Characterization of poly(styrene-co-methylacrylate)s
using gradient polymer elution chromatography-infrared detection
Kok1, 2, P.J.C.H. Cools1, T.
Hankemeier1 and P.J. Schoenmakers2
Voeding, Analytical Sciences Division, Utrechtseweg 48, 3704 HE Zeist,
of Amsterdam, Faculty of Chemistry, Nieuwe Achtergracht 166, 1018 WV
Amsterdam, The Netherlands
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|Copolymers can be characterized
according to chemical composition by gradient polymer elution
chromatography (GPEC). Reversed phase separation of random copolymers
by GPEC is mainly based on the ratio of the monomers (mol fraction)
. Since chromatography is a relative
method, selective detection and identification is necessary .
By using spectroscopic techniques e.g., infrared, the GPEC separation
can be calibrated without the use of appropriate standards.
|The aim was to study the applicability
of FT infrared for calibration of the RP-GPEC separation, e.g.
the determination of the (average) chemical composition of chromatographic
fractions. This was evaluated by means with poly(styrene-co-methylacrylate)
samples with known chemical composition.
If Beer’s law is obeyed, the absorbance ratio
should be linear to the ratios of the mol fractions if selective
spectral bands are selected for each compound to be measured
absorbance styrene / absorbance methylacrylate = k ·
(mol fraction styrene / mol fraction methylacrylate)
The molar extinction coefficients should be independent of
the copolymer composition.
Liquid Chromatography - IR coupling
|The HPLC effluent is directed to
the spraying interface (LC Transform), see Figure 1a.
After evaporation of the effluent and sample deposition on a germanium disc,
the disc is transferred to the IR optics module and spectra of the deposited
chromatogram are collected by stepwise moving the Ge-disc (Figure
Figure 1a: Diagram of LC Transform 500
nebulizer. (With permission of
LabConnections, Inc., Marlborough, MA, USA)
Figure 1b: Diagram of infrared optics
scanning module. (With permission of
LabConnections, Inc., Marlborough, MA, USA)
Optimization of LC Transform deposition
|Optimization of LC effluent deposition
was performed by optimizing the nozzle temperature at different
eluent compositions. During the gradient run, the nozzle temperature
was programmed to maintain optimum evaporating conditions during
the RP-GPEC separation.
Chemical composition determination by RP-GPEC
A plot of mol fraction styrene vs. retention time (Figure
3) results in a linear calibration curve (r = 0.9867) for the known
Figure 2: RP-GPEC ELSD
and UV (215 nm) chromatograms: poly(styrene-co-methylacrylate)s (m=35
µg). Peak assignment: a, mol fraction styrene = 0.095, b=0.2, c=0.3, d=0.4,
e=0.6, f=0.7, g=0.8, h=0.9, ? = impurity. Conditions: column, Novapak
C18 (Waters), 150 * 3.9 mm I.D.; gradient, 50:50 % (v/v) H2O/AcN
to 100 % (v/v) AcN to 100 % (v/v) THF (2 % (v/v)/min); flow, 0.5 mL/min.
Figure 3: Calibration plot of mol fraction
styrene in poly(styrene-co-methylacrylate)s vs. ELSD retention time.
Chemical composition determination by IR
LC-IR coupling is possible, but appeared to be critical
with respect to deposition, e.g., increasing deposit width on the Ge disc
during separation, or nebulizer temperature.
Possible explanations of a non-linear relationship (Figure
7) between IR-peak ratio and mol fraction for the known poly(styrene-co-methylacrylate)
samples are: (i) response and molar extinction coefficient both depend
on molecular structure, i.e., chemical composition; (ii) the absence of
selective IR absorbance peaks in the styrene spectrum, i.e., a region
where methylacrylate is not absorbing IR radiation (Correction for these
contributions was not sufficient, especially in the region where the styrene
fraction is small or high); (iii) unequal contribution of transmittance
and/or reflectance response due to inhomogeneous deposit.
Figure 4: Functional group RP-GPEC-IR chromatograms:
black trace, IR of wavenumber region 1744 - 1724 cm-1, characteristic
for C-O (MA); blue trace, IR of wavenumber region 688 - 708 cm-1,
characteristic for phenyl (S). Peak assignment, see Figure
Figure 5: RP-GPEC-IR spectra of copolymer with
varying styrene mol fraction (black trace, f=0.095; red trace, f=0.6;
blue trace, f=0.9).
Figure 6: IR contour plot of RP-GPEC separation
of copolymers with mol fraction styrene f = 0.095 - 0.9. Third dimension:
transmittance (%). For details, see Figure 2.
Figure 7: Calibration plot
of peak height ratio vs. mol fraction f(S)/f(MA).
RP-GPEC-IR has been realized. In principle,
an almost linear relationship was found between retention time
and mol fraction styrene, however, influence of molar mass is
not known, but a deviation was found for small and high mole
fractions of styrene. This was mainly assigned to interference
of the absorbance bands of styrene by the absorbance bands characteristic
for methylacrylate. Therefore, in principle IR ratio-ing has
to be improved to realize the goal of independent determination
of the chemical composition by IR.
The next step is implementation of multi-variate
data analysis. Multi-variate data analysis will be used for
background correction in GPEC-IR and for calibration of peak
height vs. molar ratio. Therefore, it will be investigated in
the near future. The use of a larger range of the IR spectra
from methylacrylate rather than a single IR absorbance peak
will improve determination of the chemical composition distribution.
This is even more true for polymer systems consisting of more
than two monomers.
- Teramachi, S., Macromol. Symp., 110 (1996) 217-229
- Cools, P.J.C.H., Thesis, Technical University of Eindhoven,
The Netherlands, 1999