Chapter 1
Quantitation in Analysis of Polymers by Multiple Detector SEC Bernd Trathnigg Institute of Organic Chemistry, Karl-Franzens-University at Graz, A-8010 Graz, Heinrichstrasse 28, Austria
It is shown, that SEC with dual detection is a powerful tool in the analysis of polymers and also of oligomers. For the latter, one has to take into account the molar mass depend ence of response factors, if just one detecto is used. Alter natively, dual detection can be applied also in this case with advantage. Besides the molar mass dependence of response factors, another source of error is the SEC calibration, which may be considerably different not only for different homopolymers, but also for polymer homologous series with the same repeating unit, but different end groups.
Introduction: In the analysis of polymers by SEC, three transformations are required from chromatographic raw data to molar mass distributions, which may be subject to different sources of error. • Step 1 (elution time to elution volume) is performed rather easily by using an internal standard for compensation of flow rate variations (assuming the flow rate remaining constant during the run). • Step 2 (elution volume to molar mass) requires either a calibration function or a molar mass sensitive detector (such as a viscometer or light scattering detector) in addition to the concentration detector(s). • Step 3 (detector response to weight fraction) is especially important in the case of oligomers and copolymers, where severe errors may result from the assumption of constant response factors over an entire peak.
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© 1999 American Chemical Society
3 Consequently, quantitation faces different problems, depending on the nature of the samples and the detectors, which can be applied. In the analysis of oligomers, corrections of response factors for their molar mass dependence have to be made. In the characterization of copolymers, multiple detection (using combinations of different concentration detectors) is generally inevitable, as it yields additional information on chemical composition and thus allows an accurate quantitation. In SEC of copolymers or polymer blends, the chemical composition may vary considerably within a peak. In this case, the use of coupled concentration detectors is inevitable. The concentration detectors most frequently used in SEC of polymers are the U V and the RI detector. Recently, two other detectors have been introduced, which are useful in the analysis of non-UV absorbing polymers: the density detector (according to the mechanical oscillator principle) and the evaporative light scattering detector (ELSD). The ELSD detects any non-volatile material, but its response depends on various parameters , , and the nature of these dependencies is rather complex. The U V detector detects UV-absorbing groups in the polymer, which may be the repeating unit, the end groups, or both. Hence one has to distinguish between polymers, in which the repeating units contain a chromophoric group, and polymers with chromophoric end groups. In the first case, the response of the U V detector represents the mass, in the second case the number of molecules in a given volume interval. Many chromatographers use this assumption, when they derivatize "nonabsorbing" polymers with UV-active reagents. Complications may, however, arise in some mobile phases, as will be discussed later on. 1 2
Copolymers: If the response factors of the detectors for the components of the polymer is sufficiently different, the chemical composition of each slice of the polymer peak can be determined from the detector signals. Basically, only very few concentration detectors may be applied: U V absorbance (UV), refractive index (RI), and density detectors. Infrared (IR) detection suffers from problems with the absorption of the mobile phase, and the evaporative light scattering detector (ELSD) is not suitable for this purpose because of its unclear response to copolymers . For UV-absorbing polymers, a combination of U V absorbance and RI detection is typically used. If the components of the copolymer have different UV-spectra, a diode-array detector can also be applied. In dual detector SEC there may be different situations in the selection of detectors: • One component can be detected in U V , the other one does not absorb UV-light. Typical examples are poly(methyl methacrylate-g-ethylene oxide) and poly(methyl methacrylate-g-dimethyl siloxane). 3
4 • Both components can be detected in the UV: in the case of poly(methyl methacrylate - b -styrene) the U V spectra of the components are significantly different, and the U V detector can be regarded as selective detector, in the case of poly(methyl methacrylate-b-decyl methacrylate) they are identical, and the UV-absorbance detector has to be regarded as a universal one. • None of the components can be detected in the U V : this is the case with poly(ethylene oxide-b-propylene oxide) and fatty alcohol ethoxylates (FAE). In the analysis of such samples a combination of density and RI detection can be applied . 4
5
6,7,8
The principle of dual detection is rather simple: when a mass mi of a copolymer, which contains the weight fractions w and w (= 1 - w ) of the monomers A and B , is eluted in the slice i (with the volume AV) of the peak, the areas Xij of the slice obtained from both detectors depend on the mass mi (or the concentration ci =mi/AV) of polymer, its composition (w ), and the corresponding response factors fj and f j , wherein j denotes the individual detectors. A
A
B
>A
Xi j = m j ( w f j A
+ w fj )
A
B
A
B
equation 1
B
The weight fractions wA and wB of the monomers can be calculated using equation 2: 1 W
(— x
x
A
*f ,A-fl,A) 2
i2 1
-= 1 r
equation 2
U *
f
(
\
f
I2,B ~ M,B ) x
i,2
and therefrom the mass of polymer in the corresponding interval x
i l 1
mj = f
w *(fi,A- i,B) A
equation 3 +
f
l,B
Once the amount of polymer in an interval and its chemical composition are known, one may calculate the corresponding molar mass (transformation 2). In the case of copolymers, the molar mass Mc of the copolymer can obtained by interpolation between the calibration lines of the homopolymers (which may be considerably different) 9
lnMç, = lnMg+ vt>^* ( l n A / ^ - l n M^)
equation 4
5 wherein M and M are the molar masses of the homopolymers, which would elute in this slice of the peak (at the corresponding elution volume V ) There is , however, still a chemical polydispersity in each slice, which means, that Mc is just the average molar mass, since w is also an average composition. Obviously, the precision as well as the accuracy of the results obtained by this technique will depend on the individual response factors. Thus, it is important to find an appropriate combination of detectors and mobile phase in order to obtain reliable results. This can, however, sometimes re quire a lot of experiments, which means also a lot of trial and error. In a recent study , we have shown, that a simple simulation procedure can considerably reduce the time required for the optimization of such a method. Basically, one has to determine the response factors of both detec tors for the homopolymers, and calculate the peak areas, which would re sult from different amounts of sample (say 1 - 10 μg) with compositions between 0 and 100 %. Then the smaller area is increased by 1 digit (thus simulating a baseline uncertainty), and the composition is again calculated for the new peak areas. The error in composition for sample sizes of 1- 5 μg is a good criterion for evaluating the suitability of a mobile phase with a given detector combination. In Figure 1, such a plot is shown for the system density + RI detector with chloroform as mobile phase applied to the analysis copolymers of ethylene oxide (EO) and propylene oxide (PO). Even though this is not the most favourite case, good results can be ob tained, as can be seen from Figures 2 - 4 , which show a chromatogram of an EO-PO block copolymer and the M M D and chemical composition cal culated therefrom. This sample obviously contains a fraction of PPG, as could be proven by two-dimensional L C . While the M M D in Figure 3 was obtained with the calibration function for PEG, the molar mass for each interval was calculated using equation 4 to yield the M M D shown in Figure 4. As can be seen, the molar mass aver ages thus obtained are considerably different ! It must be mentioned, that also the molar mass dependence of response factors has been accounted for in these results, as will be discussed in the following section. A
B
1 0
e
A
11
12
Oligomers: In the analysis of oligomers, additional problems arise from the fact, that the response of most detectors depends more or less strongly on the molar mass of the samples. The U V detector detects UV-absorbing groups in the polymer, which may be the repeating unit, the end groups, or both. Hence one has to distiguish between polymers, in which the repeating units contain a chromophoric group, and polymers with chromophoric end groups.
6
Figure 1 : Simulation of dual detector SEC of EO-PO copolymers in chloroform with coupled density and RI detection
RI ERC
density elution volume
Figure 2: Chromatogram of an EO-PO block copolymer, as obtained by SEC in chloroform with coupled density and RI detection ( Plgel 5μπι 100 Â, 600x7.8 mm)
log M * PEG
mass distribution
α
PPG
Figure 3: M M D and chemical composition of an EO-PO block co polymer (Figure 2), as determined by SEC in chloroform with density and RI detection. Calibration: PEG, Molar mass averages: M = 801 M = 680 M /M„ = 1.214 w
n
w
100
M Β •
50
%
0
9
ι
-* mass distribution
.
.7 log M * PEG
4
3
ι
ι
V • PPG
Figure 4: M M D and chemical composition of an EO-PO block copoly mer (Figure 2), as determined by SEC in chloroform with density and RI detection. Calibration: PEG+ PPG Molar mass averages: M = 845 M = 696 M /M„ =1.214 w
n
w
8 In the first case, the response of the U V detector represents the mass, in the second case the number of molecules in a given volume interval. Many chromatographers use this assumption, when they derivatize "nonabsorbing" polymers with UV-active reagents. Complications may, how ever, arise in some mobile phases, as will be discussed later on. RI and density detector measure a property of the entire eluate, which is related to a specific propertiy of the sample (the refractive index incre ment or the apparent specific volume, respectively). It is a well known fact , that specific properties are related to molar mass 3
χ. = x 1
0 0
+ M. ι
Equation 5
wherein Xi is the property of a polymer with the molar mass M x . is the property of a polymer with infinite (or at least very high) molar mass, and Κ is a constant reflecting the influence of the end groups ' . A similar relation describes the molar mass dependence of response factors for RI and density detection. i s
14 15,16
f =f + ' M. ι
Equation 6
0 0
In the case of the evaporative light scattering detector (ELSD) no such simple relation exists, and the (more volatile) lower oligomers can be lost at higher evaporator temperatures . In a plot of the response factors ξ of polymer homologous series (with de fined end groups) as a function of l/M» straight lines will be obtained. Their intercept f*, can be considered as the response factor of a polymer with infinite molar mass, or the response factor of the repeating unit, the slope Κ represents the influence of the end groups . The magnitude of K, determines the molar mass range, above which response factors can be considered as constant, which is often the case only at molar masses of several thousands. Neglecting this dependence can lead to severe errors, as has been shown in the analysis of ethoxylated fatty alcohols (FAE) by SEC in chloroform. Once and Κ have been determined, the correct re sponse factors for each fraction with the molar mass M i (which is obtained from the SEC calibration). In a previous paper , three methods were described for the determination of foo andK: 1. If a sufficient number of monodisperse oligomers is available, as is the case with PEG and PPG, one may determine the individual response factors and calculate f*, and Κ by linear regression, as is shown in Fig. 5. As has been shown previously, this approach is also possible with fatty alcohol ethoxylates (FAE). 17
18
19
20
9
~a-^
•ftXPEG) +fD(PPG) AfR(PEG) xfR(PPG)
•
* • - · .1
+
—
0,000
0,001
0,002
0,003
0,004
0.005
0,006
Figure 5: Response factors of polyethyleneglycol (PEG) and polypropyleneglycol (PPG) in chloroform for density and RI detection
10 2. If only one monodisperse oligomer is available (such as the fatty alcohol for some FAE) and can be determined otherwise (either as above or as a good approximation - from a sufficiently high molecular sample), a two-point calibration may work quite well. 3. If no monodisperse oligomer is available, but f» is known, an iteration procedure may be used, which is shown schematically in Fig. 6. The latter procedures can also be applied to macromonomers, for which no monodisperse oligomers are available. Obviously, the same approach should be applicable to U V detection of polymers with UV-absorbing end groups. Figures 7 - 9 show the response factors of density, RI and U V detection (at 260 nm) as a function of 1/M. For all polymer homologous series with the same repeating unit the same intercept must be expected, while the different end groups should result in a different slope. Linear regression was used for PEG, PEG-mono- and dimethacrylates, and methyl-PEG-monomethacrylates; the iteration procedure (approach 3) was applied in all cases, and the slopes thus obtained were in good agreement with those from linear regression. A strange phenomenon is observed with the U V detector, while the oligomers with methacrylic end groups follow equation 6 (as expected), PEGs of a molar mass larger than a few hundreds produce large peaks in U V at a wavelength, where no adsorption could be expected (260 nm)! This is especially bad news for anybody, who tries to determine the functionality of such products after derivatization. The same behaviour can also be observed with fatty alcohol ethoxylates (FAE), as can be seen in Fig. 10. The reason for these large signals is not yet clear. Because of these complications, U V detection was not applied in chloroform any more. While the response factors of the density detector are always negative in chloroform (regardless the nature of the end groups), those of the RI detector change their sign a lower molar mass, as can also be seen from Fig. 11, which shows a chromatogram of a PEG-200-monomethacrylate, obtained by SEC in CHCL3 with density and RI detection. Obviously, the lower oligomers have a negative sign in RI detection, while no problems occur in density detection. From the density data, the M M D was calculated using equation 6. Obviously, such a correction cannot be applied for the refractive index trace. Alternatively, functional oligomers may be regarded as block copolymers, consisting of one block without end groups, and another one: the end groups; in this case this means PEG and the methyl methacrylate (MMA), respectively. Using equations 2 and 3, the M M D shown in Figure 12 was calculated, which agreed perfectly with that from the density data and equation 6.
11
average response factor: f(av) = peak area /sample size
Integration without compensation: K=0
Calculation of Κ from f(av) and M(max) Repeat integration : calculate mass of peak with f = f(R) + K/M
Increase or decrease Κ
Figure 6: Iteration procedure for determination of Κ for polydisperse oligomers
PEG: y *-7β5,69χ- 15,912 R2 = 0,9775
0.000
0,001
0.002
0.003
0.004
0.005 1/M
0,006
0.007
0,008
0,009
* PEG • PEG-MA λ MePEG-MA χ PEG-DMA PEG-MA MePEG-MA PEG-DMA Et-PEG-MA
0.010
Figure 7: Response factors of PEG derivatives in chloroform for den sity detection
12
9.4
elution volume (ml)
18.8
Figure 10: Chromatogramm of a fatty alcohol ethoxylate based on 1dodecanol (Brij 30), as obtained with density and U V detection (260 nm)
elution volume
Figure 11: Chromatogram of a macromonomer, methoxy-PEG-200methacrylate, as obtained on a set of 5 columns Phenogel 5μπι (300x7.8 mm each, 2 χ 500Â + 3 χ 100 Â) in chloroform with coupled density and RI detection
14
100
9
οΕ a a g. '
50
•à •
++ l 0
ι
2 A /
ι ι ι ι ι ni '
mass distribution
ι
\
ι ι
log M + PEG
3 I
4
ι
I I Ill II
I
I
π MMA
Figure 12: M M D and chemical composition of the macromonomer in Figure 11, as determined from dual detection
15 Figure 13 shows the effect of different corrections on the molar mass aver ages for this sample. Obviously, the agreement between the values from the density data using eqn. 6 and those from dual detection is very good. Additionally, it becomes clear, that there is a big difference in the values calculated with the PEG calibration and those obtained with the correct calibration for this homologous series. As a test for the performance of the dual detection method, we have ana lyzed methoxyethyl methacrylate, which represents the oligomer 1 (containing 1 M M A + 1 EO). The result is shown in Figure 14: A nice horizontal line if found for the composition, which indicates, that as well the delay volume between the detectors is correct, and that peak dispersion between the detectors is negligible. The EO content was found to be 28.6%, which agrees quite well with the theoretical one (30.6). In Figures 15 and 16, the same comparison is given for samples with dif ferent end groups and different molar mass. As before, the correction of density data using equation 6 and the dual detector method agree very well, while the correction of RI data is not as efficient, if it is possible at all. Experimental The polyethylene glycols used as calibration standards were purchased from Polymer Laboratories (Church Stretton, Shropshire, UK), the macromonomers MePEG-200-MA and MePEG-400-MA from Polysciences (Warrington, PA), all other samples from F L U K A (Buchs, Switzerland), A l l measurements were performed on a modular SEC system comprising of a Gynkotek 300C pump equipped with a VICI injector (sample loop 100 μΐ), two column selection valves Rheodyne 7060, a density detection system DDS 70 (Chromtech, Graz, Austria) coupled with an E R C 7512 RI detector or a JASCO 875 U V absorbance detector. Data acquisition and processing was performed using the software CHROMA, which is part of the DDS 70. The following columns were used: Phenogel M (5 μπι), 600x7.6 mm, PL Microgel M (10 μπι), 600x7.6 mm, a set of four columns Phenogel (5μπι), 2x500 and 2x100 Â, 300x7.6 mm each, and a set of two columns PL Microgel (ΙΟμπι), 1 0 + 1 0 Â , 300x7.6 mm each. A l l measurements were performed at a flow-rate of 1.00 ml/min and a column temperature of 30.0°C. Sample concentrations were 3.0 -10.0 g/1. The chloroform used in this study was HPLC grade and stabilized with 2methyl-butene (Mallinckrodt). 3
4
Acknowledgement Financial support by the Austrian Academy of Sciences (Grant OWP-53) is gratefully acknowledged.
Μη
Ο
50
100
150
200
250
300
350
400
450
500
molar mass averages (Mw, Μη)
Figure 13: Molar mass averages of methoxy-PEG-200-methacrylate, as obtained on Plgel 5μιη 100 Λ, 600 χ 7.8 mm in chloroform with coupled density and RJ detection with and without correction
100
•
τ1
50
.
J
ι
ι
ι
ι
ι
ι
ι
ι
ι
ι
7
\A I \A\
mass distribution
, \\
3 ι1
ι1
ι1
ι1 ι1 ι1 ι1ι I 1
1
ι
1 ι
11_
1
1
1 1 1 1
k>gM + PEG
° MMA
Figure 14: M M D and chemical composition o f the methoxyethyl methacrylate, as determined from dual detection.
17
• Nto(RI,unc.) • IVto(D,unc.) • Mw(RI, Κ) • Mv\
