multiple detection in size-exclusion chromatography of macromolecules

Multiple Detection in Size-Exclusion Chromatography of Macromolecules

André M. StriegelFlorida State University

Chemical and physical detectors characterize the

distributions of macro-molecular parameters

that have a critical effecton the end product.

Synthesizing polymers is not as exact a science as wewould like it to be. Natural syntheses and even well-con-trolled laboratory syntheses often yield macromolecules

that vary in length, molar mass, branching, chemical compo-sition, and other properties. Characterizing these propertiesand their distributions is important because of their critical ef-fect on end-use structure–property relations and, hence, onthe end product itself. The most commonly studied proper-ties are the molar mass averages (Mn, Mw, Mz, etc.) and themolar mass distribution (MMD). Various processing charac-teristics of macromolecules can be related to the individual av-erages, for example, flow properties and brittleness (related toMn) and flex life and stiffness (related to Mz). Similarly, prop-erties such as tensile strength and abrasion resistance tend toincrease as MMD narrows, and properties such as elongationand yield strength tend to increase as MMD broadens.

During the past four decades, size-exclusion chromatogra-phy (SEC) has been established as the premier method forcharacterizing M averages and the distribution of natural and

M A R C H 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y 1 0 5 A© 2 0 0 5 A M E R I C A N C H E M I C A L S O C I E T Y

1 0 6 A A N A LY T I C A L C H E M I S T R Y / M A R C H 1 , 2 0 0 5

synthetic polymers. Equally impor-tant, or more important, is the abil-

ity of SEC to characterize the distri-butions of many other macromolecular

parameters (Table 1). This ability is imparted by the many ana-lytical techniques that serve as detection methods and that arethe primary subject of this article (1). Because of space limita-tions, this article will focus on SEC as the sole separation methodprior to detection and ignore its growing role in 2-D LC.

Detection methods will be divided into two classes. Chemicaldetectors, such as UV–vis (when not used as a concentration-sensitive detector), IR, NMR, and MS, usually combine in addi-tive fashion. Physical detectors, such as the viscometer (VISC)and light-scattering (LS) photometers, on the other hand, cancombine in synergistic fashion. Some detectors appear to bridgethe gap between classes; for example, IR and NMR can functionas either chemical or physical detectors. The type of informationprovided by each class will be explored. Because of the large num-ber of detectors covered here, it is impossible to include an expla-nation of the fundamentals of each. Many excellent referencesources exist on SEC, as well as on each of the individual topics(2, 3); a few relevant references are given where appropriate.

SEC and the single detectorSEC operates via an inverse molecular sieving mechanism thatdepends on the relative size or hydrodynamic volume of a dis-solved molecule with respect to the average pore size of the pack-ing material in the column (2). During its passage through a size-exclusion column, a polymer molecule samples a portion of theavailable pore space. Certain molecules will be too big to fit intoany of the pores and will elute together at the so-called total ex-clusion limit. Other molecules will occupy a volume so small com-

pared with the smallest pore size that they will elute together atthe total permeation limit, regardless of differences in size. Be-tween the total exclusion and total permeation limits is the sep-aration region of the column; here, the fraction of the sampledpore volume is inversely proportional to the size of the polymer,that is, inversely proportional to the hydrodynamic volume ofthe polymer. As a result, molecules occupying a larger hydrody-namic volume will elute earlier than those occupying a smallervolume. Although this behavior can correspond with larger Mpolymers eluting before those with smaller M, the elution ordercan be drastically changed by long-chain branching (LCB) andother factors.

Concentration-sensitive detectors—differential refractometer(DRI), UV–vis detector, and evaporative LS or evaporative massdetector—are by far the most widely used in SEC because theymeet the minimum detection requirement for calculating M av-erages and distributions. Generally, the values obtained usingonly a concentration-sensitive detector are highly precise but ofdubious accuracy. This dubiousness is caused by calibration curvesthat are normally constructed using narrow-polydispersity linearstandards that usually do not possess the same chemistry or ar-chitecture as the analyte. Beyond their use in the construction ofcalibration curves, concentration-sensitive detectors are also need-ed for determining the distributions of M, intrinsic viscosity, andso forth, when LS and/or VISC detection methods are used.This is because the concentration of analyte in each slice elut-ing from the SEC column(s) is a necessary datum for calculat-ing these parameters.

Light scatteringOne type of physical detector is the LS photometer, which comesin both static and dynamic varieties (4). In a static LS (SLS) ex-

periment (also referred to as total-intensity LS), thelight scattered from a dilute macromolecular solutionis detected either at a single angle or at many anglessimultaneously. At a single angle, only M informationabout the analyte is generally obtained, whereas atmany angles, both M and size are measured. “Size” isa versatile term, because a variety of macromolecularsize parameters can be measured in a multidetectorSEC experiment.

When a multi-angle LS (MALS) photometer iscoupled to an SEC setup that contains a concentra-tion-sensitive detector, the amount of incident lightscattered by each slice eluting from the column ismeasured at many angles � simultaneously (5, 6). Theweight-average molar mass Mw of each slice is obtainedby extrapolating the measurements at the different an-gles to � = 0°. Then, the M distribution of a polymeris obtained by summing over all the slices. The angu-lar dissymmetry, or difference in the light scattered bythe dilute macromolecular solution at one angle withrespect to that scattered at another, is used to deter-mine the root-mean-square radius Rg (often referredto by the misnomer “radius of gyration”) of a macro-molecule, defined as the root-mean-square distance of

Table 1. Properties of polymers, how they affect

performance, and detection modes.

Macromoleculardistribution End-use properties Detection mode

M Elongation, tensile strength, SLSand adhesion

Long-chain Shear strength, tack, and peel MALSbranching

Short-chain Haze, resistance to stress IR, NMRbranching cracks, and crystallinity

Architecture Flow modification, encapsulation, MALS/QELS/VISCand diffusion (/IR, /NMR)

Chemical Toughness, brittleness, UV–vis, NMR,heterogeneity and biodegradability IR, MS

Copolymer Dielectric properties, NMR, IRsequence miscibility, and reactivity

Polyelectrolyte Flocculation, binding Conductometrycharge of metals, and transport

M A R C H 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y 1 0 7 A

an array or group of atoms from their common center of mass.As a good rule of thumb, determining Rg can become difficultbelow ~�0/20n0, in which �0 is the vacuum wavelength of theincident radiation and n0 is the refractive index (RI) of the sol-vent, because of the lack of the necessary angular dissymmetry.

The determination of M averages and distributions by SEC/SLS is an absolute measurement that depends at a fundamentallevel only on the validity of the relationship between polarizabil-ity and RI. The accuracy of the M and Rg values, however, de-pends on the accuracy of the determination of several systemparameters, such as the calibration of the SLS photometer, thenormalization of the photodiodes of a MALS system, the inter-detector volume(s), and the measurement of the specific RI in-crement �n/�c of the polymer solution. This last term corre-sponds to the change in the RI of the solution (n) as a functionof the concentration of dissolved analyte (c); this datum is neces-sary for accuracy in both SLS and DRI measurements. Accuratedetermination of �n/�c (which depends on the solvent, temper-ature, and wavelength of the experiment in addition to thechemical identity of the analyte) can be complicated by variousfactors, such as copolymerization, a large oligomeric region inthe MMD, and polyelectrolytic behavior.

When using SEC/MALS to study sample aggregation, re-searchers take advantage of the fact that the detector’s responseis proportional to the product of M and the solution concentra-tion of the analyte. The SLS photometer is therefore referred toas an “M-sensitive” detector. Even a small amount of high-Msample will scatter a large amount of light; hence, this detectoris ideal for studying the solution aggregation behavior of macro-molecules. Figures 1a and 1b show the 90° SLS, DRI, and dif-ferential viscometer traces of the SEC separation of the poly-saccharide pullulan (7). At the given experimental conditions,pullulan aggregated in solution, as evidenced by the large, earlyelution volume peak (retention volume ~18 mL). This aggrega-tion was not detected by either DRI or VISC.

SEC/MALS can also be used to study LCB in macromole-cules, which affects processing properties such as melt viscosityand end-use properties such as tack, peel, and shear strength (8;Table 1). Quantitative determination of LCB relies upon calcu-

lating g, the ratio of the mean-square radius of a branchedpolymer to that of its linear counterpart of the same M. Becausethe radii are measured at each slice eluting from the column, anSEC/MALS experiment can measure the LCB distribution of apolymer as a continuous function of the analyte’s M. Figure 2ashows the so-called conformation log–log plot of Rg versus Mfor linear and branched poly(vinyl acetate) (PVAc; 9). At anygiven M, the Rg of sample 2 (PVAc2) is smaller than that ofPVAc1. This is because a molecule with LCB will adopt a morecompact structure in solution than will a linear molecule of thesame chemistry and M. Figure 2b shows the change in g as afunction of M for PVAc2. Applying the theory developed byZimm and Stockmayer (10) and assuming that PVAc2 is a tri-functional randomly branched polymer, Grcev et al. calculatedthe number of long-chain branches B and the branching fre-quency � (the number of long-chain branches per 1000 repeatunits of polymer) as a continuous function of M for PVAc2 (Fig-ures 2c and 2d, respectively).

Note that accurate, quantitative measurement of the LCB dis-tribution of macromolecules by SEC/MALS requires strict at-tention to four parameters (8, 10). First, it is necessary to possessa linear standard to which the branched sample can be compared.Second, the sample and standard should have the same chemicalcomposition. This can be complicated in the case of copolymers,in which the same chemical heterogeneity, chemical compositiondistribution, and so forth are also necessary. Third, the linearstandard should cover the region of interest of the MMD of thebranched sample. Finally, the functionality f, which is the num-ber of long-chain branches emanating from each branch point,should be known a priori. Other methods exist that determinethe branching distribution by comparing the intrinsic viscositiesof linear and branched samples of the same M or by comparingthe molar masses of linear and branched samples at the same elu-tion volumes in an SEC experiment. Although useful, both thesemethods depend on the “draining” properties of the polymers insolution, which can lead to inaccurate results.

Self-similarity features of macromolecules, such as their fractaldimension, can also be determined using SEC/MALS (11, 12).One meaning of “self-similar” is that, regardless of the length

0.04

0.02

0.00

10 15 20 25 30 35Volume (mL)

AUX,

90°

det

ecto

r

0.04

0.02

0.00

10 15 20 25 30 35Volume (mL)

AUX,

90°

det

ecto

r

FIGURE 1. SLS sensitivity to solution aggregation of macromolecules: Pullulan dissolved in N,N ´-dimethyl acetamide with 0.5% LiCl (DMAc/LiCl)at 80 °C.

(a) 90° SLS (pink squares) and DRI (blue crosses; AUX) signals. (b) 90° SLS (pink squares) and differential viscometer (blue crosses; AUX) signals. Unaggregatedanalyte elutes at ~20.5 mL, aggregated analyte at ~18 mL. No evidence of aggregation is observed by either VISC or DRI. The increase in sensitivity of SLS withincreasing M permits even a very small concentration of a high-M sample, such as an aggregate, to be seen with this type of detector. (Adapted from Ref. 7 .)

(b)(a)

scale used to measure Rg, this parameter shouldscale with M in a unique fashion. A change in thisscaling relationship indicates a fundamental archi-tectural change in the polymer or a fundamentalthermodynamic change in the polymer solution.The fractal dimension df of polymers is obtainedfrom the conformation log–log plot of Rg versus Mand is defined as the inverse of the slope � (Figure3). The df of polymers can provide information thatis not given by the topological dimension d T. Forexample, dilute solutions of a rigid rod, a linear ran-dom coil at good solvent and temperature condi-tions, and a linear random coil at theta conditions(the point at which the poorness of the solvent andtemperature conditions exactly compensate for theexcluded volume effect) can have vastly differentproperties. However, these differences are not ob-served when the topological dimensions are com-pared because in all three cases d T = 1. The differ-ences are borne out, however, by df: df = 1 for therigid rod, 5/3 � df � 2 for the linear random coilat good conditions, and df = 2 for the linear randomcoil at theta conditions.

A specific example of this type of application isseen in Figure 3, in which the conformation plot ofpoly(�-benzyl-L-glutamate) (PBLG) is overlaidupon those of two poly(vinyl butyral) samples, onewith native branching (PVBN) and one with bothnative and induced branching (PVBX; 12, 13). PBLGis a fairly rigid macromolecule, as its df (1.20) in-dicates. At lower molar masses, we observed thatPVBN and PVBX resemble linear random coils,with df = 1.75. At higher molar masses, df of PVBNis ~2, which is very close to that expected for a ran-domly branched polymer at good solvent and tem-perature conditions (2 � df � 2.28 for the genericcase). The relatively high df ≈ 2.4 of the branchedregion of PVBX indicates the heterogeneity ofbranching in this polymer, which contains both na-tive and induced branching and may also includebranch-on-branch-type structures.

A variation on SLS is the low-angle LS (LALS)photometer. Long used as an SEC detector bymany researchers in the 1970s and 1980s, it fell outof favor when multi-angle systems were introducedand became popular. A new low-angle instrumentwith a redesigned optical arrangement was recentlyintroduced for on-line SEC measurements, as partof a triple-detector array combining LS, VISC, andDRI (14). In an SEC/LALS experiment with � �7°, Mw of each slice was determined without ex-trapolating to an angle of 0°. Although this experi-ment can determine a polymer’s M with extreme ac-curacy, no size information by way of Rg is obtainedbecause angular dissymmetry cannot be measuredusing a single angle.

(a)

(c)

B

g

Rg

(b)

100

10

1

10

8

6

4

2

0

0.4

0.8

0.6

PVAc1

PVAc2

0.8

(d)

0.6

0.4

0.2

0

M (g/mol)

105 106 107

105 106 107

105 106 107

105 106 107

�

FIGURE 2. Determination of LCB distribution of PVAc by SEC/MALS.

(a) Conformation log–log plot of Rg versus M for linear (PVAc1) and branched (PVAc2)poly(vinyl acetate). (b) g, (c) B, and (d) � as a function of M for PVAc2. (Adapted with per-mission from Ref. 9; figure courtesy of P. Iedema and colleagues.)


MALS detectors can also be decoupled from the SEC systemfor off-line use in what are called batch-mode experiments (4–6,12). These experiments can provide information that directlycomplements that obtained by on-line SEC/MALS studies. Thebasic experiment consists of measuring the scattering at many an-gles for a series of solutions at different concentrations. The re-sults for each concentration at each angle are plotted. The angu-lar data are then extrapolated to a concentration of 0, and theslope of the extrapolated line gives Rg. The concentration dataare extrapolated to an angle of 0°, and the slope of this extrapo-lated line gives A2, the second virial coefficient, a fundamentalproperty of polymer solutions that is correlated with solubility,solvation, and conformation. From the common y intercept ofthe two extrapolated lines, Mw is obtained. Figure 4 is an exam-ple of a plot that results from this dual-extrapolation procedure(also called a Berry plot) for the PVBX sample described earlier.The results from a batch-mode MALS experiment can be used tocalculate another size parameter, the thermodynamic radius RT,which can be thought of as the radius of a sphere with the sameexcluded volume as the polymer (11, 15).

In a dilute solution or suspension, the consequence of the dis-solved particles continuously colliding with solvent molecules israndom thermal (Brownian) motion, which causes the intensityof scattered light reaching a photodiode to fluctuate with timearound some average value. In SLS, the time-averaged fluctua-tions of the scattered light are measured, whereas in quasi-elasticLS (QELS; also referred to as dynamic LS and photon correla-tion spectroscopy), it is the time-dependent fluctuations that areof interest (4, 16). Through QELS, we obtain our third size pa-rameter, the hydrodynamic or Stokes radius Rh, which may bethought of as the radius of an equivalent hard sphere that wouldfeel the same force due to flow as would a macromolecule (11,15). Determination of Rh down to a few nanometers is possiblewith QELS.

In several modern instruments, SLS and QELSare housed in a single apparatus, obviating the needto determine interdetector volumes between thetwo systems. Recently, Liu et al. used SEC withboth dual-angle SLS and QELS to determine theM, size, and conformation of several synthetic poly-mers (17 ). They used relations derived by Burchard(11) to combine Rg and Rh into a single dimen-sionless parameter � � Rg/Rh and obtain informa-tion about conformation and dilute solution ther-modynamics across the MMD of poly(dimethylsiloxane). More recently, Cotts combined the capa-bilities of MALS, QELS, VISC, and SEC to providearchitectural information, as a continuous functionof M, for structures ranging from semi-rigid rods torandom coils to stars (18).

ViscometryThe pressure drop ∆P across a capillary is related byPoiseuille’s law to the length L and radius r of thetube and to the viscosity of the solution flowingthrough the capillary at a volumetric flow rate Q by

This equation constitutes the operating principle of single-cap-illary viscometers. Although it has been used for studying manynatural and synthetic polymers, the differential viscometer isbecoming more popular. The differential viscometer is usually afluid-flow analog of the classic Wheatstone-bridge electrical cir-cuit (19). A differential pressure transducer measures the changein pressure across the bridge, and an inlet pressure transducermeasures the pressure change through the bridge. The differen-tial pressure transducer signal is proportional to the specific vis-cosity sp defined by

in which is the viscosity of the polymer solution, s is the vis-cosity of the neat solvent, and rel is the relative viscosity of thesolution (rel � /s).

Of primary interest when using VISC with SEC is the deter-mination of the intrinsic viscosity, [], and how it changes withM. The definition of [] is

in which [] is recognized as the ratio of the signal from the vis-cometer (which measures sp) to the signal from the concentra-tion-sensitive detector (which measures c) forthe same data slice, after correction for in-terdetector delay. The units of [] aredeciliters per gram, so intrinsic viscositymay be thought of as inverse density.The measurement of [] may be com-bined with that of M to define the

[] � limspcc 0

sp = rel –1= – ss

8LQ πr 4 ∆P =

Rg ~ M �

� ~ 1/df

� = 0.57

� = 0.83df = 1.20

� = 0.51df = 1.96

df = 1.75

� = 0.42df = 2.38

R g (n

m)

M (g/mol)

20105 106

30

40

50

60

70

80

90

FIGURE 3. SEC/MALS provides df.

Conformation plots of poly(�-benzyl-L-glutamate) (PBLG; green) and poly(vinyl butyral) with na-tive branching (PVBN; blue) and with both native and induced branching (PVBX; red). (Adaptedfrom Ref. 13.)



fourth size parameter, the viscometric radius R of a macromol-ecule (11).

R can be thought of as the radius of a solid sphere that candissipate (as heat) the same amount of the flowing solvent’s ki-netic energy as does the polymer, or as the radius of a solidsphere that increases the fluid’s viscosity by the same amount asdoes the polymer (15). As with Rh, determining R into the sin-gle-digit nanometer range is possible; thus, we see that SEC/VISC and SEC/QELS can measure macromolecular size with ahigh degree of accuracy and precision to a much lower limit thanSEC/MALS can. Naturally, the “size” that is measured by eachtechnique is different.

Accurate information on polymer draining is needed for thequantitative calculation of the LCB distribution. Because theMark–Houwink plot of log [] versus log M is normally lessnoisy than the conformation log–log plot of Rg versus M, SEC/VISC is generally a more sensitive method than SEC/MALS for determining the presence of LCB (8). Fig-ure 5 is an example of a Mark–Houwink plot used todetect LCB in two polyethylene (PE) samples, onelinear and one branched (20). Because LCB tends tomanifest more markedly in polymers with increasingM, the slope of the Mark–Houwink plot of thebranched PE decreases with increasing M, whereasthe slope of the linear PE remains constant as a func-tion of M. The df can also be calculated from SEC/VISC data using the relation df = 3/(1 + a), in whicha is the slope of the Mark–Houwink plot (8, 11). Thisis a useful definition, although it should be ap-proached with caution because of potential polymerdraining and coil interpenetration effects.

The four main size parameters are the Rg, Rh, R,and RT radii. The first three, along with their distri-butions, can be measured as continuous functions ofM in a single SEC/MALS/QELS/ VISC experimentusing commercially available detectors; RT is ob-tained through off-line, batch-mode MALS. Bur-chard has done extensive work on the relationship of� to polymeric architecture and dilute solution ther-

modynamics (11). For example,for a monodisperse, linear ran-dom coil, � = 1.504 at thetaconditions and 1.78 at goodsolvent and temperature condi-tions; for a tetrafunctional starat theta conditions, � = 1.33when the arms are of uniformlength but 1.534 when the armlength is polydisperse. Other ra-tios of the four radii have notbeen explored as extensively as�; the majority of the work hasbeen devoted to star polymers(11, 21, 22).

The molecular radii have alsobeen used to define the polymer

draining function ( � R/Rg) and the coil interpenetrationfunction (�* � RT/Rg); describes the penetration of the hy-drodynamic volume of the polymer by a solvent molecule, and�* describes the interpenetration of the hydrodynamic volumesof two connected polymer segments. Though both functionshave been combined in VA2 (� RT/R), its usefulness, alongwith that of the polymer draining and coil interpenetration func-tions, has thus far been restricted to star polymers (11, 12).SEC/MALS/QELS/ VISC could be used to explore the de-pendence of �, , and other ratios—along with the thermody-namic and architectural information these provide—as continu-ous functions of M for polydisperse macromolecules, but thispotential remains largely untapped.

We will next discuss interfacing SEC to chemical detectors. Asin earlier sections, we will deal only with on-line detection meth-ods or with techniques used in continuous off-line mode. The

4.00

1.96

– 0.08

– 2.12

– 4.16

– 6.204.00 4.25 4.50 4.75

log M

log

[] �

10 –1

5.00 5.25 5.50

FIGURE 4. Berry plot of PVBX from off-line, batch-mode MALS data; Mw, Rg, and A2 were determined fromscattering measurements taken simultaneously at 17 different angles from a series of dilutions of PVBX.

FIGURE 5. Mark–Houwink plot of linear (blue) and branched (red) PEs obtained bySEC/ VISC using a differential viscometer and universal calibration. The slope ofthe linear PE remains invariant as a function of M, and the slope of the branchedPE decreases as M increases, in the presence of LCB.

�(K

*c/R

�)

6.0 �10 –3

5.0 �10 –3

4.0 �10 –3

3.0 �10 –3

2.0 �10 –3

–1.0 –1.5 0.5 1.00.0

sin2 ( ) – 189*c�

2


widely used practice of collecting a fewdiscrete fractions of eluate for subsequentanalysis is not discussed, because it doesnot provide a continuous distribution ofthe measured parameter.

MS detectionESI is the softest ionization method current-ly available in MS. Soft ionization combinedwith ESI’s inherent multiple charging mech-anism have made MS an ideal tool for study-ing biopolymers and for accurately deter-mining their molar masses. Beyond itsapplication to perfectly monodisperse ana-lytes, ESI-MS has also been used in a varietyof other polymer applications (23). As anon-line detection method, ESI-MS has beenused to obtain more accurate SEC calibra-tions than those obtained via traditional ap-proaches, to obtain chemical compositiondistribution information on copolymers, andto study humic and fulvic acids. Becauseonly ~1% of the SEC effluent is needed forESI-MS, other detectors are usually used.Because of their low microliters-per-minuteflow rates, micro-SEC columns can be utilized. Deery et al. usedSEC/ESI-MS to study 5000-Da and 12,000-Da dextrans andenzymatically digested arabinoxylan polysaccharides (24). Theyalso used SEC/ESI-MSn to determine glycosidic linkage of per-methylated arabinogalactan oligomers.

In contrast to ESI-MS, multiple charging is not usually ob-served in MALDI MS; therefore, interpretation of the MALDIinformation and its use in calculating M are more straightforwardthan in ESI-MS. Fei and Murray combined SEC on-line withaerosol MALDI for the complete mass spectra, across the entireelution profile, of 1000-Da poly(ethylene glycol) and poly(pro-pylene glycol) (25).

A more popular approach is continuous off-line MALDI.Using a homemade apparatus, Lou and van Dongen electro-sprayed the SEC eluent and a coaxially added matrix materialonto a MALDI plate (26). Others have used a commerciallyavailable interface in which the SEC eluent in sprayed directlythrough a heated capillary nozzle onto a moving MALDI targetprecoated with an appropriate matrix. For polydisperse macro-molecules, a continuous track of sample is deposited onto thematrix surface of the target; spectra are obtained from differentpositions on the track. Esser et al. used this approach to studypolystyrene 32500 and poly(methyl methacrylate) (PMMA)10900 to obtain both M and copolymer composition of a di-block copolymer of n-butyl methacrylate and PMMA (27 ) .

Matrix selection is an important challenge, and currently onlya few different types of matrix materials have been coated ontocommercially available targets. Recently, Liu et al. used poly(di-methyl siloxane)s to compare continuous off-line SEC/MALDITOFMS with on-line SEC/ESI TOFMS (28). They found thatthe electrospray technique is more effective at characterizing

low-M oligomers and the MALDI tech-nique is more effective at characterizinghigh-M oligomers. Inductively coupledplasma MS (ICPMS) is a quite popular de-

tection method for the SEC quantitation ofmetals across the elution profile of polymers.

Sadi et al. listed several precautions that mustbe taken when coupling SEC to ICPMS (29).The use of organic solvents should be kept toa minimum because they affect the stability ofthe plasma. The ionic strength of the mobilephase buffers must be adjusted carefully be-cause they can help limit non-SEC behavior,but they may also denature the organometal-lic complexes of interest. In addition, the saltcontent of the mobile phase should be kept toa minimum to avoid clogging the nebulizerand to reduce wear on the sampler and skim-mer cones.

Sample preparation for this technique isalso tricky because the species of interest mustbe extracted from the sample matrix withoutaltering the nature of these species. Gardneret al. used SEC/ICPMS along with a UV de-tector to study metals in natural waters (30).

Hall et al. used SEC/ICPMS to fractionate lead-bound ligandsin human amniotic fluid and to detect and quantitate lead (31).In the realm of glycopolymers, Szpunar’s group has used SEC/ICPMS along with DRI. The concentration-sensitive detectorcharacterized the elution profiles of water-soluble and enzymati-cally digested polysaccharides, and the MS detector provided thedistribution patterns of select metals in high- and low-M frac-tions of apple and carrot samples (32).

Other MS methods have also been coupled on-line to SEC,including chemical reaction interface MS to analyze nucleicacids, proteins, and other biopolymers (33), as well as atmo-spheric-pressure chemical ionization MS to determine pesticidebinding by humic substances (34).

Spectroscopic detectorsThe IR detector can act as either a chemical or a physical detec-tor, and it can be used in on-line or continuous off-line modes.In off-line use, the same commercially available hardware usedfor SEC/MALDI can often be used for SEC/FTIR. The appli-cations of FTIR as a physical detector have thus far been on-lineand limited to characterizing the short-chain branching contentof polyolefins. Markovich et al. used SEC/FTIR to measure themethyl group content per 1000 carbons, as a function of M, ofethylene-based polyolefin copolymers (35). Figure 6 shows amore recent example by DesLauriers, who used on-line SEC/FTIR and chemometrics to quantitate the ethyl and/or butylcontent of ethylene 1-olefin copolymers as a function of M andrelated the results to trends resulting from catalysis and processchanges (36).

Cotts and Ouano used IR as a chemical detector to studypoly(vinyl butyral), which is actually a random terpolymer and is

Some detectors appear

to bridge the gap between

classes; for example, IR

and NMR can function as

either chemical or

physical detectors.


more accurately referred to as poly(vinyl butyral-co- vinyl alco-hol-co-vinyl acetate) (12, 37 ). By monitoring the OH stretch ofthe hydroxyl group and the butyral ring vibration, they foundthe vinyl alcohol content of the polymer to be independent of M.Most applications of FTIR as a chemical detector are continuousand off-line. The SEC eluent is directed to a heated nozzle forevaporation of the solvent and deposition of the analyte onto arotating germanium disk; spectra are obtained from any locationon the disk (38, 39).

To date, all SEC/NMR experiments have been performed incontinuous (not stop-flow) mode, and all reports have dealt withSEC/1H-NMR (40). The use of NMR as a physical detector ap-pears to be thus far restricted to measurement of the tacticity dis-tribution in mixtures of isotactic and syndiotactic PMMA (41). Asa chemical detector for SEC, on-line coupling of NMR has beenused to determine the MMD of uniformly isotactic PMMA (42)and to study the chemical heterogeneity of synthetic copolymers(i.e., the change in chemical composition as a function of molarmass; 43). Neiss and Cheng reported recently on the SEC/NMRanalysis of alginates, in which the microstructure of these plantpolysaccharides was determined in combination with Markov sta-tistical models, off-line 13C-NMR, and enzymology (44) .

In addition to its widespread use as a concentration-sensitivedetector, UV–vis has also been used to determine the chemicalheterogeneity of copolymers, for example, in the study ofpoly(styrene-co-methyl methacrylate)s by SEC/DRI/UV/MALS(45). The DRI served as a concentration-sensitive detector, andthe UV detector monitored � = 262 nm, where styrene presentsa strong absorption band but methyl methacrylate does not ab-sorb. The weight fraction of styrene was quantitated across theMMD of the copolymers.

Fluorescence spectroscopy is growing in popularity as a de-tection method for SEC. Fauser and colleagues separated air-borne organic molecules from bitumen by SEC, identifyingthese particles by on-line fluorescence detection (46). In study-

ing dissolved organic matter fromBaltic Sea water, Lepane isolated thehydrophobic and hydrophilic frac-tions by adsorption chromatogra-phy and then characterized them bySEC/fluorescence (47). Moon andco-workers monitored the couplingof fluorescence-labeled anhydride-functional polystyrene and PMMAin dilute polymer blends with SEC/fluorescence (48). Maliakal et al.used the same method to measurepolymer–polymer chain-end reac-tion rate constants (49).

Edwards et al. attempted to useSEC/FT-Raman MS with a low-vol-ume flow cell to characterize the mi-crostructural variation in polybutadi-ene polymers as a function of elutionvolume (50). As an on-line detector,the Raman spectrometer lacked the

necessary sensitivity to provide quantitative information for theparticular analytes being examined because of its low sensitivityat the different excitation wavelengths. This setup is still promis-ing, however, provided the proper experimental conditions canbe found for the analyte solutions so that they exhibit a high scat-tering intensity relative to the solvent or mobile phase.

The use of conductivity detection in SEC has been rathersparse, despite its great potential for the analysis of polyelec-trolytes. Rinaudo et al. used SEC/DRI/VISC/conductivity tomeasure the charge distribution, which was superimposed uponthe MMD, for a series of sodium carboxymethylcelluloses (51).

ConclusionsFor the purposes of determining both fundamental and end-useproperties of macromolecules, multidetector SEC has come ofage (1). The current focus is thus on obtaining the proper com-bination of detectors to characterize the physicochemical prop-erties of interest. Physically, SEC/DRI/MALS/ VISC has beenshown to be extremely powerful and can now be augmented byon-line QELS. The combination of chemical detectors has alsobecome impressive with the recent report of characterizing poly-mer additives using SEC/UV/1H-NMR/ESI-MS/FTIR—thefirst three detectors were on-line, and the FTIR operated in con-tinuous off-line mode (52). The constant conversion of analyti-cal techniques into SEC detection methods provides a growingchoice of parameters that can be measured.

André M. Striegel is an assistant professor at Florida State University. Hisresearch interests include the study of natural and synthetic polymers—in particular, applying multidetector SEC and related techniques todetermine structure–property relationships of macromolecules andelucidating fundamental aspects of separation science. Address cor-respondence about this article to Striegel at Department of Chem-istry & Biochemistry, Florida State University, Tallahassee, FL 32306-4390 ([email protected]).

1.2

0.8

0.4

03 4 5

log M

SEC

dW/d

(log

M )

SCB

/ 100

0 TC

(1 M

e CE

)

6 70

10

20

30

FIGURE 6. Short-chain branching in polyolefins by SEC/FTIR.

Distribution of the number of short-chain branches (SCB) with one methyl chain-end (1 Me CE) per 1000 totalcarbons (TC) across the MMD of an ethylene 1-olefin copolymer. Each type of symbol represents one runthrough the column. (Adapted from Ref. 36; figure provided courtesy of Paul DesLauriers, Chevron Phillips.)

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