Size Exclusion Chromatography Calibration Research Articles

Conversion of calibration curves for accurate estimation of molecular weight averages and distributions of polyether polyols by conventional size exclusion chromatography

Accurate measurement of molecular weight averages (M¯n,M¯w,M¯z) and molecular weight distributions (MWD) of polyether polyols by conventional SEC (size exclusion chromatography) is not as straightforward as it would appear. Conventional calibration with polystyrene (PS) standards can only provide PS apparent molecular weights which do not provide accurate estimates of polyol molecular weights. Using polyethylene oxide/polyethylene glycol (PEO/PEG) for molecular weight calibration could improve the accuracy, but the retention behavior of PEO/PEG is not stable in THF-based (tetrahydrofuran) SEC systems. In this work, two approaches for calibration curve conversion with narrow PS and polyol molecular weight standards were developed. Equations to convert PS-apparent molecular weight to polyol-apparent molecular weight were developed using both a rigorous mathematical analysis and graphical plot regression method. The conversion equations obtained by the two approaches were in good agreement. Factors influencing the conversion equation were investigated. It was concluded that the separation conditions such as column batch and operating temperature did not have significant impact on the conversion coefficients and a universal conversion equation could be obtained. With this conversion equation, more accurate estimates of molecular weight averages and MWDs for polyether polyols can be achieved from conventional PS-THF SEC calibration. Moreover, no additional experimentation is required to convert historical PS equivalent data to reasonably accurate molecular weight results.

Improving the accuracy of hyaluronic acid molecular weight estimation by conventional size exclusion chromatography.

There is an unreasonably high variation in the literature reports on molecular weight of hyaluronic acid (HA) estimated using conventional size exclusion chromatography (SEC). This variation is most likely due to errors in estimation. Working with commercially available HA molecular weight standards, this work examines the extent of error in molecular weight estimation due to two factors: use of non-HA based calibration and concentration of sample injected into the SEC column. We develop a multivariate regression correlation to correct for concentration effect. Our analysis showed that, SEC calibration based on non-HA standards like polyethylene oxide and pullulan led to approximately 2 and 10 times overestimation, respectively, when compared to HA-based calibration. Further, we found that injected sample concentration has an effect on molecular weight estimation. Even at 1g/l injected sample concentration, HA molecular weight standards of 0.7 and 1.64MDa showed appreciable underestimation of 11-24%. The multivariate correlation developed was found to reduce error in estimations at 1g/l to <4%. The correlation was also successfully applied to accurately estimate the molecular weight of HA produced by a recombinant Lactococcus lactis fermentation.

Coupling of size-exclusion chromatography with electrospray ionization charge-detection mass spectrometry for the characterization of synthetic polymers of ultra-high molar mass.

Coupling size-exclusion chromatography (SEC) with mass spectrometry (MS) allowed generation of a SEC calibration curve based on the analyte itself, which is more reliable than calibration based on non-related standards and faster than the use of the multiple detection mode. However, such SEC/MS couplings were limited to rather small synthetic polymers. Based on the concept of image current detection, charge-detection mass spectrometry (CDMS) coupled to electrospray ionization (ESI) is a useful method for weighing macro-ions from compounds with masses higher than one megadalton (MDa). Using columns designed to allow analysis of synthetic polymers of over 15 million Dalton in mass, performance of the SEC/ESI-CDMS coupling was evaluated for polyacrylamide (PAM, 5-6 MDa) and a poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS, 2 MDa). The SEC/ESI-CDMS profiles were first compared with SEC-UV profiles: the systematic shift in retention time was assigned to the slightly different geometries of the two instrumental systems. The SEC/ESI-CDMS data were then compared with results obtained after the direct infusion of each sample into the ESI source. Both the shape of the molecular weight distribution and the mass values were similar with or without separation prior to ESI, and these values were consistent with data provided by the sample supplier. SEC/MS incorporating an online ESI-CDMS coupling was shown to be a rapid and efficient technique for the analysis of water-soluble synthetic polymers with ultra-high molecular mass in the megadalton range. The coupling also afforded an attractive solution for SEC calibration without the use of external standards.

Aqueous size exclusion chromatography in semimicro and micro-columns by newly synthesized monodisperse macroporous hydrophilic beads as a stationary phase

A new class of monodisperse macroporous beads in the hydrophilic form were synthesized by seeded microsuspension copolymerization of two acrylic crosslinking agents, glycerol dimethacrylate (GDMA) and glycerol-1,3-diglycerolate diacrylate (GDGDA). The monodisperse porous poly(glycerol dimethacrylate- co-glycerol-1,3-diglycerolate diacrylate), poly(GDMA- co-GDGDA) beads were highly hydrophilic in nature due to hydroxyl functionality resulting from both crosslinking agents. The beads with different particle sizes between 4.5 and 6.7 μm and with different porous properties were obtained by changing the seed latex to monomer ratio. The bead size decreased, the average pore size increased and the specific surface area decreased with increasing seed latex to monomer ratio. Poly(GDMA- co-GDGDA) beads were slurry packed in microbore and semimicro-HPLC columns and successfully used as a stationary phase in aqueous size exclusion chromatography (SEC) mode in a micro-liquid chromatography system. The aqueous SEC runs were performed by using dextran standards in the molecular weight range of 1000–670,000 Da. SEC calibration curves exhibiting linearity in a wider range of molecular weight were obtained with the semi-micro and micro-HPLC columns packed with the poly(GDMA- co-GDGDA) beads synthesized with the seed latex to monomer ratios of 0.038 and 0.058 g/mL. The dextran standards could be eluted in an analysis time shorter than 2 min using micro or semi-micro columns packed with poly(GDMA- co-GDGDA) beads as stationary medium. These packings are suitable for molecular weight determination between 5 × 10 3 and 5 × 10 5 Da in the aqueous medium by using mobile phase flow rates in the range of 25–250 μL/min. The average molecular weight determinations of different water soluble polymers, an ionic polymer, poly(2-dimethylaminoethyl methacrylate), a zwitterionic polymer, poly([2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide), and a non-ionic polymer, poly(vinyl alcohol) were performed on a semimicro-column packed with the poly(GDMA- co-GDGDA) beads. Satisfactory results were obtained in the molecular weight determination of hydrophilic polymers by aqueous SEC.

The Impact of Band Broadening on Molar‐Mass Determination of Narrow‐Distribution Polymer by Size‐Exclusion Chromatography

AbstractA systematic study of the effects of band broadening (BB) in size‐exclusion chromatography (SEC) on Poisson molar‐mass distributions (MMDs) is carried out. Such distributions apply for polymer standards used for SEC calibration, and they are a good model for the MMD from any quasi‐living polymerization, including reversible‐deactivation radical polymerization (RDRP). BB is simulated using the exponentially modified Gaussian model, which is shown to work excellently for actual polymer standards. A series of simulations with this model revealed how BB parameter values affect number‐average degree of polymerization and polydispersity index from SEC. It is shown that the skewness parameter is particularly important in these considerations. Many of the obtained trends echo ones from RDRP, suggesting that BB has been making a hitherto unsuspected – indeed uninvestigated – contribution to deviations from ideal behavior in such systems.magnified image

Molecular Weight Distribution Evaluation of Polysaccharides and Glycoconjugates Using Analytical Ultracentrifugation

We review here the advances that have been made in methodology using the analytical ultracentrifuge for characterising polydisperse polymer systems with a quasi-continuous distribution of molecular weight. These advances include improved ways of defining the weight and z-average molecular weights of a distribution from sedimentation equilibrium experiments and hence the ratio Mz/Mw, off-line calibration of size exclusion chromatography by sedimentation equilibrium and conversion of a sedimentation coefficient distribution into a molecular weight distribution. Although these methods are applicable to other polymeric systems, we focus on polysaccharides, mucins and other glycoconjugates, systems which particularly benefit from the availability of recently available long optical path length cells for the minimisation of complications through thermodynamic non-ideality.

Determination of Propagation Rate Coefficients of a Family of Acrylates with PLP‐MALDI‐ToF‐MS

AbstractThe combination of MALDI‐ToF‐MS with pulsed laser polymerization (PLP) has one big advantage over the combination of size exclusion chromatography (SEC) with PLP. MALDI‐ToF‐MS is an absolute measurement which does not need calibration. Especially in the field of acrylates, this is an important advantage over the conventional use of SEC, since low polydispersity standards are not readily available for acrylates. Moreover, acrylates suffer from branching. Literature shows that since branched polymers have a different hydrodynamic volume than linear polymers, this can affect the calibration of SEC. The determination of the Arrhenius parameters for a family of acrylates is performed with PLP‐MALDI‐ToF‐MS. The results clearly demonstrate that an increase of the ester side group indeed results in an increase of the propagation rate coefficient. Whether this is due to an entropic or enthalpic effect cannot be derived from the results.magnified image

Determination of Critical Conditions of Adsorption for Chromatography of Polymers

Liquid chromatography (LC) at critical conditions of adsorption was used to separate various poly(ethylene oxides), poly(propylene oxides) and their copolymers. For the first time, the determination of the critical conditions by means of Ultra Performance Liquid Chromatography (UPLC) coupled to Electrospray Ionization Time-of-flight Mass Spectrometry (ESI-TOF MS) is reported. In contrast to established, mostly laborious routines to find suitable chromatographic separation conditions, this coupling enables a very fast adjustment of parameters. Similar to LC Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (LC/MALDI MS) coupling, a two-dimensional analysis of homo- and copolymers regarding its functionality type and molecular weight distribution, as well as copolymer composition, can be performed simultaneously. Furthermore, there is no need for using polymer standards for the determination of critical conditions or Size Exclusion Chromatography calibration.

Probing Size Exclusion Mechanisms of Complex Hydrocarbon Mixtures: The Effect of Altering Eluent Compositions

This paper describes the application of size exclusion chromatography (SEC) to petroleum residue samples that are incompletely soluble in 1-methyl-2-pyrrolidinone (NMP). The limitations arising from the use of tetrahydrofuran, pure chloroform, and pure NMP have been reviewed. The SEC of polycyclic aromatic hydrocarbons (PAHs) in pure chloroform was observed to give longer elution times with increasing molecular mass, the opposite of the expected trend. Because the smallest molecules of coal tars are known to be PAHs, SEC can only be of use in investigating the larger molecules of unknown mass if the elution behavior of the smaller standard PAH relates to the calibration using polymers. The work relies on the observation that the NMP/chloroform mixtures dissolved both the heavier fractions of a coal tar pitch and the whole of a petroleum asphaltene, completely at the low concentrations needed for SEC. The first tests of the new SEC calibration have been carried out using a Mixed-E column and pure NMP, pure...

Size-exclusion chromatography using deuterated mobile phases

The effect of deuterated solvents in size-exclusion chromatography (SEC) was studied by comparing intrinsic viscosity measurements, SEC calibration curves, and column efficiency using water-soluble polymers. For aqueous SEC, the use of deuterium oxide slightly increases the SEC elution volume. To verify that adsorption onto the packing was absent, data from exclusion experiments were compared at 35 and 50 °C. Our results indicate that adsorption is not occurring for pullulan or polyethylene glycol (PEG)/poly(ethylene oxide) (PEO); for the latter, however, the elution volume increased using both D 2O and H 2O, indicative of slight hydrodynamic volume contraction of PEG/PEO at higher temperatures. A moderate increase in band broadening (moderate decrease in column efficiency) was observed using D 2O. Finally, the effects of chloroform versus deuterated chloroform were evaluated, but no hydrodynamic volume changes were observed.

SEC/MALDI-TOF Mass spectrometry study of hydroxy-, carboxy- and methyl ester-terminated aliphatic polyesters

AbstractPoly(ethylene adipate) (PEA) and poly(ethylene dodecanedioate) (PED) with dicarboxy-, dihydroxy-, dimethyl ester- and hydroxy-carboxy end-groups were synthesized and characterized by 13C NMR and MALDI-TOF MS. Size-exclusion chromatography (SEC) fractions of these polyesters were analyzed by MALDI-TOF MS and used as standards to obtain SEC calibration curves leading to the absolute molar masses of the unfractionated polyesters. PEA and PED calibration curves were different from each other, but the same calibration curve was obtained for each type of polyester, irrespective of the nature of end-groups, showing the predominant role of the nature of repeating units on the hydrodynamic volume of oligoesters. The study also shows that the conventional SEC calibration method using polystyrene standards leads to a significant overestimation of polyester average molar masses.

The high‐mass component (&gt;m/z 10 000) of coal tar pitch by matrix‐assisted laser desorption/ionisation mass spectrometry and size‐exclusion chromatography

The size-exclusion chromatography (SEC) of acetone-soluble, pyridine-soluble and pyridine-insoluble fractions of a coal tar pitch indicates a bimodal distribution in each fraction. The proportion of high-mass material excluded from the SEC column porosity increases with solvent polarity. The polymer calibration of SEC shows the mass range of the small molecules to be from approximately 100 u to approximately 6000 u, with the mass range of the large excluded molecules above 200 000 u and up to several million u. In contrast, matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) shows a similar low-mass range of ion abundances (< m/z 6000), but with a smaller range of high-mass ion abundances, from approximately m/z 10 000 to 100 000. The large molecules may have three-dimensional structures to allow molecules of relatively low mass to behave as if they are of large size in SEC. Laser desorption mass spectrometry of the acetone- and pyridine-soluble fractions produced molecular ions of polycyclic aromatics that can be related to the known compositions from gas chromatography (GC) mass spectrometry. The experimental conditions used to generate the bimodal distribution by MALDI-MS involve reducing the ion signal intensities to avoid overload of the detector and enable detection of the high-mass ions, by reducing the high-mass detector voltage (i.e. sensitivity) and increasing the laser power.

A Study of the Separation Principle in Size Exclusion Chromatography

Size exclusion chromatography (SEC) separates polymer molecules according to their size in dilute solution, but what size to use has been a matter of debate for 35 years. In 1967, Benoit and co-workers found an excellent correlation between elution volume and a dynamically based molecular size, the hydrodynamic volume VH, for a wide range of species and large-scale molecular architectures. However, both theory and simulations assume a thermodynamic separation principle. This assumption is based on experimental observations that elution volumes are independent of flow rates. Thus, one might surmise that the radius of gyration Rg is a more appropriate size measure for purposes of universal SEC calibration, although the calculations of Casassa and co-workers on star polymers suggest some other thermodynamically based size may be required. In an attempt to resolve this dilemma, we undertook an extensive study of SEC with three groups of nearly monodisperse polymerslinear polystyrenes, linear polyethylenes, an...

Matching average masses of pitch fractions of narrow polydispersity, derived from matrix‐assisted laser desorption ionisation time‐of‐flight mass spectrometry, with the polystyrene calibration of SEC

AbstractA coal tar pitch has been fractionated by preparative and analytical size exclusion chromatography (SEC) to provide narrow time‐elution fractions, expected to have relatively low molecular dispersities. The fractions were characterised by analytical SEC, by UV‐fluorescence spectroscopy, and by laser‐desorption MS and by MALDI‐MS using sinapinic acid as matrix. The MALDI spectra were evaluated by measuring the peak‐intensity mass, Mp, and by calculating number (Mn) and weight (Mw) average masses. The high mass limits of the spectra were determined by three methods based on (i) truncating the spectrum when the signal fell below 5 times the standard deviation of signal at the instrument limit, (ii) taking the upper mass as defined by a minimum slope of the spectrum from baseline, and (iii) taking all of the high mass signal as from sample. The laser desorption spectra were evaluated by Mp values only; the results below m/z 1,000 were in excellent agreement with the polymer calibration. The MALDI mass spectra gave a good fit between Mp values below m/z 3,000 and the calibration of SEC by polymers. Working with Mp gave better results than the Mn and Mw values from Method (i). Results from Methods (ii) and (iii) were significantly worse. Reasons for the lack of agreement between results above m/z 3,000 by MALDI and above m/z 1,000 by LD‐MS have been discussed.

Critically Evaluated Rate Coefficients for Free‐Radical Polymerization, 4

AbstractPropagation rate coefficients, kp, which have been previously reported by several groups for free‐radical bulk polymerizations of cyclohexyl methacrylate (CHMA), glycidyl methacrylate (GMA), benzyl methacrylate (BzMA), and isobornyl methacrylate (iBoMA) are critically evaluated. All data were determined by the combination of pulsed‐laser polymerization (PLP) and subsequent polymer analysis by size‐exclusion chromatography (SEC). This so‐called PLP‐SEC technique has been recommended as the method of choice for the determination of kp by the IUPAC Working Party on Modeling of Polymerisation Kinetics and Processes. The present data fulfill consistency criteria and the agreement among the data from different laboratories is remarkable. The values for CHMA, GMA, and BzMA are therefore recommended as constituting benchmark data sets for each monomer. The data for iBoMA are also considered reliable, but since SEC calibration was established only by a single group, the data are not considered as a benchmark data set. All kp data for each monomer are best fitted by the following Arrhenius relations: CHMA: $k_{\rm p} = 10^{6.80} \;{\rm L} \cdot {\rm mol}^{ - 1} \cdot {\rm s}^{ - 1} \exp \left( {{{ - 23.0\;{\rm kJ} \cdot {\rm mol}^{ - 1} } \over {{\rm R} \cdot T}}} \right)$, GMA: $k_{\rm p} = 10^{6.79} \;{\rm L} \cdot {\rm mol}^{ - 1} \cdot {\rm s}^{ - 1} \exp \left( {{{ - 22.9\;{\rm kJ} \cdot {\rm mol}^{ - 1} } \over {{\rm R} \cdot T}}} \right)$, BzMA: $k_{\rm p} = 10^{6.83} \;{\rm L} \cdot {\rm mol}^{ - 1} \cdot {\rm s}^{ - 1} \exp \left( {{{ - 22.9\;{\rm kJ} \cdot {\rm mol}^{ - 1} } \over {{\rm R} \cdot T}}} \right)$, iBoMA: $k_{\rm p} = 10^{6.79} \;{\rm L} \cdot {\rm mol}^{ - 1} \cdot {\rm s}^{ - 1} \exp \left( {{{ - 23.1\;{\rm kJ} \cdot {\rm mol}^{ - 1} } \over {{\rm R} \cdot T}}} \right)$. Rather remarkably, for the methacrylates under investigation, the kp values are all very similar. Thus, all data can be fitted well by a single Arrhenius relation resulting in a pre‐exponential factor of 4.24 × 106 L · mol−1 · s−1 and an activation energy of 21.9 kJ · mol−1. All activation parameters refer to bulk polymerizations at ambient pressure and temperatures below 100 °C. Joint confidence intervals are also provided, enabling values and uncertainties for kp to be estimated at any temperature.95% joint confidence intervals for Arrhenius parameters A and EA for cyclohexyl (CHMA), glycidyl (GMA), benzyl (BzMA), and isobornyl (iBoMA) methacrylate; for details see text.magnified image95% joint confidence intervals for Arrhenius parameters A and EA for cyclohexyl (CHMA), glycidyl (GMA), benzyl (BzMA), and isobornyl (iBoMA) methacrylate; for details see text.

Controlled Radical Polymerization ofn-Butylα-Fluoroacrylate. 1. Use of Atom Transfer Radical Polymerization as the Polymerization Method

Atom transfer radical polymerization (ATRP) of n-butyl α-fluoroacrylate with CuCl and CuBr as the catalyst and 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA) as the ligand has been studied. Ethyl 2-bromoisobutyrate (2-EBiB) and 3-(trimethylsilyl)propyl 2-bromopropionate (2-SBP) were used as initiators. The latter was used as a probe for 1H NMR analysis in order to determine the true molecular weight up to about 10 000. In these conditions at T = 90 °C, polymerization was controlled as indicated by a plot of Mn= f(αM), and the effect of the halogen of the catalyst perfectly agrees with the current knowledge of ATRP. In addition, analysis of the CFBr end groups by 19F NMR further confirmed that the polymerization follows the ATRP mechanism. By calibration of size exclusion chromatography with standards prepared with the 2-SBP initiator, it was shown that the experimental molecular weights were close to the theoretical values. The results were better than those where the molecular weights were determi...

Determining the molar mass of a plasma substitute succinylated gelatin by size exclusion chromatography–multi-angle laser light scattering, sedimentation equilibrium and conventional size exclusion chromatography

The clinical effectiveness of succinylated gelatin as a plasma substitute depends strongly on its molar mass, determined conventionally by size exclusion chromatography (SEC). This study evaluates different SEC calibration standards in comparison with two independent “absolute” methods for determining the weight average molar mass ( M w) of a succinylated gelatin sample. SEC calibrated using succinylated gelatin fractions correlated well with size exclusion chromatography–multi-angle laser light scattering (SEC–MALLS) and sedimentation equilibrium whereas SEC calibrated with unmodified gelatin, sodium polystyrene sulfonates or pullulans overestimated M w by over 20%. Universal calibration was equivocal. The problems associated with the preparation of succinylated gelatin fractions suggest that an absolute method such as SEC–MALLS may be a more suitable choice for determining the M w in succinylated gelatins.

Comparison of different methods for the determination of the molar mass and molar mass distribution of poly(ethylmethylsiloxane)

The application of a poly(dimethylsiloxane) (PDMS) calibration curve for size exclusion chromatography (SEC) to poly(ethylmethylsiloxane) (PEMS) leads to molar masses, which are 15–40% higher than expected compared to measurements of the absolute molar mass values. As for the analysis of physical properties of PEMS, the exact knowledge of the molar mass is fundamental, therefore PEMS calibration curves for both SEC and viscometry were established. Eleven narrow distributed linear PEMS samples in the range between 4 000 and 110 000 g/mol were synthesised by anionic ring-opening polymerisation of a cyclic monomer. The molar masses of the PEMS samples were determined by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), light scattering, membrane osmometry, vapour pressure osmometry, and by viscometry. The polydispersities calculated from MALDI-TOF-MS spectra were lower compared to the SEC results. With the help of universal calibration, a SEC calibration curve for PEMS was established. Small angle neutron scattering (SANS) experiments on a 50%/50% protonated/deuterated PEMS blend match exactly the molar mass obtained from the PEMS calibration curve for SEC.

Polystyrene‐graft‐poly(ethylene oxide) copolymers prepared by macromonomer technique in dispersion. I. Liquid chromatographic separation of product mixtures

Products of the radical dispersion copolymerization of methacryloyl-terminated poly(ethylene oxide) (PEO) macromonomer and styrene were separated and characterized by size exclusion chromatography (SEC), full adsorption-desorption (FAD)/SEC coupling and eluent gradient liquid adsorption chromatography (LAC). In dimethylformamide, which is a good solvent for PEO side chains but a poor solvent for polystyrene (PS), amphiphilic PS-graft-PEO copolymers formed aggregates, which were very stable at room temperature even upon substantial dilution. The aggregates disappeared at high temperature or in tetrahydrofuran (THF), which is a good solvent for both homopolymers and for PS-graft-PEO. FAD/SEC procedure allowed separation of homo-PS from graft-copolymer and determination of both its amount and molar mass. Effective molar mass of graft-copolymer was estimated directly from the SEC calibration curve determined with PS standards. Presence of larger amount of the homo-PS in the final graft-copolymer products was also confirmed with LAC measurements. The results indicate that there are at least two or maybe three polymerization loci; namely the continuous phase, the particle surface layer and the particle core. The graft copolymers are produced mainly in the continuous phase while PS or copolymer rich in styrene units is formed mostly in the core of monomer-swollen particles. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2284–2291, 2000

Polystyrene-graft-poly(ethylene oxide) copolymers prepared by macromonomer technique in dispersion. I. Liquid chromatographic separation of product mixtures

Products of the radical dispersion copolymerization of methacryloyl-terminated poly(ethylene oxide) (PEO) macromonomer and styrene were separated and characterized by size exclusion chromatography (SEC), full adsorption-desorption (FAD)/SEC coupling and eluent gradient liquid adsorption chromatography (LAC). In dimethylformamide, which is a good solvent for PEO side chains but a poor solvent for polystyrene (PS), amphiphilic PS-graft-PEO copolymers formed aggregates, which were very stable at room temperature even upon substantial dilution. The aggregates disappeared at high temperature or in tetrahydrofuran (THF), which is a good solvent for both homopolymers and for PS-graft-PEO. FAD/SEC procedure allowed separation of homo-PS from graft-copolymer and determination of both its amount and molar mass. Effective molar mass of graft-copolymer was estimated directly from the SEC calibration curve determined with PS standards. Presence of larger amount of the homo-PS in the final graft-copolymer products was also confirmed with LAC measurements. The results indicate that there are at least two or maybe three polymerization loci; namely the continuous phase, the particle surface layer and the particle core. The graft copolymers are produced mainly in the continuous phase while PS or copolymer rich in styrene units is formed mostly in the core of monomer-swollen particles.