elsewhere [1-3], use o f evaporative light- organic mobile phases in the RPHPLC o f low-M com-
scattering detection (ELSD) has proved a reliable and pounds, although it continues to be used as the "pri-
powerful tool for the detection o f non-chromophoric mary" organic modifier in some applications, for ex-
samples such as the poly(butylene glycols). This is pri- ample analysis o f bisphenol-A-diglycidylether [8, 9]
marily because o f its insensitivity to changes in mobile- and o f polyester oligomers [10-16]. This limited use is
phase composition in gradient chromatography. ELSD preponderantly attributable to the presence in the sol-
is also the method o f choice when solvents which absorb vent o f autoxidation-related impurities, the most abun-
strongly in the usual U V wavelength range, e.g. ketones dant o f which, ~-butyrolactone, is much more strongly
or aromatic compounds, are used as mobile phase com- U V absorbing than pure THF and thus contributes sub-
ponents. Because o f the relatively low sensitivity, how- stantially to baseline drift when THF gradients are used.
ever, 1-2 orders o f magnitude lower than that o f U V For this reason, to minimize baseline deterioration, the
detection [ 1], the native samples were also converted to use o f freshly distilled THF is recommended; this is a
their cqco-bis(1-naphthyl)urethane derivatives and sig- tedious and time-consuming procedure for long-term
nal responses were measured at 280 nm. Although de- applications. The use o f THF is generally restricted to

Original Chromatographia Vol. 51, No. 11/12, June 2000 661

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Figure 4
ChromatogramsobtainedfromPBG 650 (a), PBG 1000 (b), PBG 2000 (e), and PBG 3000 (d) on a 125 mm x 4.6 mm i.d. Nucleosil 5C18column
with a ternaryacetonitrile-THF-watergradient and evaporativelight-scatteringdetection(ELSD).

size-exclusion chromatography (SEC), because it is PBG 1000, PBG 2000 and PBG 3000, the separation
well-suited to the complete suppression of solute-sta- efficiency on its considerably more hydrophobic C18
tionary phase interactions, both because of its dissolu- counterpart merits re-evaluation by"fine-tuning" of the
tion power for many polar or non-polar solutes within a binary acetonitrile-water mobile phase used hitherto.
wide molecular-weight range and because of its ex- This investigation was performed primarily because the
cellent eluent strength, even when used with the strongly interactive surface of C18 phases far exceeds that of
non-polar polymeric column supports preferred in clas- short-chain alkyl- or aromatic-substituted silica gel
sic non-aqueous SEC. materials and, therefore, better separation of oligomers
Nevertheless, because of the properties of THF as an should be possible by choice of an appropriate "co-
excellent solvent, its eluent strength and separating modifier" with solvation properties different from those
power were studied for the relatively non-polar PBG of of acetonitrile and/or methanol. The starting conditions
widely differing in M on hydrophobic column supports, of the gradients were adapted to the eluent strengths of
both as component of a binary gradient with water and the different "primary" organic modifiers to enable
as a "co-modifier" in ternary acetonitrile (or metha- elution of the first oligomers with retention times com-
nol)-water gradients. parable with those obtained by use of acetonitrile and
It has been shown elsewhere [ 1, 2] that elution of all the methanol. In addition, although the new gradients were
constituents even of PBG 1000 from strongly hydro- designed on the basis of experience obtained from for-
phobic octadecylsilyl silica gel supports cannot be mer investigations [1-3], gradient times were usually
achieved with acetonitrile or methanol as organic substantially reduced compared with those, to enable
modifier, whereas complete "release" and satisfactory sufficient column equilibration, in particular when THF
signal resolution ofPBG 1000 oligomers are much more was used as the "co-modifier", as an indispensable
likely from stationary phases of markedly lower hydro- prerequisite for either efficient elution or separation of
phobicity, for example C4 and Cphenyl- Although the high-M sample constituents.
chromatographic performance of the C4 stationary Although a binary THF-water gradient almost achieved
phase, in particular, seems satisfactory for PBG 650, baseline separation of native PBG 650 oligomers

662 Chromatographia Vol. 51, No. 11/12, June 2000 Original

 0.4
o9 T

0,35 t

0.3

1 I IL~
a
~ ' il :>
....I iM,
i ~i :'J'
i~ !:~ 9 9~ ~ 9 ~:~~~ ~ ~ ~'~:~:~:~~~ )~;', .,;:?,;::~'~,'~:~v~,
~.'~.~,~......

~
t I,
, j! ,, J!i
0 5 10 15 2o 3{] ~0 so 40 ~o
Retention t i m e (rain) Retention time (rain) C

o.oe
!
i
o.s
I I I

~ i l ~

| o.2
:
Ii IL
I
| oos
ec
o.15 :> : : , III
i: i ~; ~ il i~ 0o2 .... ~r,%:.,, ,;, ,:> . .......
I ~, ~; ~!~;~iiill'l;',~i,'t',l!~,li~::~,,~''~''~ 9
i

Hs

!i "~'~ 5 ,j~ j~ j: ~ ~ ~,,~:~'~ ~'~/~" "~

,o ,; .0 ~ 00 o 1o 2o ao 4o 5o
Retention time (rain) ~) Rstentlon time (rain)
d
Figure 5
Chromatograms obtainedfromPBG 650 (a), PBG 1000(b), PBG 2000 (e), and PBG 3000 (d), after derivatization with 1-naphthylisocyanate,on
a 125 mm x 4.6 rnm i.d. Nucleosil 5C18column with a ternary acetonitrile-THF-watergradient and UV-detectionat 280 nm.

(Figure 1a) and even PBG 1000 was resolved into many phase on its surface. The distribution equilibrium is,
homologues (Figure lb), the peaks of higher-M oligo- therefore, highly dependent on the different system
mers of PBG 2000 (Figure lc) and PBG 3000 (Figure properties, for example the hydrophobicity and/or the
1d) totally merge into a common and broad signal en- polarity of: (i) the chromatographic support, (ii) the
velope. Essentially similar behaviour was observed for mobile phase, and (iii) the analyte, and also on the
the corresponding ~,(o-bis(1-naphthyl)urethane deriva- solvation properties of the organic modifier. In this re-
tives (Figure 2). spect, although a hydrophobic column matrix has been
used, it is easily conceivable that the low-M PBG
When the amount (%) o f THF is calculated from the
homologues, owing to their moderate interactive sur-
composition of the gradient at the retention time (te,) of
face, are eluted at relatively low THF concentrations but
the most strongly retained but still sufficiently resolved
still with satisfactory resolution. Because of this still
oligomers of PBG 650 in Figure la, a value of approxi-
"sub-critical" amount of "good" solvent, by means of
mately 75 % is obtained. It is obvious that at concentra-
which, however, the sample is kept completely in solu-
tions of THF just above this level no further separation
tion, and the concomitant sufficiently high concentra-
into individual oligomers occurs, irrespective of the
tion of water as the "non-solvent" which prevents pre-
PBG sample chromatographed (Figures 1c, d). In con-
mature elution, they can interact optimally with the non-
trast, even for 80:20 ( % v/v) acetonitrile-THF (i.e. a
polar stationary phase. In contrast, above a distinct "le-
total of 100 % organic modifier) separation into in-
vel" of organic modifier concentration, in this instance
dividual oligomers is still achieved (Figures 4c, d).
approximately 75 % THF (see above), the equilibrium is
A hypothesis intended to explain this behaviour is pre- substantially shifted towards the modifier. As a con-
sented below. Firstly, it is reasonable to assume that the sequence, oligomers are swept rapidly from the hydro-
separation mechanism in RPHPLC is governed by dis- phobic network of the column support and eluted with
tribution of the analyte molecules between the bulk the bulk mobile phase.
mobile phase and the hydrophobic network of octade- Compared with the low-M PBG oligomers, the high-M
cylsilyl chains grafted on to the solid column support members are much less soluble at eluent compositions
and forming a thin layer of a quasi-liquid stationary of approximately 75 : 25 (% v/v) THF-water (see above)

Original Chromatographia Vol. 51, No. 11/12, June 2000 663

 09

!
O.5 J

o9

== o., ': =
i i; ' t~ o9
w
;
(
i iil!'
I il i!

o.2 ,= , ,, : , = ~ ,
i
iI
09
~ ii i ~, i ~= ~!)l,,j=/~f)~,~=~ ~

ooos Y- 2 . . . . . . . . " " -- . . . . .

0 ~ 10 15 20 25 30 35 40 45 50 0 10 20 30 4o 50 60 70
Retention tirae (rnin) Retention time (rain}

I
o,I
f
: i

! i
i , !
I
( ~ r! r

~I ii !!i 1
i i !' ~ w
=i :
d [I ?,
~ o*os

!1 ~i ~ ":",':.~7~J~
]. : ' , q / ~ 9i ~iJ
09 j ii ; i i; ! !!
I I

r f
oos ~ , , i ; , , , , , , i 4

oi .... ,i=l _ .....

o.oos . . . . .
o lO eo 3o 4o o lo 2o ao 40 so 6o 70
Retention time (mini
b Retention time (min)
d
Figure 6
ChromatogramsobtainedfromPBG 650 (a), PBG 1000 (b), PBG 2000 (e), andPBG 3000 (d) on a 125 mm • 4.6 mm i.d. Nucleosil 5C18column
with a ternarymethanol-THF-watergradientand evaporativelight-scattering detection(ELSD).

and are still retained at this solvent-non-solvent com- tive and derivatized sample constituents might be re-
position. For this reason, more THF is required to sweep sponsible for the observed behaviour, even if the con-
them into the bulk mobile phase. In this respect, the tribution of the end-groups to these effects is expected to
solvation properties of THF as the "good" solvent are decrease with increasing PBG chain length.
expected to play an essential role, which, in turn, will be
associated with conformational changes and possible Because separation of polyethylene glycols on either
self-association of the oligomers, i.e. effects which have octadecylsilyl silica gel or bare silica gel with binary
a major impact on the areas of contact with the sta- water-acetone gradients proved superior to that ob-
tionary phase for high-M sample constituents. For this tained with acetonitrile or methanol as the organic
reason they will not have sufficient time to adopt a modifier [17] and, furthermore, acetone has excellent
suitable conformation for optimum interaction with the solvent properties both for a wide variety of low-M
network of octadecylsilyl chains bound to the silica gel solutes of widely differing polarity and for polymers, it
base material and will, therefore, be eluted in a bulk of was also tested as an alternative to THE As demon-
unresolved sample constituents despite their obviously strated by Figure 3, however, the resolution was no bet-
higher interactive surface towards the stationary phase. ter that that obtained with THF, and even for PBG 650
(Figure 3a) chromatographic resolution of the high-M
There is, nevertheless, a marked discrepancy between homologues was markedly less than that obtained by use
resolution of the otigomers of native PBG 3000 (Figure of THF as organic modifier (Figure la). Nevertheless,
1d) on the one hand and of their 0~,co-bis(1-naphthyl)ur- the markedly similar chromatographic fingerprints ob-
ethane derivatives (Figure 2d). This is also true, but to a tained for the four PBG investigated is indicative that a
lesser extent, for the oligomers of native PBG 2000
retention mechanism comparable with that previously
(Figure 1c) and their derivatives (Figure 2c). It should,
discussed might also be true for acetone.
however, be remembered that substitution of both ~,co-
hydroxyl groups by 1-naphthylcarbamoyl functions In contrast with the use of either THF or acetone as
confers substantial hydrophobicity on the PBG mole- organic modifier in binary aqueous-organic gradient
cules and therefore, conformational effects invoked by systems to effect complete elution ofhigh-M oligomers,
the differential solvation properties of THF towards na- few oligomers were eluted from Cls even with 100 %

664 Chromatographia Vol. 51, No. 11/12, June 2000 Original

 0~ i [ i

0.~ 4

= w

o i
|
~o o9 =r o.1
=>
=>
0~2. ! i [ ,
t ,

o.1
=' !!!:!:,, o9 I,=.
I, "1:i

o .... : o , :~' :. . . . . . . . ~= =" . . . . " ' " 2 " "

o 15 20 25 30 35 40 0 10 20 3o 40 so
Retention t i m e (rain) Retention time (min)

0.4S o.o5

il o04s

i: oo4

I I ,I ,I
o03s
o.3! i ~ ,i!ii !
i ,
= : j =r 0.03

~ o.02s .... :: :/lll!!!~ I

> .... i
o,15 ! i I :~ : ~ ~ ~i~ii ~
) o~ols
i I
I :~ ~ !:! ,!!
o.ol

o.oo~

o ~- 9 ~ 9 o ....
~5 ~0 2S Z0 3S 4O ~S o 1~ 2D ao 50
Retention time (mln) Retention time (rain)

Figure 7
ChromatogramsobtainedfromPBG 650 (a), PBG 1000 (b), PBG 2000 (c), andPBG 3000 (d), afterderivatizationwith 1-naphthylisocyanate,on
a 125 mm x 4.6 mm i.d. Nucleosil 5C18column with a ternarymethanol-THF-watergradientand UV-detectionat 280 ran.

acetonitrile, irrespective of the PBG sample applied; the From the observation that few PBG oligomers are eluted
eluent strength of methanol as organic modifier was from a strong hydrophobic C18 stationary phase by a
much stronger [ 1-3 ]. This behaviour has been explained binary acetonitrile-water gradient, and because higher-
[ 1] in terms of strong non-polar interactions of hydro- M homologues are lumped into an unresolved signal
phobic PBG samples with the network of C18 chains, envelope when THF is used as organic modifier, it was
which could not be efficiently counterbalanced by the assumed that a combination of acetonitrile as the "pri-
aprotic modifier acetonitrile. In contrast with the eluo- mary" organic modifier and THF as the "co-modifier"
tropic ranking order in RPHPLC, the still more polar would have a pronounced influence on the resolution of
solvent methanol effected elution of more than twice as the oligomers of high-M sample constituents. For this
many PBG oligomers as acetonitrile, a behaviour which reason, marked improvement of oligomer resolution in
was ascribed to its capacity to form hydrogen-bonds the "elution region" of high-M homologues should be
with the ether oxygens of the analyte and a concomitant expected, although it had always to be taken into account
solubility shift away from the non-polar chromato- that Rs is limited and strongly depends on the ratio Mo/
graphic support towards the bulk mobile phase [1]. A Mr(n), where M0 is the mass o f the repeat unit (72.1 for
corresponding conclusion was also drawn by Jandera et PBG) and Mt is the total mass of the PBG oligomer
al. for dodecyl alcohol ethoxylates [ 18]. These authors composed ofn repeat units. Taking this into account it is
explained different the chromatographic behaviour easily recognizable that the higher the molar mass the
when acetonitrile or methanol were used as organic lower the corresponding difference between M for con-
modifier in binary gradients with water in terms of the secutive oligomers, which is directly related to the cor-
capability of the protic solvent and th e non-capability of respondingly small differences between the interactive
the aprotic solvent to form hydrogen-bonds with ether surfaces ofoligomers ni and ni + 1.
oxygen atoms.

Original Chromatographia Vol. 51, No. 11/12, June 2000 665

As was hoped, a substantial improvement of oligomer
resolution was obtained by use of an optimized ternary
mobile phase comprising acetonitrile, THF and water, as
is impressively demonstrated in Figures 4 and 5, which
show the chromatograms obtained from PBG 650, PBG
1000, PBG 2000 and PBG 3000 native samples and
from their 7,co-bis(1-naphthyl)urethane derivatives, re-
spectively; the oligomers of PBG 650 and PBG 1000 are
resolved almost to baseline. Although, as expected, the
high-M oligomers of the native samples of PBG 2000
and, particularly, PBG 3000, are much less well resolved
and increasingly merge into a broad and poorly resolved
peak, more than 70 individual oligomers are still re-
cognizable in both chromatograms. For the oligomer
with n = 70, for which Mt = 5056 and M0 = 72.1 for the
repeat unit, the relative weight differences relative to
oligomers with n = 69 or n = 71 are only approximately
1.4%. Thus even very small differences in M between
consecutive homologues are associated with sufficient
differences between their interactive surfaces, and oli-
gomer resolution is still achieved. In this context it is
worthy of note that even 80:20 (v/v) acetonitrile-THF
effected signal resolution into a larger number of in-
dividual oligomers, whereas for THF-water ca 75:25 (v/
v) no further separation into PBG homologues occurs
(see above). Thus the large ameliorating effect of THF
as "co-modifier" on oligomer resolution is im-
pressively demonstrated.
As previously reported, methanol proved a substantially
better eluent than acetonitrile for higher-M PBG; this
beneficial effect can be ascribed to its capacity for hy-
drogen-bond formation with ether oxygens [ 1]. For this
reason, it is not at all surprising that the amount of THF
needed for oligomer elution was much less than was
used in a ternary gradient ofacetonitrile, THF and water.
In this respect, the final concentration of THF in the
mobile phase with methanol as the "primary" modifier
could be substantially reduced from 30 and 40 % when
used with acetonitrile to only 15 and 20 % for the pair
PBG 650 and PBG 1000 and the pair PBG 2000 and
PBG 3000, respectively. In contrast, however, signal
resolution, for the high-M sample constituents, in parti-
cular, is markedly reduced compared with that afforded
by acetonitrile, as is readily apparent from comparison
of Figures 4 and 6 on the one hand (HPLC-ELSD traces)
and Figures 5 and 7 (HPLC-UV traces) on the other. In
addition, it is conspicuous that compared with acetoni-
trile as the organic modifier much more methanol is
required in the starting mobile phase (55 % compared
with 30 and 40 % acetonitrile, respectively) to obtain
comparable retention of " l o w " - M oligomers. This ob-
servation is, therefore, in accordance with sample elu-
tion in the usual ranking order of solvents in RPHPLC,
i.e. solute retention decreases with decreasing overall
mobile-phase polarity. In contrast with this "normal"
behaviour, however, a hydrogen-bonding-mediated
"solubility shift" seems to dominate for the high-M
PBG fractions [ 1]. The influence of this is, nevertheless,
markedly less than that of THF, although there is a
666 Chromatographia Vol. 51, No. 11/12, June 2000
synergistic effect which, as an unwanted consequence,
yielded substantially lower Rs for the high-M oligomers.
The most striking discrepancy between the chromato-
graphic patterns of native PBG obtained by ELSD (Fig-
ures 1, 4 and 6) and by UV detection of their c~,og-bis(1-
naphthyl)urethane derivatives (Figures 2, 5 and 7) oc-
curs in the "elution regions" of high-M oligomers. Re-
sponses of high-M constituents of the native samples in
the HPLC-ELSD traces proved much more intense than
in the corresponding HPLC-UV traces obtained from
the c~,co-bis(1-naphthylurethane) derivatives. It should
be emphasized, however, that such large differences be-
tween signal responses for oligomers with comparable
degrees of polymerization cannot be attributed the dif-
ferent modes of detection alone, i.e. concentration de-
tection by UV and "bulk property" detection by ELSD
measurements (and there is also evidence that ELSD
deviates, perhaps substantially, from the "bulk prop-
erty" detection principle [ 19, 20]). In contrast, it is to be
expected that the differences will be relatively small
only and, for this reason, other possible causes of this
unexpected behaviour must be discussed.
Incomplete elution of high-M oligomers of to the e,co-
bis(1-naphthylurethane) derivatives can be effectively
ruled out as a possible reason because, as is apparent
from previous investigations, PBG 1000 oligomers de-
rivatized with 3,5-dinitrobenzoyl chloride are much
better eluted from stationary phases widely differing in
hydrophobicity (e.g., C18, C8, C4, Cphenyl,C1 matrices)
than are the native analogues [ 1].
To provide a reasonable explanation it should be re-
membered that the intensity of ELSD responses is re-
ported to be highly dependent on sample size and on the
individual physicochemical properties of either solute
or mobile phase, for example the melting point and
refractive index of the analyte and the surface tension
and viscosity of the mobile phase. The mobile phase in
particular plays an important role and its boiling point,
refractive index and solution behaviour markedly influ-
ence the light-scattering properties of the resulting
aerosol [21-27]. In this respect the secondary distribu-
tion maxima appearing in Figures 4c, d and 6c, d can
possibly be simply explained as artefacts of the ternary
gradient profiles used. It should be noted that these
maxima appear in the second gradient region where the
concentration of the "primary" modifier, acetonitrile,
decreases as the concentration of the "co-modifier"
THF, increases, whereas in contrast, the concentration
of both "primary" and "co-modifier" increase simul-
taneously in the first region of the gradient. This ob-
viously leads to different effects on oligomeric selec-
tivity in thetwo gradient regions resulting in a "second
distribution pattern".
Fluorescence detection of the ~,co-bis(1-naphthyl)ur-
ethane derivatives of PBG 2000 eluted with acetonitrile-
THF-water (Figure 9a) and methanol-THF-water (Fig-
ure 9b) gradients not only yielded markedly "cleaner"
chromatograms compared with the corresponding
Original
 revealed in yet unpublished investigations [7], the main
advantage of UV-detection compared with signal mon-
itoring by measurement of FD responses is its much
better suitability for quantitative purposes; FD is,
therefore, primarily used when the sensitivity of UV-
detection is insufficient.

Conclusions
Efficient separation of the oligomers of the two ,low,-M
Figure 8
samples PBG 650 and PBG 1000 was achieved on a C18
The chemicalstructureof the 1-naphthylisocyanatedimer. stationary phase with THF-water or acetone-water bin-
ary gradients or acetonitrile-water-THF or methanol-
water-THF ternary gradients. The native samples were
O.lS -
monitored by ELSD and their ~,o~-bis(1-naphthyl)ur-
ethane derivatives by UV-detection.
I
0.14 !
Whereas separation of the "high"-Moligomers of PBG
I I '
2000 and PBG 3000 by both binary gradients was either

mo.,t
0,12 -
very poor or absent, highly improved separations were
achieved by use of ternary acetonitrile-THF-water gra-
.... j dients and, to a lesser extent, ternary methanol-THF-
eo~ i!l:
.i i I
water gradients. Although oligomers present in the
0.o4 , " h i g h " - M fractions of PBG 2000 and PBG 3000 were
i, still not completely resolved, at least when ELSD was
ooe [
ii i; ~ used, resolution o f more than 70 oligomers was accom-
o•
o lO 20 30 50 plished with both ternary gradients, that prepared from
Retention time (mln)
acetonitrile, water and THF proving the more efficient.
The separation achieved with the latter mobile phase
proved substantially better than that obtained by use of
I '
0.1 +
acetonitrile-water binary gradients on less hydrophobic
, !]I C8, C4, Cpheny~and C1 matrices [1] and by use of binary
i~ I i gradients prepared from acetonitrile, methanol, ethanol,
or 2-propanol and water on a C18 stationary phase [2]
and acetonitrile or methanol and water on C4 supports
[3]; complete elution of all sample constituents was
effected.
oo4- i
] Fluorescence detection has some advantages over UV
detection not only in terms of detection sensitivity but
also in selectivity, whereas UV detection is better suited
i P;', for quantitative purposes. Although separation of the
I',
high-M fractions of PBG 2000 and PBG 3000 was not
1o 20 ao 40
Retention time ( r a i n ) sufficient, at least when ELSD was used for signal
Figure 9 monitoring, the novel optimized procedure could be
Chromatogramsobtained from PBG 2000 after derivatizationwith used successfully for chromatographic fingerprinting of
1-naphthylisocyanate,on a 125 mm • 4.6 mm i.d. Nucleosil 5C18 PBG of widely differing molecular weight.
columnwith ternary acetonitrile-water-THF(a) and methanol-wa-
ter-THF (b) gradients and fluorescencedetectionat 2~x= 232 nm
and )~em= 358 nrn.
References
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(1993).
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approximately 50-fold increase in detection sensitivity 654, 309 (1993).
[3] K. Rissler, U Fuchslueger, J. Liq. Chromatogr., 17, 2791
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822, 89 (1998).
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668 Chromatographia Vol. 51, No. 11/12, June 2000 Original