Coupled Liquid Chromatographic Techniques in Molecular Characterization · 2013-03-19 · Coupled...

Coupled Liquid Chromatographic Techniques in MolecularCharacterization

Peter Kilz and Harald Pasch

inEncyclopedia of Analytical Chemistry

R.A. Meyers (Ed.)pp. 7495–7543

John Wiley & Sons Ltd, Chichester, 2000

COUPLED LIQUID CHROMATOGRAPHIC TECHNIQUES IN MOLECULAR CHARACTERIZATION 1

Coupled LiquidChromatographic Techniquesin Molecular Characterization

Peter KilzPSS Polymer Standards Service, Mainz, Germany

Harald PaschDeutsches Kunststoffinstitut, Darmstadt, Germany

1 Introduction 1

2 Coupled Techniques in Polymer Analysis 3

3 Coupling of Liquid Chromatography withInformation-rich Detectors 43.1 Introduction 43.2 Coupling with Molar Mass-sensitive

Detectors 63.3 Coupling with Mass Spectroscopy 113.4 Coupling with Fourier Transform

Infrared Spectroscopy 183.5 Coupling with Nuclear Magnetic

Resonance Spectroscopy 22

4 Multidimensional Liquid Chromatography 254.1 Introduction 254.2 Experimental Aspects of Multi-

dimensional Separations 284.3 Separation Techniques for the First

Dimension 314.4 Separation Techniques for the

Second Dimension 344.5 State-of-the-art of On-line Coupled

Two-dimensional Chromatography 364.6 Conclusions and Future

Developments 43

List of Symbols 44

Abbreviations and Acronyms 44

Related Articles 45

References 45

Complex polymers are distributed in more than one direc-tion of molecular heterogeneity. In addition to the molarmass distribution (MMD), they are frequently distributedwith respect to chemical composition, functionality, andmolecular architecture (see Size-exclusion Chromatogra-phy of Polymers). For the characterization of the differenttypes of molecular heterogeneity it is necessary to usea wide range of analytical techniques. Preferably, these

techniques should be selective towards a specific type ofheterogeneity. The combination of two or more selectiveanalytical techniques is assumed to yield multidimensionalinformation on the molecular heterogeneity.

The present review presents the fundamental ideas ofcombining liquid chromatography (LC) with other ana-lytical techniques in multidimensional analysis schemes(see Size-exclusion Chromatography of Polymers; GasChromatography in Analysis of Polymers and Rubbers;Field Flow Fractionation in Analysis of Polymers andRubbers). The capabilities and limitations of different cou-pling techniques are discussed and a number of relevantapplications are given. It is shown that multidimensionalstructural information can be obtained when differ-ent chromatographic techniques are combined. Anotherapproach is the hyphenation of LC with information-richdetectors. These detectors include molar mass-sensitivedetection systems, such as on-line viscometry (VISC)and light scattering (LS). Information on the chemicalcomposition of complex polymers can be obtained whenspectroscopic techniques, like Fourier transform infrared(FTIR) (see Infrared Spectroscopy in Analysis of PolymerStructure–Property Relationships), nuclear magnetic res-onance (NMR) or mass spectrometry (MS) are coupledto LC.

The basics and applications of multidimensional LCare addressed rather extensively. A brief introduction todifferent separation mechanisms is given and the particularrequirements for the first and second dimensions arediscussed. In conclusion, state-of-the-art examples for on-line coupled two-dimensional (2-D) chromatography aredemonstrated, and future developments are reviewed.

1 INTRODUCTION

Today’s polymeric materials are designed to meetvery specific requirements defined by the application.Therefore, most synthetic polymers are highly complexmulticomponent materials. They are composed of macro-molecules varying in chain length, chemical composition,and architecture. By definition, complex polymers areheterogeneous in more than one distributed property(for example, linear copolymers are distributed in molarmass and chemical composition).

In general, the molecular structure of a macromoleculeis described by its size, its chemical structure, and itsarchitecture. The chemical structure characterizes theconstitution of the macromolecule, its configuration andits conformation. For a complete description of theconstitution the chemical composition of the polymerchain and the chain ends must be known. In addition tothe type and quantity of the repeat units their sequence

Encyclopedia of Analytical ChemistryR.A. Meyers (Ed.) Copyright John Wiley & Sons Ltd

2 POLYMERS AND RUBBERS

of incorporation must be described (alternating, random,or block in the case of copolymers). Macromolecules ofthe same chemical composition can still have differentconstitutions due to constitutional isomerism (1,2- versus1,4-coupling of butadiene, head-to-tail versus head-to-head coupling, linear versus branched molecules).Configurational isomers have the same constitution butdifferent steric patterns (cis- versus trans configuration;isotactic, syndiotactic and atactic sequences in a polymerchain). Conformational heterogeneity is the result of theability of fragments of the polymer chain to rotate aroundsingle bonds. Depending on the size of these fragments,interactions between different fragments, and a certainenergy barrier, more or less stable conformations maybe obtained for the same macromolecule (rod-like versuscoil conformation).

Depending on the composition of the monomer feedand the polymerization procedure, different types ofheterogeneities may become important. For example,in the synthesis of tailor-made polymers frequentlytelechelics or macromonomers are used. These oligomersor polymers usually contain functional groups at thepolymer chain end. Depending on the preparationprocedure, they can have a different number of functionalendgroups, i.e. be mono-, or bifunctional and so forth. Inaddition, polymers can have different architectures, i.e.they can be branched (star- or comb-like), and they canbe cyclic.

The structural complexity of synthetic polymers can bedescribed using the concept of molecular heterogeneity,see Figure 1, meaning the different aspects of MMD,chemical composition distribution (CCD), functional-ity type distribution (FTD) and molecular architecture

Molecular heterogeneity

Molar massdistribution

Functionality typedistribution

Molecular architecture

Distribution ofchemical

composition

Figure 1 Schematic representation of the molecular hetero-geneity of complex polymers.

distribution (MAD). They can be superimposed oneon another, i.e. bifunctional molecules can be linearor branched, linear molecules can be mono- or bifunc-tional, copolymers can be block or graft copolymers etc.In order to characterize complex polymers it is nec-essary to know the MMD within each other type ofheterogeneity.

Using the traditional methods of polymer analysis, suchas NMR, one can determine the type and concentrationof monomers or functional groups present in the sample.However, the determination of functional endgroups iscomplicated for long-chain molecules because of lowconcentration. On the other hand, these methods donot yield information on how different monomer units orfunctional groups are distributed in the polymer molecule.Finally, these methods in general do not provide molarmass information.

With respect to methods sensitive to the size of themacromolecule, one can face other difficulties. Size exclu-sion chromatography (SEC), which is most frequentlyused to separate polymer molecules from each otheraccording to their molecular size in solution, must beused very carefully when analyzing complex polymers.The molecular size distribution of macromolecules canin general be unambiguously correlated with MMD onlywithin one heterogeneity type. For samples consistingof a mixture of molecules of different functionality,the distribution obtained represents a sum of distribu-tions of molecules having a different functionality and,therefore, cannot be attributed to a specific functional-ity type without additional assumptions. For the analysisof copolymers by SEC either the chemical compositionalong the molar mass axis must be known or detectorsmust be used which, instead of a concentration informa-tion, can provide molar mass information. To this end,SEC has to be coupled to composition-sensitive or molarmass-sensitive detectors.

Another option for the analysis of complex polymersis the separation with respect to chemical compositionor functionality by means of interaction chromatography.In this case, functionally or chemically homogeneousfractions are obtained which then can be subjected tomolar mass determination.

To summarize, for the complete analysis of complexpolymers a minimum of two different characterizationmethods must be used. It is most desirable that eachmethod is sensitive towards a specific type of heterogene-ity. Maximum efficiency can be expected when, similarto the 2-D distribution in properties, 2-D analytical tech-niques are used. A possible approach in this respect isthe coupling of different chromatographic modes in 2-Dchromatography or the coupling of a separation techniquewith selective detectors, such as molar mass-sensitive orspectroscopic detectors.


2 COUPLED TECHNIQUES IN POLYMERANALYSIS

Coupled techniques (also termed hyphenated techniques)are very frequently used in low molar mass organic chem-istry. Using high-resolution chromatographic techniques,such as capillary gas chromatography (GC), gradienthigh-performance liquid chromatography (HPLC) andcapillary electrophoresis (CE), complex mixtures are sep-arated into single components which are then identifiedby MS. By hyphenated GC/MS, HPLC/MS, and CE/MSup to several hundreds of different components can beseparated and identified in one run with very high sensitiv-ity. This is particularly important for environmental andbiological samples, where frequently only very limitedsample amounts are available.

Polymers are typically complex mixtures in which thecomposition depends on polymerization kinetics andmechanism and process conditions. To obtain polymericmaterials of desired characteristics, polymer processingmust be carefully controlled and monitored. Further-more, one needs to understand the influence of molecularparameters on polymer properties and end-use perfor-mance. MMD and average chemical composition may nolonger provide sufficient information for process and qual-ity control nor define structure–property relationships.Modern characterization methods now require multi-dimensional analytical approaches rather than averageproperties of the whole sample..1/

Different from low molar mass organic samples,where single molecules are to be determined, forcomplex synthetic polymers, the analytical task is thedetermination of a distributed property. The molecularheterogeneity of a certain complex polymer can bepresented in either a three-dimensional (3-D) diagram ora so-called ‘‘contour plot’’. For a telechelic polymer thesepresentations are given in Figure 2. Using appropriateanalytical methods, the type and concentration of thedifferent functionality fractions must be determined and,within each functionality, the MMD has to be obtained.To do this, two different methods must be combined,each of which preferably is selective towards one type ofheterogeneity. For example, a chromatographic methodseparating solely with respect to functionality could becombined with a molar mass selective method. Anotherapproach would be the separation of the sample intodifferent molar mass fractions which are then analyzedwith respect to functionality.

For copolymers, in particular random copolymers,instead of discrete functionality fractions a continu-ous drift in composition is present, see Figure 3. Todetermine this chemical composition drift in interre-lation with the MMD, a number of classical methodshave been used, including precipitation, partition, and

W

R−R A–R A–A

R–R

A–R

A–A

0 1 2

Mola

r mas

s

Molar mass

Functionality

Fun

ctio

nalit

y

(a)

(b)

Figure 2 Representation of the molecular heterogeneity of atelechelic polymer in a 3-D diagram (a) and a contour plot (b).

Composition(A in copolymer AB)

Com

posi

tion

Molar mass

W

Mi

Ai

Mol

ar m

ass

(a)

(b)

Figure 3 Representation of the molecular heterogeneity of arandom copolymer in a 3-D diagram (a) and a contour plot (b).

cross-fractionation..2/ The aim of these very laborioustechniques was to obtain fractions of narrow compositionand/or MMD which are then analyzed by spectroscopyand SEC.

During the last 20 years a number of techniques havebeen introduced in organic chemistry and applied to


polymer analysis, combining chromatographic separationwith spectroscopic detection..3/ GC/MS has been usedin polymer analysis,.4 – 11/ but, due to the low volatilityof high molar mass compounds it is limited to theoligomer region. The combination of pyrolysis and gaschromatography/mass spectrometry (GC/MS), however,is of great value for polymer characterization..12,13/ Itprovides for the analysis of complex polymers withrespect to chemical composition. For a number of polymersystems characteristic low molar mass pyrolysis productsare obtained, which yield information of the averagecomposition and the ‘‘blockiness’’ of the polymer chain.Molar mass information, however, is not available frompyrolysis-GC/MS.

Much more important for polymer analysis thanGC are the different techniques of LC. Using SEC,liquid adsorption chromatography (LAC), or liquidchromatography at the critical point of adsorption(LC/CC) polymers can be fractionated with respect todifferent aspects of molecular heterogeneity, includingmolar mass, functionality, and chemical composition. Theadvantage of these techniques over GC is that intactmacromolecules are separated and analysed. As will beshown in the next sections, LC can be efficiently coupledto infrared (IR) spectroscopy,.14 – 19/ to MS, and to NMRspectroscopy..20,21/

Another most efficient approach is the chromato-graphic separation of complex polymers by combiningdifferent separation mechanisms. This can be done bycoupling two chromatographs in an off-line or on-linemode. Each of these chromatographs must operate in amode which is selective towards one type of molecularheterogeneity. This 2-D chromatography has been termed‘‘orthogonal chromatography’’ assuming the selectivity ofeach separation method with respect to one distributionfunction, e.g. MMD, FTD, or CCD..22/ The first trulyautomated 2-D chromatography set-up for polymer anal-ysis was proposed by Kilz et al.,.23/ who coupled gradientHPLC and SEC.

The need for such analysis protocols results from thefact that in complex polymers in addition to chemicalheterogeneity of the first kind, another type of chemicalheterogeneity may exist: chemical heterogeneity of thesecond kind, in which polymers of different compositionand chain length have similar hydrodynamic volumes and,hence, co-elute in SEC, see Figure 4.

A possible separation protocol for a complex polymermixture is presented in Figure 5. The sample underinvestigation comprises molecules of different chemicalcompositions (different colors) and different sizes. In afirst separation step this mixture is separated accordingto composition, yielding fractions which are chemicallyhomogeneous. These fractions are transfered to a size-selective separation method and analyzed with respect to

(a)

(b)

AAAAABBABABABABBB

Log M

Pol

ymer

con

cent

ratio

n

Figure 4 SEC fractionation showing composition or architec-ture at a given retention volume. (Reproduced by permissionfrom Barth..1/)

molar mass. As a result of this 2-D separation, informationon both types of molecular heterogeneity is obtained.

3 COUPLING OF LIQUIDCHROMATOGRAPHY WITHINFORMATION-RICH DETECTORS

3.1 Introduction

LC of polymers is often understood to be synonymouswith SEC. SEC separates polymers according to the size ofthe macromolecules by entropic interactions and enablesthe MMD of a sample to be evaluated. However, inaddition to size exclusion phenomena, other types ofinteraction of the macromolecules and the stationaryand mobile phases of a chromatographic system canbe used for separation. LAC uses enthalpic interactionsto separate substances such as copolymers according tochemical composition. Finally, LC/CC can be used forfunctionality type separation by balancing entropic andenthalpic interactions in the chromatographic system.


1

2

1

2

Vr

Figure 5 Schematic separation protocol for the analysis of a complex polymer mixture.

SEC is the premier polymer characterization methodfor determining MMD. In SEC, the separation mecha-nism is based on molecular hydrodynamic volume. Forhomopolymers, condensation polymers and strictly alter-nating copolymers, there is a correspondence betweenelution volume and molar mass. Thus, chemically sim-ilar polymer standards of known molar mass can beused for calibration. However, for SEC of random andblock copolymers and branched polymers, no simple cor-respondence exists between elution volume and molarmass because of possible compositional heterogeneity ofthese materials. The dimensional distribution of macro-molecules can, in general, be unambiguously correlatedwith MMD only within one heterogeneity type. Forsamples consisting of molecules of different chemicalcomposition, the distribution obtained represents an aver-age of dimensional distributions of molecules having adifferent composition and, therefore, cannot be attributedto a certain type of macromolecule.

The inadequacy of using SEC without further precau-tion for the determination of MMD of polymer blendsor copolymers results from the following consideration:for a linear homopolymer distributed only in molar mass,fractionation by SEC results in one molar mass beingpresent in each retention volume. The polymer at eachretention volume is monodisperse. If a blend of two linearhomopolymers is fractionated then two different molar

masses can be present in one retention volume. If nowa copolymer is analyzed then a multitude of differentcombinations of molar mass, composition, and sequencelength can be combined to give the same hydrodynamicvolume. In this case, fractionation with respect to molec-ular size is completely ineffective in assisting the analysisof composition or MMD.

Three on-line methods are used to try to charac-terize copolymers by SEC with respect to MMD andcomposition:

ž conventional SEC utilizing multiple concentrationdetection

ž on-line analysis of SEC fractions with a LS detector.ž VISC.

The experimentally simplest approach is the combi-nation of SEC with multiple concentration detectors.If the response factors of the detectors for the com-ponents of the polymer are sufficiently different, thechemical composition of each slice of the elution curvecan be determined from the detector signals. Typically,a combination of ultraviolet (UV) and refractive index(RI) detection is used; another possibility is the use ofa diode-array detector. In the case of non-UV absorb-ing polymers, a combination of RI and density detectionyields information on chemical composition..24 – 26/


The principle of dual detection is rather simple: whena mass mi of a copolymer, which contains the weightfractions wA and wB (D1� wA) of the monomers A andB, is eluted in the slice i (with the volumeV) of the peak,the area xi,j of slice i obtained from detector j dependson the mass mi (or the concentration ci D mi/V) ofpolymer in the slice, its composition (wA), and thecorresponding response factors fj,A and fj,B, wherein jdenotes the individual detector, as in Equation (1) below:

xi,j D mi.wAfj,A C wBfj,B/ .1/

The weight fractions wA and wB of the monomers can becalculated using Equation (2) below:

1wAD 1�

{.x1/x2/f2,A � f1,A

.x1/x2/f2,B � f1,B

}.2/

Once the weight fractions of the monomers are known,the correct mass of polymer in the slice can be calculatedusing Equation (3) as follows:

mi D xi

wA.f1,A � f1,B/C f1,B.3/

and the molar mass MC of the copolymer is obtainedby interpolation between the calibration lines of thehomopolymers.27/ which is given in Equation (4):

ln MC D ln MB C wA.ln MA � ln MB/ .4/

wherein MA and MB are the molar masses of thehomopolymers, which would elute in this slice of thepeak (at the corresponding elution volume Ve).

It is clear that the interpolation between the calibrationlines cannot be applied to mixtures of polymers (polymerblends): if the calibration lines are different, differentmolar masses of the homopolymers will elute at thesame volume. The universal calibration is not capable ofeliminating these errors, either, which originate from thesimultaneous elution of two polymer fractions with thesame hydrodynamic volume, but different compositionand molar mass.

The architecture of a copolymer (random, block, graft)has also to be taken into account, as Revillon.28/ hasshown by SEC with RI, UV, and viscosity detection.Intrinsic viscosity varies largely with molar mass accord-ing to the type of polymer, its composition, and the natureof its components. Tung.29/ found that for block copoly-mers in good SEC solvents the simpler first approach(Equation 4) is more precise.

Further information on quantitative aspects of SECwith dual detection can be obtained from Trath-nigg et al..30/ Different applications of dual detectionSEC in the analysis of segmented copolymers,.31/

block copolymers,.32,33/ star polymers,.34/ and polymerblends.35,36/ are also available. The limitation of SEC

with dual detection is that only binary combinations ofmonomers can be investigated successfully. In the case ofternary combinations, more than two detectors must beused or one of the detectors must be able to detect twocomponents simultaneously.

To overcome the problems related to classical SECof complex polymers, molar mass-sensitive detectors arecoupled to the SEC instrument. Since the response of suchdetectors depends on both concentration and molar mass,they have to be combined with a concentration-sensitivedetector. The following types of molar mass-sensitivedetectors are used frequently:.37 – 40/

ž differential viscometerž low angle laser light scattering (LALLS) detectorž multiangle laser light scattering (MALLS) detector.

3.2 Coupling with Molar Mass-sensitive Detectors

As has been pointed out, for SEC of complex polymersno simple correspondence exists between elution volumeand molar mass. It is, therefore, useful to determinethe molar mass not via a calibration curve but directlyfrom the SEC effluent. This can be done by using molarmass-sensitive detectors based on Rayleigh LS or intrinsicviscosity measurements..41/

In a LS detector, the scattered light of a laser beampassing through the cell is measured at angles differentfrom zero. The (excess) intensity of the scattered light atthe angle (R.)) is related to the weight-average ofmolar mass Mw as expressed by Equation (5):

KŁcR./

D 1MwP./

C 2A2c .5/

wherein c is the concentration of the polymer, A2 is thesecond virial coefficient, and P./ describes the scatteredlight angular dependence. KŁ is an optical constantcontaining Avogadro’s constant NA, the wavelength l0,the RI n0 of the solvent, and the RI increment dn/dc of thesample. Their relationship is described by Equation (6):

KŁ D 4p2n20.dn/dc/2

l40NA

.6/

In a plot of KŁc/R./ versus sin2./2/, Mw can beobtained from the intercept and the radius of gyration(Rg) from the slope. A multiangle measurement providesadditional information.

In most cases the injected concentration is small andA2 can be neglected. Thus, if the optical properties (n0

and dn/dc) of the polymer solution are known, the molarmass at each elution volume increment can be determinedas expressed by Equation (7):

Mw,i D R./iKŁP./ici

.7/


If a low-angle LS instrument is used, P./ is close tounity and Mw,i can be calculated directly. For a multiangleLS instrument, the mean-square radius of gyration hR2

giat each elution volume can also be obtained from P./as shown in Equation (8):

1P./i

D 1C q2hR2gii

3.8/

q D(

4pl0

)sin(

P./2

)In practice, however, the radius of gyration can only bedetermined for molecules larger than 20 nm in diameter.By measuring radius of gyration as a function of Mw,insight into the molecular conformation of the polymercan be obtained..1/

Molar mass determination requires the knowledge ofthe specific RI increment dn/dc which in the case ofcomplex polymers depends on chemical composition.Copolymer RI increments .dn/dc/copo can be calculatedaccurately for chemically monodisperse fractions, ifcomonomer weight fractions wi and homopolymer valuesare known, as described in Equation (9):(

dndc

)copoD∑

wi

(dndc

)i

.9/

However, in some cases additional effects on .dn/dc/copo

must be considered. Due to cooperative interactionsbetween the monomer units in the polymer chain, copoly-mer RI increments may deviate from the summationscheme. As a result of different sequence length distri-butions, different .dn/dc/copo can be obtained for thesame gross composition. Copolymer .dn/dc/copo valuescan be obtained by multiple detection SEC providing thechemical composition at each slice of the elution curve.

Unfortunately, LS investigations of copolymers arecomplicated even further by the fact that SEC doesnot separate into chemically monodisperse fractions.Accordingly, due to compositional heterogeneity theRI increment of a particular scattering center may bedifferent from the total dn/dc of the corresponding SECslice. Therefore, in general, only apparent molar massesfor copolymers can be measured..34/ Another influencingfactor is the RI of the solvent. As has been shownby Kratochvil,.42/ the solvent RI should be significantlydifferent from the values of the copolymer fractions andthe corresponding homopolymers.

The evaluation of LS detectors for SEC was conductedby Jeng et al. with respect to precision and accuracy.43/

and the proper selection of the LS equation..44/ Theresults obtained for polystyrene (PS) and polyethylenewere compared for a low-angle and a multiangle LSinstrument. The application of SEC/LS has been discussedin a multitude of papers. In addition to determining

Mw values, the formation of microgels has been studiedby Pille and Solomon..45/ Mourey and Coll investigatedhigh molar mass PS and branched polyesters, anddiscussed the problems encountered in molar massand radius of gyration determination..46,47/ Grubisic-Gallot et al. proved that SEC/LS is useful for analysingmicellar systems with regard to determining molar masses,qualitative evaluation of the dynamics of unimer-micellesre-equilibration, and revealing the mode of micelleformation..48 – 50/

Another very useful approach to molar mass informa-tion of complex polymers is the coupling of SEC to aviscosity detector..51 – 56/ The viscosity of a polymer solu-tion is closely related to the molar mass (and architecture)of the polymer molecules. The product of polymer intrin-sic viscosity [h] times molar mass is proportional to thesize of the polymer molecule (the hydrodynamic vol-ume). Viscosity measurements in SEC can be performedby measuring the pressure drop P across a capillary,which is proportional to the viscosity h of the flowing liq-uid (the viscosity of the pure mobile phase is denoted ash0). The relevant parameter [h] is defined as the limitingvalue of the ratio of specific viscosity (hsp D .h� h0//h0)and concentration c for c! 0, as shown by Equation (10):

[h] D limh� h0

h0cD lim

hsp

cfor c! 0 .10/

The viscosity of a polymer solution as compared to theviscosity of the pure solvent is measured by the pressuredropP across an analytical capillary-transducer system.The specific viscosity is obtained from P/P, where P isthe inlet pressure of the system. As the concentrationsin SEC are usually very low, [h] can be approximated byhsp/c.

A simple approach using one capillary and onedifferential pressure transducer will not work very well,because the viscosity changes h D h� h0 will typicallybe very small compared to h0, which means that onehas to measure a very small change of a large signal.Moreover, flow-rate fluctuations due to pulsations of areciprocating pump will lead to much greater pressuredifferences than the change in viscosity due to the elutedpolymer. Instruments of this type should be used with apositive displacement pump.

A better approach is the use of two capillaries (C1and C2) in series, each of which is connected to adifferential pressure transducer (DP1 and DP2), and asufficiently large holdup reservoir (HR) in between. Withthis approach, one measures the sample viscosity h fromthe pressure drop across the first capillary, and the solventviscosity h0 from the pressure drop across the secondcapillary. Pulsations are eliminated in this set-up, becausethey appear in both transducers simultaneously. Anotherdesign is that of the differential viscometer, in which four


Fromcolumn

Fromcolumn

C1

C2

DP1

DP2

HR

P

C2

C4C3

C1

HDP

(a) (b)

Figure 6 Schematic representation of differential viscometers.P is inlet pressure transducer: C1–C4 are flow restrictioncapillaries.

capillaries are arranged in a manner similar to that of aWheatstone bridge. In Figure 6, both designs are shownschematically.

In the ‘‘bridge’’ design, a holdup reservoir in front of thereference capillary (C4) makes sure that only pure mobilephase flows through the reference capillary, when thepeak passes the sample capillary (C3). This design offersconsiderable advantages: the detector actually measuresthe pressure difference P at the differential pressuretransducer (DP) between the inlets of the sample capillaryand the reference capillary, which have a common outlet,and the overall pressure P at the inlet of the bridge. Thespecific viscosity hsp D h/h0 is thus obtained fromP/P.One concern with this type of detector is that the flow mustbe divided in the ratio of 1 : 1 between both arms of thebridge. This is achieved by capillaries C1 and C2, whichmust have a sufficiently high back pressure. Nevertheless,when a peak passes through the sample capillary, a slightdeviation of the 1 : 1 ratio will be observed. A problemof flow-rate variations exists also in a single capillaryviscometer: when the polymer peak passes through themeasuring capillary, the increased back pressure leads toa peak shift..57/

Being able to determine [h] as a function of elutionvolume, one can now compare the hydrodynamic volumes(Vh) for different polymers. The hydrodynamic volumeis, through Einstein’s viscosity law, related to intrinsicviscosity and molar mass by Vh D [h]M/2.5. Einstein’s lawis, strictly speaking, valid only for impenetrable spheresat infinitely low volume fraction of the solute (equivalentto concentration at very low values). However, it canbe extended to particles of other shapes, defining theparticle radius then as the radius of a hydrodynamicallyequivalent sphere. In this case Vh is defined as the molarvolume of impenetrable spheres which would have the

same frictional properties or enhanced viscosity to thesame degree as the actual polymer in solution.

Assuming the validity of this approach and in agree-ment with the SEC mechanism, similar elution volumescorrespond to similar hydrodynamic volumes, as shownin Equation (11):

Ve,1 D Ve,2 ��!M1[h]1 DM2[h]2 .11/

In a plot of log (M[h]/ versus Ve identical calibrationlines should be found for the two polymers 1 and 2, irre-spective of their chemical composition. This ‘‘universalcalibration’’ approach has been predicted and experimen-tally proved by Benoit et al..58/ As a consequence, usingthe universal calibration curve established with knowncalibration standards (for example PS), one can obtainthe SEC-molar mass calibration for an unknown polymersample.

The intrinsic viscosity is a function of molar massgiven by the Mark–Houwink relationship (Equation 12),wherein K and a are coefficients for a given polymer in agiven solvent at a given temperature.

[h] D KMa .12/

This leads to Equation (13):

K1Ma.1/C11 D K2Ma.2/C1

2 .13/

If a column has been calibrated with polymer 1 (e.g.PS), the calibration line for polymer 2 can be calculated,provided that the coefficients K and a are known forboth polymers with sufficient accuracy. This is shown byEquation (14):

ln M2 D(

11C a2

)ln(

K1

K2

)C(

1C a1

1C a2

)ln M1 .14/

Thus, the concept of universal calibration provides anappropriate calibration also for polymers for which nocalibration standards exist. The limiting factor of thisapproach is the accuracy of determining K and a. Thereare very high variations in the values reported in theliterature..59,60/ Even for such common polymers as PSand polymethyl methacrylate (PMMA) the values maydiffer considerably.

If the Mark–Houwink coefficients are not available, auniversal calibration curve is established using PS cali-bration standards and the SEC–viscometer combination.The basic steps involved in the MMD analysis are sum-marized in Figure 7. First, the universal calibration curveof the SEC separation system has to be established byusing narrow molar mass standards as indicated by the toparrow pointing to the right. Once the universal calibrationcurve is established, one can then reverse the procedure,by going from right to left following the bottom arrow, to


Log

M

Log

[η]

Log

[η] M

PS

PS

Molar mass-calibration [η]-Calibration(from viscosity detector)

Universal

To obtain absolute molar mass calibration (unknown polymer)

To obtain universal calibration (PS standard )

VR VR VR

Figure 7 Determination of absolute molar masses via universal SEC calibration.

obtain the molar mass calibration curve of any unknownpolymer. The calibration curve is obtained literally bysubstracting the [h] calibration curve of the unknownsample from the universal calibration curve. The [h] cal-ibration curve for the unknown sample is obtained fromthe on-line viscometer..61/

The application of RI and differential viscometerdetection in SEC has been discussed by a number ofauthors..62 – 64/ Lew et al. presented the quantitative anal-ysis of polyolefines by high-temperature SEC and dualRI–viscosity detection..65/ They applied a systematicapproach for multidetector operation, assessed the effectof branching on the SEC calibration curve, and useda signal averaging procedure to define better intrinsicviscosity as a function of retention volume. The combi-nation of SEC with RI and viscosity detectors was usedto determine molar mass and functionality of polytetra-hydrofurane simultaneously..66/ Long chain branching inethylene–propylene–diene rubber (EPDM) copolymersby SEC–viscometry was analysed by Chiantore et al..67/

One of the difficult problems in characterizing copoly-mers and polymer blends by SEC–viscometry is theaccurate determination of the polymer concentrationacross the SEC elution curve. The concentration detec-tor signal is a function of the chemical drift of thesample under investigation. To overcome this problem,Goldwasser proposed a method where no concentra-tion detector is required for obtaining number-averagemolar mass (Mn) data..68/ In the usual SEC–viscometryexperiment, the determination of the intrinsic viscosity ateach slice of the elution curve requires a viscosity and aconcentration signal as shown by Equation (15):

[h]i D(

ln hrel

c

)i

.15/

where ln hrel is the direct detector response of theviscometer. One calculates the molar mass averages by theexpressions given in Equation (16) and in Equation (17):

Mn D∑

ci∑[ci/.Vh,x/[h]/i]

.16/

Mw D∑

ci.Vh,x/[h]/i∑ci

.17/

where Vh,x D [h]xMx is the data retrievable from the uni-versal calibration curve. By rearranging Equation (17)using Equation (16) the following expression (Equa-tion 18) is obtained:

Mn D∑

ci∑.ln hrel/Vh,x/i

or

Mn D sample amount∑.ln hrel/Vh,x/i

.18/

The sample amount can be determined easily from theinjection volume and the sample concentration and noinformation from a concentration detector is required.With this approach, the Mn value of any polymer samplecan be determined by SEC using only the viscositydetector. Other molar mass averages, however, cannotbe determined. The advantage of the Goldwasser Mn

method is that it can access much wider molar massranges than other existing methods like osmometry orendgroup methods.

Due to the problems encountered with SEC/LALLSand SEC–viscometry, a triple-detector SEC technologyhas been developed, where three on-line detectors are


used together in a single SEC system. In addition tothe concentration detector, an on-line viscometer anda LALLS instrument are coupled to the SEC: thisarrangement is known as TriSEC. With TriSEC, abso-lute molar mass determination is possible for polymersthat are very different in chemical composition andmolecular conformation. The usefulness of the TriSECapproach has been demonstrated in a number of appli-cations. It was shown by Pang and Rudin that only byusing both viscometer and LS detection are accurateMMDs obtained..69/ Wintermantel et al. have developeda custom-made multidetector instrument and demon-strated that it has great potential not only for absolutemolar mass determinations but also for structure char-acterization of linear flexible, semiflexible, and branchedpolymers..70/ Degoulet et al. characterized polydispersesolutions of branched PMMA,.71/ while Jackson et al.investigated linear chains of varying flexibility in orderto prove universal calibration..72/ Yau and Arora dis-cussed the advantages of TriSEC for the determination ofMark–Houwink coefficients, long-chain branching, andpolymer architecture..73/

Finally, several attempts have been made to developan absolute molar mass detector based on osmotic pres-sure measurements. Commercially available membraneosmometers are designed for static measurements, andthe cell design with a flat membrane is not suited for con-tinuous flow operation. Yau.61,74/ developed a detectorwhich is different from the conventional design; it mea-sures the flow resistance of a column caused by osmoticswelling and deswelling of soft gel particles used for thepacking, see Figure 8. With a microbore gel column anMn sensitive detector with a fast response was obtainedwhich could be coupled to the SEC equipment. However,since the change in flow resistance could not easily berelated to the osmotic pressure of the solution, absolutecalibration was lost.

Recently, an osmometer based on a concentric designwith a capillary-shaped membrane has been developedby Kohler et al..75/ and Lehmann et al..76/ The flow cellvolume is 12.2 µL, the response time approximately 15 s,and the molar mass cut-off is below 5.000 g mol�1. Thedesign of the cell is given in Figure 9. The cylindersymmetry and stiffness of the osmometer and thefavorable properties of the membrane were combined tomeet the requirements for on-line detection. Testing theinstrument in both batch and continuous flow operationwith PS standards yielded reproducible results and goodagreement with the nominal molar masses. However, theosmometer still caused a certain peak broadening, andthe pressure noise level still strongly exceeded the noiseof the concentration detector.

As has been discussed, the combination of SEC andmolar mass-sensitive detectors is a powerful tool for the

��

��

��

��

Solvent Polymer solution

Solvent Solution

High∆Pgel

Low∆Pgel

Osmotic effects of polymer solution:

Shrinkage of soft gel particles

More open flow channels

Lower flow resistance

Lower pressure drop ∆Pgel

Figure 8 Differential pressure measurement of osmotic effecton a soft gel column.

��

��

��

��

��

��

��

��

��

��

Pressure transducer

Out

Flush

Flush

GasketMembrane

SolventSolution

SealGlass tube

Holder

Figure 9 Design of a concentric osmometric flow cell. (Repro-duced by permission from Lehmann et al..76/)

analysis of complex polymers. However, it is importantto distinguish between claimed versus actual capabilitiesand between potential expectations and demonstratedperformance. Tables 1 and 2 below, taken from acritical review of different techniques summarize theinformation content and additional details of SEC/LS andSEC–viscometry coupling..61/ The information content isclassified into two categories. ‘‘Primary’’ information is ofhigh precision and accuracy, insensitive to SEC operationvariables, and does not require molar mass or universalcalibration. ‘‘Secondary’’ information is less precise andrequires calibration.


Table 1 SEC analysis using molar mass-sensitive detectors

Method Information content

Primary Secondary

Conventional SEC MMDSEC/LALLS MMDSEC/MALLS MMD Rg distributionSEC/VISC [h] distribution MMD

Rg distributionCopolymer Mn

SEC/VISC/LS [h] distribution Copolymer MnMMDRg distribution

Table 2 Generalization of molar mass-sensitive detectors

Intended LALLS/MALLS Viscometermeasurements

MMD Requires precise nand dn/dc values

Not affected bynonexclusioneffects

Requires universalcalibration andK, a-parameters

[h] distribution Directly fromexperiment

Not affected bynonexclusioneffects

Rg distribution MALLS only Calculable from[h]M

Chainconformationand branching

Rg vs M plot,MALLS only

[h] vs M plot, Rg vsM plot

Chemicallyheterogeneouspolymeranalysis

Limited Better

Noise,particulates,bubbles

Strongly affected Less affected

In addition, the complex procedures related to SEC/LSand SEC–viscometry coupling are a potential source oferror. According to Jackson and Barth.77/ these include:

1. Accuracy of the universal calibration curve.

2. Detector configuration: arrangement of multipledetectors in series or in parallel can cause additionalpeak broadening, flow rate variations, back pressurevariations.

3. Interdetector volume: detectors are placed at dif-ferent physical positions and their signals must bealigned very precisely.

4. Detector sensitivity: LS and viscosity detectors arevery sensitive towards higher molar masses, while theRI detector is most sensitive at lower molar masses.

5. Low molar mass fractions: polymer moleculesmay not adopt random coil conformation, theMark–Houwink coefficients become functions ofmolar mass.

To summarize, although the principal limitation ofSEC separating according to hydrodynamic volume andnot molar mass cannot be overcome, the advantages ofmultidetector SEC in the accurate characterization ofcomplex polymers are significant. However, in orderto generate reproducible and accurate results on aroutine basis, special care must be taken regarding theadded complexity of the instrumentation. In additionto improving the design of multidetector SEC set-ups, important advances are expected from methodsfor determining the chemical composition across theMMD by interfacing SEC with FTIR spectroscopy, MS,and NMR.

3.3 Coupling with Mass Spectroscopy

From the very early stages of development of modernMS, the value of its combination with chromatographywas quickly recognized. The coupling of GC with MSwas a natural evolution since they are both vaporphase techniques, and very quickly GC/MS has beenaccepted as a standard component of the organicanalytical laboratory. It has taken considerably longerto achieve a satisfactory and all-purpose mode ofHPLC/MS coupling. The difficulties with HPLC/MS wereassociated with the fact that vaporization of typically1 mL min�1 from the HPLC translates into a vapor flow-rate of approx. 500–1000 mL min�1. Other difficultiesrelated to the eluent composition as a result of thefrequent use of nonvolatile modifiers, and the ionizationof nonvolatile and thermally labile analytes. However,during the past several years commercial interfaces havebeen developed which have led to a broad applicabilityof HPLC/MS..78 – 80/ The techniques necessary for thesuccessful introduction of a liquid stream into a massspectrometer are based on the following principles:electrospray ionization (ESI),.81/ atmospheric pressurechemical ionization,.82/ thermospray ionization,.83/ andparticle beam ionization..84/

From the point of view of polymer analysis, a massspectrometric detector would be a most interestingalternative to the conventional detectors because thisdetector could provide absolute molar masses of polymercomponents..85,86/ Provided that fragmentation does notoccur, intact molecular ions could be measured. The mea-sured mass of a particular component could then be cor-related with chemical composition or chain length. How-ever, the major drawback of most conventional HPLC/MStechniques is the limited mass range, preventing higher


oligomers (molar mass above 2000–3000 g mol�1) to beionized without fragmentation..87 – 89/

The use of MS for detailed polymer analysis hasbecome increasingly established due to the introductionof soft ionization techniques that afford intact oligomeror polymer ions with less fragmentation..90 – 93/ Oneof these techniques, ESI/MS, has been widely appliedin biopolymer analysis. Proteins and biopolymers aretypically ionized through acid–base equilibria. When aprotein solution (the effluent from an HPLC separation) isexposed to an electrical potential it ionizes and dispersesinto charged droplets. Solvent evaporation upon heattransfer leads to the shrinking of the droplets and theformation of analyte ions. Larger molecules acquiremore than one single charge, and, typically, a mixtureof differently charged ions is obtained.

Unfortunately, up to now ESI/MS has had limitedapplication in polymer analysis..94,95/ Unlike biopolymers,most synthetic polymers have no acidic or basic functionalgroups that can be used for ion formation. Moreover, eachmolecule gives rise to a charge distribution envelope, thuscomplicating the spectrum further. Therefore, syntheticpolymers that can typically contain a distribution of chainlengths and have a variety in chemical composition orfunctionality furnish complicated mass spectra, makinginterpretation nearly impossible.

To overcome the difficulties of ESI/MS, Prokai andSimonsick added sodium cations to the mobile phaseto facilitate ionization..96,97/ To simplify the resultingESI spectra, the number of components entering theion source was reduced. Prokai et al. implementedmicrocolumn SEC for the separation of polydisperse

mixtures prior to ESI detection..98/ They used a 250ð0.5 mm internal diameter SEC column for the molar massseparation of octylphenoxy polyethylene oxide (PEO).Applying a flow of 4 µL min, they were able to supply theeffluent from the column directly into the ESI source. Topromote ionization, a sheath liquid of sodium iodide inmethanol was delivered to the ESI interface. Figure 10shows a representative chromatogram and mass spectrumfrom the SEC/ESI analysis. The mass spectrum wasobtained by averaging between 6.9 and 9.2 min. It showssingly and doubly charged molecules in the molar massrange of 1000–2000 g mol�1.

The analysis of PEOs by SEC/ESI/MS with respect tochemical composition and oligomer distribution was dis-cussed by Simonsick..99/ In a similar approach, aliphaticpolyesters,.100/ phenolic resins,.101/ methyl methacrylatemacromonomers.101/ and polysulfides.102/ have been anal-ysed. The detectable mass range for different species,however, was well below 5000 g mol�1, indicating that thetechnique is not really suited for polymer analysis.

The quantitative analysis of PMMA-butyl acrylatecopolymers by coupled LC and particle beam MS hasbeen described by Murphy et al..103/ For separationwith respect to chemical composition gradient HPLCwas used. The copolymer composition was determinedby monitoring several low-mass fragments formed bythermal decomposition and electron impact ionization inthe particle beam interface.

Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) MS is one of the newest softionization techniques that allows desorption and ioniza-tion of very large molecules even in complex mixtures. In

00

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1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

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600 800 1000 1200 1400 1600 1800 2000

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m/z

787

831

875919 1007

10511095

13741462 1550 1638 1726 1815 1902 1990

Figure 10 Micro-SEC/ESI analysis of octyloxy PEO, (a) TIC chromatogram, (b) averaged mass spectrum. (Reproduced bypermission from Prokai et al..98/)


polymer analysis, the great promise of MALDI/TOF/MSis to perform the direct identification of mass-resolvedpolymer chains, including intact oligomers within anMMD, and the simultaneous determination of structureand endgroups in polymer samples. This most promis-ing method for the ionization of large molecules andanalysis according to their molar mass and functional-ity has been introduced by Karas and Hillenkamp.104,106/

and by Beavis and Chait..105/ Compared to other MStechniques, the accessible mass range has been extendedconsiderably, and the technique is fast and instrumentallyvery simple. Moreover, relatively inexpensive commer-cial instrumentation has become accessible. In principle,the sample to be investigated and a matrix solution aremixed in such a ratio that matrix separation of the sam-ple molecules is achieved. After drying, a laser pulse isdirected onto the solid matrix to photo-excite the matrixmaterial. This excitation causes the matrix to explode,resulting in the expulsion and soft ionization of the sam-ple molecules. Once the analyte is ionized, it is acceleratedand analysed in a TOF mass spectrometer. As a result,the analyte is separated according to the molar mass of itscomponents, and in the case of heterogeneous polymersadditional information on chemical composition may beobtained. In a number of papers it was shown that poly-mers may be analysed up to relative molar masses of about500 000 Da..107 – 111/ It was shown in a number of applica-tions that functionally heterogeneous polymers can beanalysed with respect to the degree of polymerizationand the type of functional groups..112 – 115/

The on-line combination of LC and MALDI/TOF/MSwould be of great value for polymer analysis. Inparticular, for chemically or functionally heterogeneouspolymers LC could provide separation with respect tochemical composition while MALDI/TOF would analysethe fractions with respect to oligomer distribution ormolar mass. Unfortunately, MALDI/TOF is based onthe desorption of molecules from a solid surface layerand, therefore, a priori not compatible with LC. Inan attempt to take advantage of the MALDI/TOFcapabilities, a number of research groups carried outoff-line LC separations and subjected the resultingfractions to MALDI/TOF measurements. Although thisis laborious, it has the advantage that virtually any typeof chromatographic separation can be combined withMALDI/TOF.

The different options for using MALDI/TOF as anoff-line detector in LC have been discussed by Paschand Rode..116/ In SEC of low molar mass samples theseparation into individual oligomers and the quantitativedetermination of the MMD via an oligomer calibrationcould be achieved, see Figure 11 for oligo(caprolactone).The lower oligomers appeared as well separated peaksat the high retention time end of the chromatogram.

For the analysis of the peaks, i.e. the assignment ofa certain degree of polymerization (n) to each peak,MALDI/TOF/MS was used. The SEC separation wasconducted at the usual analytical scale and the oligomerfractions were collected, resulting in amounts of 5–20 ngsubstance per fraction in tetrahydrofuran (THF) solution.The solutions were directly mixed with the matrixsolution, placed on the sample slide and subjected tothe MALDI experiments. As a large number of fractionsmay be introduced into the mass spectrometer at onetime, sample preparation and MALDI/MS measurementstake a very short period of time. In total, nine fractionswere collected from SEC and measured by MALDI/MS.For the lower oligomers the spectra consisted of anumber of peaks of high intensity, having a peak-to-peak mass increment of 114 Da, which equals themass of the caprolactone repeating unit. These peaksrepresented the MCNaC molecular ions, whereas thepeaks of lower intensity in their vicinity were due tothe formation of MCKC molecular ions. MCNaC andMCKC molecular ions were formed due to the presenceof small amounts of NaC and KC ions in the samples and/orthe matrix. Further peaks of low intensity indicated afunctional heterogeneity in the samples. From the massesof the MCNaC peaks the degree of polymerizationof the corresponding oligomer was calculated. By thisprocedure, the first peak in the chromatogram wasassigned to n D 1, the second peak to n D 2, and so on.From the elution time and the degree of polymerizationof each oligomer peak an oligomer calibration curve oflog molar mass vs elution time was constructed. Theconventional calibration curve based on PS standardsdiffered remarkably from this oligomer calibration curve.

A much more demanding task is the analysis of fractionsfrom LC not only with respect to molar mass but alsowith respect to chemical structure. The separation of atechnical fatty alcohol ethoxylate (FAE) by LC, underconditions where the chain length as well as the endgroupsdirect the separation, is presented in Figure 12. Usingthis chromatographic technique, the FAE was separatedinto three main fractions, the first fraction appearingas one peak at a retention time of about 60 s and thesecond and third fractions showing oligomer separations.Fraction 1 was collected in total, whereas for fractions 2and 3 the individual oligomer peaks were collected.The MALDI/MS spectra of all three fractions gave apeak-to-peak mass increment of 44 Da, thus indicatingthat all fractions consisted of species with an ethyleneoxide-based polymer chain. From the masses assigned tothe peaks and the peak-to-peak mass increment of theethylene oxide repeating unit the mass of the endgroupfor the different fractions was calculated. Provided thesample was a pure FAE, the endgroups of fractions1–3 could be identified as being polyethylene glycol


30

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e (m

in)

40

50(a)

(b) 200 600

n = 2

n = 3

n = 4

n = 4

56

7

8

9

m/z (Da)1000 1400

Figure 11 SEC of oligo(caprolactone) and MALDI/TOF analysis of fractions (a) and SEC calibration graphs (b). (Reproduced bypermission from Pasch and Rode..116/)

(PEG) (a,w-dihydroxy endgroups), C13-terminated PEO(a-tridecyl-w-hydroxy endgroups) and C15-terminatedPEO (a-pentadecyl-w-hydroxy endgroups), respectively.Using MALDI/TOF the oligomer distribution of the PEGfraction was measured directly. For fractions 2 and 3 bydetermining the degree of polymerization of the oligomerpeaks oligomer calibration curves were obtained, whichwere used for the molar mass calculation of the fractions.Thus, by combining LC and MALDI/MS detection,complex samples can be analysed with respect to chemicalstructure and molar mass.

Other examples of successful off-line combinationsof LC and MALDI/TOF were given by Kruger et al.,

separating linear and cyclic fractions of polylactidesby LC/CC..117/ Just and Kruger were able to separatecyclic siloxanes from linear silanols and to charac-terize their chemical composition..118/ The calibrationof an SEC system by MALDI/TOF was discussed byMontaudo et al..119/ Polydimethyl siloxane (PDMS) wasfractionated by SEC into different molar mass frac-tions. These fractions were subjected to MALDI/TOFfor molar mass determination. The resulting peak max-imum molar masses were combined with the elutionvolumes of the fractions from SEC to give a PDMScalibration curve log M vs. Ve. The calibration of SECby MALDI/TOF/MS for PMMA, polyvinyl acetate and


n = 9

n = 7

n = 5

n = 9

n = 7

n = 5

n = 5

C13H27O(CH2OCH2O)nH

C15H31O(CH2CH2O)nH

PEG

7

9

57

9

0

300

600

900

1200

t (s)

400 500 600 700

500 600 700

m/z (Da)

Figure 12 Separation of a technical PEO by LC and analysis of fractions by MALDI/TOF, peak assignment indicates degree ofpolymerization n. (Reproduced by permission from Pasch and Rode..116/)

vinyl acetate copolymers has been discussed by Daniset al. In addition to obtaining proper calibration curves,band broadening of the SEC system was detected..120/

The analysis of random copolyesters has been describedrecently by Montaudo et al..121/

To overcome the difficulties of the off-line analysisof SEC fractions, recently interfaces were introducedwhere the SEC effluent was sprayed onto a movingmatrix-coated substrate. Kassis et al. used a modifiedLC-Transform SEC/FTIR interface,.122/ while Nielenapplied a robotic interface of Bioanalytical Instrumentswhere the effluent was spotted on the MALDI target..123/

A novel interface for coupling SEC and MALDI/TOFhas been developed recently by Lab Connections Inc..124/

In this interface, the effluent from the SEC is sprayed

through a heated capillary nozzle continuously on a slowlymoving MALDI target precoated with the appropriatematrix, resulting in a uniform surface layer of samplefraction and matrix. The matrix can be depositedmanually or automatically on the MALDI target froman appropriate solution. When necessary, a salt is addedto the matrix solution.

The characterization of PMMA by SEC/MALDI/TOFis shown in Figure 13..125/ Prior to fraction depositionthe target was precoated with the matrix dithranol anda small amount of LiCl to enhance the formation ofMC LiC molecular ions. Since the fraction depositionwas carried out through a heated capillary nozzle, a solidfraction/matrix film was obtained on the MALDI/TOFtarget. The MALDI/TOF target had a length of 70 mm


3000 5000 7000 9000

5000 7000 9000

7000 9000 11 000

9000 11 000

10 000

4000

12 000

13 00011 000

13 000

11 000

Pulses 351–400

Pulses 151–200

Pulses 551–600

Pulses 751–800

Pulses 951–1000

Pulses 1151–1200

Mass/Charge

Figure 13 MALDI/TOF spectra of PMMA fractions obtained from SEC/MALDI/TOF analysis. (Reproduced by permission fromPasch..125/)

and was scanned continuously with 3500 laser pulses.Every 50 pulses were summarized to give a completeMALDI/TOF spectrum. With SEC as the preseparationtechnique, low positions on the target correspond to highmolar masses, while high positions are equivalent to lowmolar masses. Selected spectra from different positionsof the polymer/matrix track of the PMMA sample aregiven in Figure 13. In the present experiment, a sampleamount of 10 µg (100 µL of a 0.1 mg mL�1 solution) wasinjected into the SEC. An amount of 10% of the totaleffluent was sprayed onto the MALDI target, resultingin a total amount of deposited sample of 1 µg. As canbe seen, for all fractions high quality spectra wereobtained giving the oligomer distributions of the differentfractions.

Depending on the complexity of a specific sam-ple, MALDI/TOF is more or less capable of resolvingdifferent chemical structures. While this technique is

very powerful in determining different endgroups inmacromonomers and telechelics, it has its limitationswhen it comes to analysing copolymers. Due to the factthat the number of possible oligomers increases expo-nentially with the degree of polymerization, even for lowmolar masses very complex product mixtures are obtainedwhich cannot be analysed solely by MALDI/TOF. Inthese cases it is unavoidable to combine a chromato-graphic prefractionation with a MALDI/TOF analysis.The usefulness of such a combination shall be demon-strated for a diblock copolymer of n-butyl methacrylateand methyl methacrylate, i.e. poly-n-butyl methacrylate(PnBMA)-b-PMMA.

The sample under investigation was prepared by grouptransfer polymerization (GTP) resulting in structure (1)which follows and is typical.

Typical spectra for fractions of different molar massesobtained from the SEC/MALDI/TOF experiment are


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Figure 14 MALDI/TOF spectra of fractions obtained from SEC/MALDI/TOF analysis of a PMMA/PnBMA block copolymer.(Reproduced by permission from Pasch..125/)

H CH2 CCH3

COOC4H9

CH2 CCH3

COOCH3

H

X Y

(1)

given in Figure 14. The higher molar mass fractions inFigure 14(a), (b) and (c) are characteristic for copolymerstructures exhibiting typical mass increments of 100 Dafor the MMA repeat unit and 142 Da for the nBMArepeat unit. Even these narrow disperse fractions exhibit amultitude of different mass peaks (usually more than 100)indicating the high complexity of the fractions. The lowermolar mass fraction in Figure 14(d) is very uniform with

respect to composition and thus differs from the molarmass fraction in Figure 14(a), (b) and (c). For the fractionin Figure 14(d), only peak-to-peak mass increments of142 Da were observed which are typically for PnBMA.The chemical composition of the block copolymer wasstudied in detail by analysing the different mass peaks (seezoomed part of the spectrum in the insert of Figure 14b).Each peak in the spectrum could be assigned to oneindividual oligomer composition (nBMA)X(MMA)Y .

Considering the potential of MALDI/TOF in terms ofversatility and sensitivity, the direct interfacing of LC andMALDI/TOF would be a highly attractive possibility.Given the experiences with the direct introduction ofsmall matrix-containing liquid streams into high-vacuum


Solventsyringe pump

Injectionvalve

GPCcolumn

Matrixsyringe pump

TEE

Nebulizer

Reflectron

Detector Oscilloscope

Computer

Nd: yttrium aluminumlaser(a)

51 min

49 min

47 min

45 min

43 min

600 800 1000 1200 1400 1600

(b) m /z (u)

Figure 15 Aerosol MALDI apparatus configured for on-lineSEC/MS (a) and five spectra obtained during the separation ofPEG 1000 (b). GPC, gel permeation chromatography; TEE,T-shaped flow-splitter. (Reproduced by permission from Feiand Murray..128/)

instruments, it took surprisingly long before a devicefor liquid introduction to MALDI was described. Insome recent papers Murray and Russell.126,127/ andFei and Murray.128/ discussed the on-line coupling ofSEC and MALDI/TOF/MS. In an aerosol MALDI/SECexperiment, the effluent from the SEC column wascombined with a matrix solution and sprayed directly intoa TOF/MS. Ions were formed by irradiation of the aerosolparticles with pulsed 355 nm radiation from a frequency-tripled Nd : yttrium aluminum garnet laser. The ionswere mass separated in a two-stage reflectron TOFinstrument, and averaged mass spectra were stored every11 sec throughout the SEC/MS experiment. Well-resolvedMALDI/TOF spectra were obtained from commercialPEG 1000 and poly(propylene glycol) (PPG) 1000, seeFigure 15.

3.4 Coupling with Fourier Transform InfraredSpectroscopy

When analysing a complex polymer, very frequentlythe first step must be the determination of the grosscomposition. Only when the chemical structures ofthe polymer components are known can sophisticatedseparation techniques such as gradient HPLC or LC/CCbe adapted to a specific analysis.

The most frequently used techniques for a ‘‘flash’’analysis are IR spectroscopy and SEC. IR spectroscopyprovides information on the chemical substructurespresent in the sample, while SEC gives a first indicationof the molar mass range. Information on both molarmass and composition is obtained when SEC or acomparable chromatographic method is combined withan IR detector. In the past, numerous workers havetried to use IR detection of the SEC column effluentin liquid flow cells. The problems encountered relateto obtaining sufficient signal-to-noise (S/N) ratio evenwith FTIR instruments, flow-through cells with minimumpath lengths and mobile phases with sufficient spectralwindows. Attempts to use FTIR detection with liquidflow-through cells and high performance columns havenot been very successful due to the requirement ofconsiderably less sample concentration for efficientseparation..129 – 136/

A rather broad applicability of FTIR as a detector inLC can be achieved when the mobile phase is removedfrom the sample prior to detection. In this case the samplefractions are measured in pure state without interferencefrom solvents. Experimental interfaces to eliminatevolatile mobile phases from HPLC effluents have beentried with some success.137 – 139/ but the breakthroughtowards a powerful FTIR detector was achieved only byGagel and Biemann, who formed an aerosol from theeffluent and sprayed it on a rotating aluminum mirror.The mirror was then deposited in an FTIR spectrometerand spectra were recorded at each position in the reflexionmode..140 – 142/

Recently, Lab Connections Inc. introduced the LC-Transform, a direct HPLC/FTIR interface based onthe invention of Gagel and Biemann and discussed itsfirst applications in polymer analysis..143 – 145/ The designconcept of the interface is shown in Figure 16. The systemis composed of two independent modules, the samplecollection module and the optics module. The effluentof the LC column is split with a fraction (frequently10% of the total effluent) going into the heated nebulizernozzle located above a rotating sample collection disc.The nozzle rapidly evaporates the mobile phase whiledepositing a tightly focused track of the solute. When achromatogram has been collected on the sample collectordisc, the disc is transferred to the optics module in the


SpectraSample identification

FTIR-spectrophotometer

Optics module

RI-detector

GPC/HPLC

Collection module

Sample separation

Figure 16 Schematic representation of the principle of coupled LC and FTIR spectroscopy.

FTIR detector for analysis of the deposited sample track.A control module defines the sample collection discposition and rotation rate in order to be compatible withthe run time and peak resolution of the chromatographicseparation. Data collection is readily accomplished withsoftware packages presently used for GC/FTIR. Thesample collection disc is made from germanium whichis optically transparent in the range 6000–450 cm�1. Thelower surface of the disc is covered with a reflectingaluminum layer.

As a result of the investigation a complete FTIRspectrum for each position on the disc and, hence,for each sample fraction is obtained. This spectrumbears information on the chemical composition ofeach sample fraction. The set of all spectra can bearranged along the elution time axis and yields a3-D plot in the coordinates elution time–FTIR fre-quency–absorbance.

One of the benefits of coupled SEC/FTIR is theability to identify directly the individual componentsseparated by chromatography. A typical SEC separationof a polymer blend is shown in Figure 17..146/ Twoseparate elution peaks 1 and 2 were obtained, indicatingthat the blend contained at least two components ofsignificantly different molar masses. A quantificationof the components with respect to concentration andmolar mass, however, could not be carried out aslong as the chemical structure of the components isunknown.

The analysis of the chemical composition of the samplewas conducted by coupled SEC/FTIR using the LC-Transform. After separating the sample with respectto molecular size, the fractions were deposited on thegermanium disc and FTIR spectra were recorded con-tinuously along the sample track. In total, a set ofabout 80 spectra was obtained which was presentedin a 3-D plot, see Figure 18. The projection of the

20 22 24

RI-

resp

onse

1

26 28 30

2

Ve (mL)

Figure 17 SEC separation of a binary blend, stationary phase:Ultrastyragel 2ð linearC 105 A, eluent: THF.

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Figure 18 SEC/FTIR analysis of a binary blend, ‘‘Waterfall’’representation.


0.010 0.015 0.022 0.033 0.049 0.073 0.109 0.162 0.242 0.360 ABS

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1

Figure 19 SEC/FTIR analysis of a binary blend, ‘‘Contour plot’’ representation.

3-D plot on the retention time–IR frequency coordi-nate system yielded a 2-D representation, where theintensities of the absorption peaks were given by acolor code. Such a ‘‘contour plot’’ readily providesinformation on the chemical composition of each chro-matographic fraction, see Figure 19. It was obvious thatthe chromatographic peaks 1 and 2 had different chem-ical structures. By comparison with reference spectrawhich are accessible from corresponding data bases, com-ponent 1 could be identified as PS, while component 2was polyphenylene oxide. With this knowledge, appro-priate calibration curves could be used for quantifyingthe composition and the component molar masses of theblend.

Coupled SEC/FTIR becomes an inevitable tool whenblends comprising copolymers have to be analysed. Veryfrequently components of similar molar masses are used inpolymer blends. In these cases the resolution of SEC is notsufficient to resolve all component peaks: see Figure 20 for

a model binary blend containing an additive. The elutionpeaks of the polymer components 1 and 2 overlappedand, thus, the molar masses could not be determineddirectly. Only the additive peak 3 at the low molar massend of the chromatogram was well separated and couldbe quantified.

A first indication of the composition of the presentsample could be obtained from the contour plot inFigure 21. Component 3 showed typical absorption peaksof a phenyl benzotriazole and could be identified asa UV stabilizer of the Tinuvin type. Component 2exhibited absorption peaks which were characteristicfor nitrile groups and styrene units, while component 1showed a strong ester carbonyl peak and peaks ofstyrene units. In agreement with the peak patternof literature spectra, component 2 was identified asstyrene–acrylonitrile (SAN) copolymer. Component 1could have been a mixture of PS and PMMA or astyrene–methyl methacrylate copolymer. Since the FTIR


20

1

2

3

22 24

Ve (mL)

Rl-r

espo

nse

26 28 30

Figure 20 SEC separation of a blend of two copolymers andan additive, chromatographic conditions (see Figure 17).

spectra over the entire elution peak were uniform, it ismore likely that component 1 was a copolymer.

One important feature of the SEC/FTIR software isthat from the contour plot specific elugrams at one absorp-tion frequency can be obtained. Taking the elugram at2230 cm�1, which is specific for the nitrile group, theelution peak of the SAN copolymer could be presentedindividually. For the presentation of component 1 theelugram at the carbonyl absorption frequency was drawn.Thus, via the ‘‘chemigram’’ presentation the elution peakof each component is obtained, see Figure 22.

In a relatively short period of time the LC-Transform

system found its way into a large number of laboratories.Applications of the technique have been discussedin various fields. Willis and Wheeler demonstratedthe determination of the vinyl acetate distributionin ethylene–vinyl acetate copolymers, the analysis ofbranching in high-density polyethylene, and the analysisof the chemical composition of a jet oil lubricant..147/

Provder et al..148/ showed that in powder coatings alladditives were positively identified by SEC/FTIR throughcomparison of the known spectra. Even biocides couldbe analysed in commercial house paints. The comparison

3

2

1

20

3000

0.010 0.015 0.021 0.031 0.045 0.066 0.097 0.141 0.206 0.300 ABS

2500 2000

Wavenumber (cm−1)1500 1000

22

24

26

28

30

Tim

e (m

in)

Figure 21 Contour plot of the SEC/FTIR analysis of a blend of two copolymers and an additive.


200

5

10

15

20

25

30

Chemigram: 3103−2817 cm−1 (Total)Chemigram: 1761− 1662 cm−1 (PS-b-PMMA)Chemigram: 2242−2228 cm−1 (SAN)Chemigram: 808−804 cm−1 (Tinuvin 326)

35

22 24

Retention time (min)

Inte

nsity

26 28 30 32

Figure 22 Chemigrams taken from the contour plot inFigure 21.

of a PS/PMMA blend with a corresponding copolymergave information on the chemical drift. In the analysisof a competitive modified vinyl polymer sample bySEC/FTIR some of the components of the binder couldbe identified readily (vinyl chloride, ethyl methacrylateand acrylonitrile), and an epoxidized drying oil additivewas detected..148/ The analysis of styrene–butadienecopolymers by combining interaction chromatographyand FTIR has been demonstrated..149,150/ By using LC/CCit was possible to separate block copolymers and technicalrubber mixtures with respect to chemical composition.The determination of the styrene : butadiene ratio and thefine structure of the butadiene units (cis/trans-, 1,2/1,4-units) was achieved by FTIR spectroscopy.

The quality of the results from SEC/FTIR stronglydepend on the surface quality of the deposited samplefractions. Cheung et al. demonstrated that the surface-wetting properties of the substrate dominate the depositmorphology.151/ and the spectra fidelity, film quality,resolution and polymer recovery were considered..152/

For different interface designs it was found that themorphology of the deposited polymer film was a keyparameter for quantitative measurements.

3.5 Coupling with Nuclear Magnetic ResonanceSpectroscopy

NMR spectroscopy is by far the most powerful spec-troscopic technique for obtaining structural informationabout organic compounds in solution. Its particularstrength lies in its ability to differentiate between moststructural, conformational and optical isomers. NMRspectroscopy can usually provide all necessary informa-tion to identify unambiguously a completely unknown

compound. The NMR detection technique is quantitativewith individual areas in spectra being proportional to thenumber of contributing nuclei. The major drawback ofNMR is the relatively low sensitivity in comparison to MS,another is the fact that structure elucidation of mixturesof unknown compounds with overlapping NMR signalsis difficult and may be nearly impossible in cases withovercrowding signals in a small chemical shift region ofthe NMR spectrum. Therefore, in many cases it would beuseful that a separation is performed prior to the use ofNMR. For more efficient procedures, a direct coupling ofseparation with NMR detection would be the method ofchoice..153/

The direct coupling of LC with proton NMR hasbeen attempted numerous times. Early experiments ofcoupled HPLC-1H-NMR were conducted in a stop-flowmode or with very low flow rates..154 – 156/ This wasnecessary to accumulate a sufficient number of spectra persample volume in order to improve the S/N ratio. Otherproblems associated with the implementation of on-line HPLC/NMR have included the need for deuteratedsolvents. However, with the exception of deuterium oxidethe use of deuterated eluents is too expensive for routineanalysis. Therefore, proton-containing solvents such asacetonitrile (ACN) or methanol must be used. To get ridof the solvent signals in the spectra, the proton NMRsignals of the solvents have to be suppressed.

Recent rapid advances in HPLC/NMR provide evi-dence that many of the major technical obstacles havebeen overcome..157,158/ With the development of morepowerful NMR spectrometers combined with new NMRtechniques for solvent suppression it became much easierto obtain well-resolved spectra in an on-flow mode. Inparticular, very efficient solvent-suppression techniquessignificantly improved the spectra during the HPLC/NMRrun..159,160/ These techniques combine shaped radio fre-quency pulses, pulsed-field gradients, and selective 13Cdecoupling to acquire high-quality spectra at on-flow con-ditions even with high HPLC gradients. Recently, eventhe direct coupling of supercritical fluid chromatography(SFC) with 1H-NMR.161 – 163/ together with the monitoringof supercritical fluid extraction.164/ as well as the couplingof CE and 1H-NMR.165 – 167/ have been reported. Anoverview of the applications of on-line HPLC-1H-NMRin organic chemistry was given by Albert..153/

The first steps of polymer analysis into coupled LC-1H-NMR were performed by Hatada et al..168/ Theylinked a size exclusion chromatograph to a 500 MHzproton NMR spectrometer and investigated isotacticPMMA. Using deuterated chloroform as the eluentand running the chromatography at a rather low flow-rate of 0.2 mL min�1 they were able to accumulate wellresolved proton spectra. From the intensities of the protonsignals of the endgroups and the monomer units they


determined the number-average molar mass across theelution curve. In further investigations they developed anabsolute calibration method for direct determination ofmolar masses and MMDs by on-line SEC-1H-NMR..169/

Ute reported on the chemical composition analysis ofEPDM copolymers as a function of molar mass, and themonitoring of stereocomplex formation for isotactic andsyndiotactic PMMA..170/

The analysis of a technical PEO with respect tochemical composition and degree of polymerizationhas been performed by Pasch and Hiller..171/ Thisinvestigation has been conducted under conditions whichare common for HPLC separations, i.e. sufficiently highflow-rate, moderate sample concentration, and on-flowdetection. Using an octadecyl-modified silica gel as thestationary phase and an eluent of ACN–deuterium oxide50 : 50 (v/v) the sample was separated into differentfunctionality fractions, see Figure 23. The major fractionof the sample eluting between 14 and 25 min exhibited apartial oligomer separation.

For structural identification of the fractions, the 1H-NMR spectrometer was directly coupled via capillarytubing to the HPLC system. The injection of the sampleinto the HPLC system was automatically initiated bythe NMR console via a trigger pulse when starting toacquire NMR data. Using an appropriate pulse sequence,

both solvent resonances (ACN at 2.4 ppm and waterat 4.4 ppm) could be suppressed simultaneously. As aresult of the on-line HPLC/NMR experiment a contourplot of 1H chemical shift vs retention time could begenerated, see Figure 24. Owing to the efficient solventsuppression, the obtainable structural information relatesto the entire chemical shift region. From the contour plot,four different elution peaks could clearly be identifiedand analysed with respect to chemical composition. Theremarkable feature of this investigation was that even thelow concentration components in peaks 1–3 could clearlybe identified in the contour plot.

Detailed structural information could be obtained fromthe individual NMR spectra of the fractions at the peakmaximum, see Figure 25. This also gave the relevantstructures (2 and 3). The first peak was identified as beingPEG while the other fractions were alkylphenoxy PEOs.From the intensities of the endgroups and the ethyleneoxide repeat units the average degree of polymerizationfor each fraction was calculated. Based on the totalintensity distribution, a calculated chromatogram (orchemigram) was generated from the NMR contourplot. Comparing the real chromatogram (Figure 23) withthe chemigram (Figure 24) an excellent agreement wasobtained even recalling the oligomer separation patternof the major fraction.

0.8

0.6

0.4

0.2

0.050 10 15 20 25

1.65

91.

238

2.68

53.

208

5.14

0

8.07

47.

796

10.1

629.

903

10.4

37

15.0

3315

.341

15.7

6116

.263

16.8

4917

.578

18.2

7819

.110

20.0

53

21.4

12

23.1

61

24.9

16

26.1

44

UV

-res

pons

e


Figure 23 HPLC chromatogram of a technical PEO. Stationary phase: RP-18; eluent: ACN–deuterium oxide 50 : 50 (v/v).(Reproduced by permission from Schlotterbeck et al..171/)


8 7 6 5 4 3 2 1

Ret

entio

n tim

e (s

)

400

800

1200

1600

2000

δ (ppm)

4

3

2

1

ACN

12

34

Figure 24 Contour plot of chemical shift vs. retention time and chemigram of the on-line HPLC/NMR analysis of a technical PEO.(Reproduced by permission from Pasch and Hiller..171/)

The analysis of FAE based surfactants by on-line HPLC-1H-NMR has been described by Schlotter-beck et al..172/ Using a reversed stationary phase andACN–deuterium oxide as the eluent, surfactant mix-tures were separated with respect to the fatty alcoholendgroups. 1H-NMR detection revealed the number ofcomponents, the chemical structure of the components,endgroups, and the chain length.

Finally, the investigation of the tacticity of oligostyrenesby on-line HPLC-1H-NMR has been reported by Paschet al..173/ The oligomer separation was carried out byhydrophobic interaction chromatography using isocraticelution with ACN on a reversed phase (RP) columnRP-18. The chromatogram of an oligostyrene is shown inFigure 26. The first oligomer peak could be identified asbeing the dimer (n D 2), the next peak was identified asthe trimer (n D 3) and, accordingly, the following peakscould be assigned to the tetramer, pentamer etc. Thedimer peak appeared uniform, whereas for the followingoligomers a splitting of the peaks was obtained. For n D 3and n D 4 a splitting into two peaks was observed. Forn D 5 and further, a splitting into three or more peaksoccurred, which could be attributed to the formation ofdifferent tactic isomers.

The analysis of the isomerism of the oligomers byHPLC/NMR is given in Figure 27..173/ In this experimentconventional HPLC grade ACN was used as the eluentand no deuterium lock was applied. These conditionsrequired high stability of the NMR instrument and avery efficient solvent-suppression technique since 100%

ACN must be suppressed. The obtainable structuralinformation related to the entire chemical shift region:however, residual signals of the eluent were obtained at1.8–2.4 ppm and 1.3 ppm due to ACN and its impurities.The contour plot clearly revealed two signal regions,which could be used for analysis. These were the regionof the methyl protons of the sec-butyl endgroup at0.6–0.8 ppm and the aromatic proton region of thestyrene units at 6.5–8.0 ppm. For the generation ofthe contour plot every 8 seconds a complete spectrumwas produced by co-adding 8 scans. Accordingly, forthe structural analysis 128 spectra were available overthe entire retention time range. For the analysis of aseparated oligomer, a minimum of four spectra could beused. These spectra bear selective information on thetacticity, even without completely separating the tacticisomers chromatographically.

As has been shown recently by Kramer et al., on-line coupled SEC-1H-NMR can be used to monitor thechemical composition of random copolymers across themolar mass axis..174/ They investigated high-conversionpoly(styrene-co-ethyl acrylate)s using dichloromethaneas the solvent for SEC. The contour plot for a typicalsample with a styrene ethyl acrylate (EA) ratio of40 : 60 indicates that all characteristic spectral regions areaccessible for analysis: see Figure 28. Residual solventsignals at 5.0–5.5 ppm do not overlap with resonancesof the polymer molecules. The chemical compositionacross the elution peak of the copolymers is shownin Figure 29. The data were calculated from the peak


12345678

(a)

(b)

(c)

(d)

δ (ppm) δ (ppm)

δ (ppm) δ (ppm)12345678 12345678

12345678

HO(CH2CH2O)nH

O(CH2CH2O)nH

CH3– CH3

CH3

CH3 C

–CH2O –

–CH2OH

H2O

ACN

××

3

4

21

Ar−H

76 5

1 2 3 4

7

3

4

6

5

21

Ar−H

CH2CCCH3

CH3

CH3

CH3

CH3

OCH2 CH2O (CH2CH2O)m CH2 CH2OH

Figure 25 Individual fraction spectra taken from Figure 24. (a) NMR spectrum obtained from peak 1 in Figure 24; (b) NMRspectrum from peak 2 in Figure 24; (c) NMR spectrum from peak 3 in Figure 24; (d) NMR spectrum from peak 4 in Figure 24.

areas of the aromatic protons (d D 6.5–7.3 ppm) andthe oxymethylene protons (d D 3.5–4.2 ppm) which wererecorded during the SEC/NMR experiment. Owing tothe low S/N ratio at the start and the end of the SECelution curves, the chemical composition determinationis less accurate than in the peak maximum. As can beseen, most of the copolymers exhibit constant chemicalcomposition across the elution curves. This correspondsto the average chemical composition of the bulk sample.An exception is the EA-richest copolymer (styrene-EA10.95), where at the high molar mass end of the elutioncurve an increased EA content is detected. At the sametime, at the low molar mass end of the elution curve astrong tailing and an increased EA content is obtained.This strongly indicates that at high concentrations of EAin the monomer feed, copolymers with increased chemicaland molar mass heterogeneity are obtained.

4 MULTIDIMENSIONAL LIQUIDCHROMATOGRAPHY

4.1 Introduction

Despite the fact that substantial progress has beenachieved in recent years in size-exclusion and interactionmodes of polymer chromatography, the need and use formultidimensional separation systems has increased. Themain reason for that is the fact that nowadays most classesof macromolecules posses property distributions in morethan one parameter (e.g. molar mass and chemical compo-sition at the same time). It is obvious that n independentproperties require n-dimensional methods for accurate(independent) characterization of all those parameters.Moreover, the separation efficiency of any single separa-tion method is limited by the efficiency and selectivity of


3.0

2.5

2.0

1.5

1.0

0.5

0

0 5 10 15


UV

(26

0nm

)

20 25

2.47

32.

611

2.28

72.

869

3.38

33.

635

4.07

74.

998

6.04

26.

192

7.80

9

11.5

4912

.747

12.8

85

10.8

6910

.324

8.67

98.

823

8.17

4

13.2

07

4.39

3

5.50

45.

339

6.54

8 6.70

26.

795

Figure 26 HPLC chromatogram of an oligostyrene PS 530. Sta-tionary phase: RP-18; eluent: ACN. (Reproduced by permissionfrom Pasch et al..173/)

80

100

200

300

400

500

600

700

800

900

1000

7 6

F2 (ppm)

Ret

entio

n tim

e (s

)

5 4 3 2 1 0

Heptamer

Hexamer

Pentamer

TetramerTrimerDimer

Figure 27 Contour plot of chemical shift vs retention time ofthe on-line HPLC/NMR analysis of PS 530. (Reproduced bypermission from Pasch et al..173/)

the separation mode, i.e. the plate count of the columnand the phase system selected. Adding more columns willnot overcome the need to identify more components ina complex sample, due to the limitation of peak capaci-ties. The peak capacity in an isocratic separation can bedescribed, following Grushka,.175/ as in Equation (19):

n D 1Cp

N4

lnVp

V0.19/

The corresponding peak capacity in an n-dimensionalseparation is considerably higher due to the fact that

7 6 5 4 3 2 1

0

44

88

ppm

min

Figure 28 Contour plot of chemical shift vs retention timeof the on-line SEC/NMR analysis of a random copolymer ofstyrene and EA. (Reproduced by permission from Krameret al..174/)

each dimension contributes to the total peak capacity asa factor and not as an additive term for single dimensionmethods as described in Equation (20):

ntotal D∏

ni sin.i�1/ #i .20/

where ntotal represents the total peak capacity, ni thepeak capacity in dimension i and #i is the ‘‘angle’’between two dimensions. The angle between dimensionsis determined by the independence of the methods; a 90°angle is obtained by two methods, which are completelyindependent of each other and will, for example, separatetwo properties solely on a single parameter withoutinfluencing themselves.

In the case of a 2-D system the peak capacity is givenby Equation (21):

n2D D n1n2 sin#

D(

1Cp

N1

4ln

Vp,1

V0,1

)(1Cp

N2

4ln

Vp,2

V0,2

)sin#

.21/This effect is schematically illustrated in Figure 30.

Multidimensional chromatography separations canbe done in planar systems or coupled-column sys-tems. Examples of planar systems include 2-D thin-layer chromatography (TLC),.176,177/ where succes-sive 1-D TLC experiments are performed at 90°angles with different solvents, and 2-D electrophore-sis, where gel electrophoresis is run in the firstdimension followed by isoelectric focusing in thesecond dimension..178 – 180/ Hybrids of these systemswhere chromatography and electrophoresis are used ineach spatial dimension were reported nearly 40 yearsago..181/

The main problem using planar methods is the difficultyin detection and collection of fractions among other

COUPLED LIQUID CHROMATOGRAPHIC TECHNIQUES IN MOLECULAR CHARACTERIZATION 27In

tens

ity P

DA

EA

in c

opol

ymer

(m

ol-%

)14

(c)

(b)

(a) 16

SEA 10.95

SEA 40.98

18 20 22 24 2650

60

70

80

90

100

12 14 16 18 20 22 28262450

60

70

80

90

100

SEA 90.99


14 16 18 20 22 24 2826

0

10

20

30

40

Figure 29 Chemical composition as a function of molar massfor random styrene–EA copolymers of different average com-position. (Reproduced by permission from Kramer et al..174/)

less critical problems, such as homogeneous preparationof chromatographic media. However, the detectionproblem exists also for the coupled-column methods,mainly because of fraction dilution by each stage in amultidimensional separation system. Another aspect isthe adjustment of chromatographic time bases betweenthe different dimensions so that first dimension peaksmay be sampled an adequate number of times by thenext dimension separation system. This aspect has beenrecently studied in detail..182/

In 2-D column chromatography systems an aliquotfrom a column or channel is transferred into the nextseparation method in a sequential and repetitive manner.Storage of the accumulating eluent is typically providedby sampling loops connected to an automated valve.Many variations on this theme exist which use variouschromatographic and electrophoretic methods for one ofthe dimensions. In addition, the simpler ‘‘heart cutting’’mode of operation takes the eluent from a first dimensionpeak or a few peaks and manually injects this into anothercolumn during the first dimension elution process. Apartial compilation of these techniques has been given inseveral places..182 – 190/

The use of different modes of LC facilitates the sep-aration of complex samples selectively with respect todifferent properties like hydrodynamic volume, molarmass, chemical composition or functionality. Using thesetechniques in combination, multidimensional informa-tion on different aspects of molecular heterogeneitycan be obtained. If, for example, two different chro-matographic techniques are combined in a ‘‘cross-fractionation’’ mode, information on CCD and MMDcan be obtained. Literally, the term ‘‘chromatographiccross-fractionation’’ refers to any combination of chro-matographic methods capable of evaluating the distribu-tion in size and composition of copolymers. An excellentoverview on different techniques and applications involv-ing the combination of SEC and gradient HPLC waspublished by Glockner..60/

In SEC mode the separation occurs according to themolecular size of a macromolecule in solution, whichis dependent on its chain length, chemical composition,solvent and temperature. Thus, molecules of the samechain length but different composition may have differenthydrodynamic volumes. Since SEC separates accordingto hydrodynamic volume, SEC in different eluents canseparate a copolymer in two diverging directions. Thisprinciple of ‘‘orthogonal chromatography’’ was suggestedby Balke and Patel..191 – 193/ The authors coupled two SECinstruments together so that the eluent from the first oneflowed through the injection valve of the second one. Atany desired retention time the flow through SEC 1 couldbe stopped and an injection made into SEC 2. The firstinstrument was operated with THF as the eluent and PSgel as the packing, whereas for SEC 2 polyether bonded-phase columns and THF–heptane were used. Bothinstruments utilized SEC columns. However, whereasthe first SEC was operating so as to achieve conventionalmolecular size separation, the second SEC was used tofractionate by composition, utilizing a mixed solvent toencourage adsorption and partition effects in addition tosize exclusion. Consequently, independent informationon both MMD and CCD could not be obtained from suchan experiment.


Resolution enhancement by 2-D separation

2. Dimension peak capacity: 3

1. D

imen

sion

pea

k ca

paci

ty:

4

2-D peak capacity: 12

ϑ

Figure 30 Schematic contour map representation of increased resolution and peak capacity in 2-D separations (peaks in eachdimension are indicated by bars at the axes).

Since ‘‘orthogonality’’ requires that each separationtechnique is totally selective towards an investigatedproperty, it seems to be more advantageous to usea sequence of methods, in which the first dimensionseparates according to chemical composition. In this wayquantitative information on CCD can be obtained andthe resulting fractions eluting from the first dimension arechemically homogeneous. These homogeneous fractionscan then be analyzed independently in SEC modein the second dimension to get the required MMDinformation. In such cases, SEC separation is strictlyseparating according to molar mass, and quantitativeMMD information can be obtained.

Examples illustrating the potential of multidimensionalseparations will be given in section 4.5.

4.2 Experimental Aspects of MultidimensionalSeparations

Setting up a 2-D chromatographic separation systemis actually not as difficult as one might think at first.As long as well-known separation methods exist foreach dimension the experimental aspects can be handledquite easily in most cases. Off-line systems just requirea fraction-collection device and something or someonewho reinjects the fractions into the next chromatographicdimension. In on-line 2-D systems the transfer of fractionsis preferentially done by automatic injection valves as wasproposed by Kilz et al..23,188,194/ Figure 31 shows a generalset-up for an automated 2-D chromatography system.

The focal point in 2-D chromatography separations isthe transfer of fractions eluting from the first dimensioninto the second dimension. This can be done in variousways. The most simplistic approach is by collecting

Column 1

Col

umn

2

Detector 1

Detector 2

Inj1 TV To waste

To waste

First dimension(horizontal)

TV: transfer valve

Solventdeliverysystem 2

Solventdeliverysystem 1

Sec

ond

dim

ensi

on (

vert

icta

l)Figure 31 General experimental set-up for an 2-D chromatog-raphy system.

fractions from one separation and manually transferringthem into the second separation system. Obviously,this approach is prone to many errors, labor intensiveand quite time-consuming. A more efficient way offraction transfer can be achieved by using electrically(or pneumatically) actuated valves equipped with twoinjection loops. Such a set-up allows one fraction tobe injected and analyzed from one loop while the nextfraction is collected at the same time in the second loop(see Figure 32). Total mass transfer from the first to thesecond dimension can be guaranteed by proper selectionof flow rates in both dimensions..195/ This is a verybeneficial situation as compared to heart-cut transfers,since by-products and trace-impurities can be separatedeven if they are not visible (VIS) in the first dimensionseparation. Table 3 shows a summary of potential fractiontransfer options.


HP

LC

SE

C

HP

LC

SE

C

THF

Waste

THF

Waste

Position INJECT

Position LOAD

Figure 32 Fraction transfer between chromatography dimen-sions using a dual-loop 8-port valve.

There are some other important aspects which have tobe considered for optimum 2-D experiment design.

4.2.1 Selection of Separation Techniques

Despite the fact that, historically, planar chromatographyplayed an important role in multidimensional separations,

this article will not discuss these aspects because theyrepresent the past. The foreseeable future is with column-based techniques, which allow a well-controlled transferof samples between different methods.

Obviously, destructive methods like GC and SFC,which destroy the chromatographic phase system, playa more limited role in multidimensional separations asthey can only be used in the last separation step.

Schure recently published a theoretical paper.196/ whichdiscussed different chromatographic method combina-tions on the basis of efficiency, sample dilution anddetectability. He investigated CE, GC, LC, SEC andfield-flow fractionation (FFF) in detail, while omittingother methods, which are potential candidates for methodhyphenation, such as SFC and temperature-rising elutionfractionation (TREF).

Schure highlights several universal experimental fac-tors (including plate count, injection volume, injectedmass and injection band dilution), which should betaken into account when designing multidimensionalseparations. Table 4 summarizes Schure’s results forthe applicability of a given method in a multidimen-sional experiment. It is obvious that a low resolution,low injected mass method with high dilution of the

Table 3 Summary of 2-D transfer injection options

Transfer Mode Advantages Disadvantages Example

Manual Off-line Very simpleFast set-up

Time-consumingNot for routine workNot preciseNo correlation of fraction

elution to transfer timeNot quantitative

Test tube

Automatic Off-line SimpleEasyFast set-up

Less preciseNo correlation of fraction

elution to transfer timeNot quantitative

Fraction collectorstorage valve

Single-loop On-line Correct concentrationsCorrect transfer timesAutomation

Transfer not quantitativeSet-up time

Injection valve (withactuation)

Dual-loop On-line Correct concentrationsCorrect transfer timesQuantitative transfer

automation

Set-up timeSpecial valve

8-port actuated valveCombination of 2

conventional 6-portinjection valves

Table 4 Synopsis of typical conditions and dilution factors in 1-D separations (Schure.196/)

Separation Experimental parameters Resultmode dimensions (mm) dp (µm) N (1 col�1) Vinj (µl) Dilution f

GC 2500ð 0.25 n/a 125 000 1 26.2LC 250ð 4.6 5 10 000 10 25.1SEC 300ð 8.0 5 10 000 100 6.54FFF 600ð 2ð 0.002 n/a 651 2 1340CE 400ð 0.05 n/a 100 000 4.9e�4 12.7


Table 5 Calculated dilution factors for 2-D separations(Schure.196/) assuming splitless transfer injections(experimental conditions similar to those of Table 4)

2-D GC LC SEC FFF CEa

1-D

GC 226 n/a n/a n/a n/aLC 217 141 113 4120 43.7SEC 56.4 36.6 29.4 1070 11.4FFF 11 600 7500 6030 220 000 2230CEb 28 18.2 14.6 533 5.7

a Splitless transfer injections very difficult.b Transfer concentrations very small.

injection band is a poor candidate for a multidimensionalexperiment.

Schure also calculated parameters to estimate thepotential of 2-D method combinations. Results for split-less transfer injections are given in Table 5. CE, LCand SEC were rated best. This theoretical result agreesvery well with experimental results and the actual num-ber of published papers on 2-D separations. The mostwidely used method combination currently is that of LCwith SEC.

4.2.2 Sequence of Separation Methods

This is an important aspect in order to get the bestresolution and most accurate determination of propertydistributions. It is advisable to use the method with highestselectivity for the separation of one property as the firstdimension. This ensures the highest purity of elutingfractions being transfered into the subsequent separation.In the case of gradient HPLC and GPC as separationmethods, authors of early publications.191 – 193,197,198/ usedGPC as the first separation, because it took much longerthan a subsequent HPLC analysis. This is not the bestset-up, however, because the GPC fractions are onlymonodisperse in hydrodynamic volume, not in molarmass, chemical composition, etc. On the other hand,HPLC separations can be fine-tuned using gradients tofractionate only according to a single property, which canthen be characterized for molar mass without any bias.

In many cases, interaction chromatography as thefirst dimension separation method is the best and mostadjustable choice. From an experimental point of view,high flexibility is required for the first chromatographicdimension. In general, this is also more easily achievedwhen running the interaction chromatography mode inthe first dimension, because:

(i) More parameters (mobile phase, mobile phasecomposition, mobile phase modifiers, stationaryphase, temperature etc.) can be used to adjust the

separation according to the chemical nature of thesample.

(ii) Better fine-tuning in interaction chromatographyallows for more homogeneous fractions.

(iii) Sample load on such columns can be much higheras compared to SEC columns, for instance.

4.2.3 Detectability and Sensitivity in the SecondDimension

Because of the consecutive dilution of fractions,detectability and sensitivity become important criteriain 2-D experiment design. If byproducts and trace impu-rities have to be detected, only the most sensitive and/orselective detection methods can be employed. Evapora-tive light scattering detection (ELSD), despite severaldrawbacks, has been used mostly due to its high sensitiv-ity for compounds which will not evaporate or sublimeunder detection conditions. Fluorescence and diode arrayUV/VIS are also sensitive detection methods, which canpick up samples at nanogram level. Mass spectrometershave a high potential in this respect too: however, theyare currently not developed to a state where they wouldbe generably usable.

Only in rare cases has RI detection, otherwise very pop-ular in SEC, been used in multidimensional separations,because of its low sensitivity and strong dependence onmobile phase composition.

As a general rule, the higher the inject band dilutionof a given separation method the more sensitive asubsequent detection method has to be. Such type ofmodel calculations can be done easily; refer to Table 4 insection 4.2.1 and the paper by M. Schure.196/ for furtherdetails.

4.2.4 Other Experimental Factors AffectingMultidimensional Separations

Depending on the specific type of the multidimensionalexperimental set-up, there are a number of otherparameters to control and care about. Some are listedhere, but because they are specific to the methodcombination, this list reflects only those techniques inmost common use.

4.2.4.1 Influence of Eluent Transfer from First to SecondDimension A very important aspect in multidimen-sional chromatography design is the compatibility ofmobile phases which are transferred between the dif-ferent dimensions. It is a necessity that the mobile phasesin two consecutive stages in multidimensional separationsare completely miscible. Otherwise the separation in thesecond method is dramatically influenced and the fractiontransfer is restricted or completely hindered. In gradient


systems, this requirement has to be verified for the totalcomposition range.

In SEC separations the transfer of mixed mobilephases can affect molar mass calibration. In order toget proper molar mass results, the calibration curveshave to be measured using the extremes of mobile phasecomposition and tested for changes in elution behaviorand pore-size influence in the SEC column packing. Thebetter the thermodynamic property of the SEC eluent,the less influence is expected on the SEC calibration whenthe transfer of mobile phase from the previous dimensionoccurs. It has been shown to be advantageous to use theSEC eluent as one component of the mobile phase in theprevious dimension to avoid potential interference andmobile phase incompatibility.

4.2.4.2 Time Consumption Time is an importantissue when designing multidimensional experiments. Set-up time itself plays only a lesser role, but the time neededfor the multidimensional separations themselves can beconsiderable. This is especially true for 2-D separationsusing quantitative mass transfer via tandem-loop transfervalves. Heart-cut experiments require much less timeand are often sufficient to check out the applicabilityof the approach. Cutting down on time consumptionfor multidimensional experiments is currently a heavilyinvestigated topic. Several approaches are investigatedand allow investigators to be optimistic and reduceexperiment times by a factor of about 10 for completemass transfer experiments using optimized column setsand flow conditions.

Another time requirement in multidimensional sep-arations is that needed to reduce the amount of dataand present them in an instructive way. With severaldozen transfers between dimensions, data reduction andpresentation can be very time-consuming and has beena real burden for those who performed the first cross-fractionation experiments..191 – 193,198/ There is a clearneed for specialized multidimensional software whichdoes all the data acquisition, fraction transfer, valveswitching, data reduction, data consolidation and pre-sentation of results. Currently, there is only one 2-Dchromatography system commercially available.23,199/

which is widely used. A few laboratories use in-housesolutions, which are specific to their own chromatogra-phy and data capture hardware and specific also to resultcalculation and report creation.

4.3 Separation Techniques for the First Dimension

For an in-depth description of individual separationtechniques used for multidimensional separations, pleaserefer to the respective sections in this encyclopedia. Thischapter deals with the specific aspects of the separation

methods, which will help the reader to understand howto select one of them for a given multiple separationexperiment.

4.3.1 Liquid Chromatography

This is the most often used technique for multidimen-sional separations. LC can be performed in normal phaseor RP systems using isocratic or gradient elution. Thereis an abundance of stationary phases with different typesof surface modifications of different polarities. This flex-ibility in experimental parameters is a very importantconsideration when using LC as a first dimension method,since it can be fine-tuned to separate according to a givenproperty more easily than most other chromatographictechniques.

Gradient HPLC has been useful for the characteriza-tion of copolymers..200 – 204/ In such experiments carefulchoice of separation conditions is imperative. Otherwise,low resolution for the polymeric sample will obstructthe separation. On the other hand, the separation inHPLC, dominated by enthalpic interactions, perfectlycomplements the entropic nature of the SEC retentionmechanism in the characterization of complex polymerformulations.

LC separation is based on an enthalpic interactionbetween the solute and the ‘‘surface’’ of the stationaryphase. In pure interactive LC separations entropiccontributions to the retention are absent. The distributioncoefficient Kd can be derived from basic thermodynamicsand can be measured from the activity of the analyte inthe mobile (am) and stationary (as) phase as shown inEquation (22):

Kd.LC/ D as

amD e�

HRT .22/

The enthalpy change of the analyte corresponds todispersion, polarization and charge-transfer interactionsas well as H-bonding and ion exchange. Obviously,the distribution coefficient is larger than unity in LCseparations.

The retention in pure LC separations can be calculatedaccording to Equation (23):

Ve D V0 CKd.LC/Vpore .23/

The absence of entropic contributions to the separationis only possible if the stationary phase consists ofnonporous beads or if the analyte molecules cannotpenetrate into any pore in the stationary phase becauseof their size or interaction energy (e.g. ionic repulsion). Ingeneral, it will not be possible to avoid entropy changes inLC experiments with samples of different molar massesor sizes. In such cases it is best to select either a columnwhich has very small or very large pores, which will force


the molecules to be excluded totally from the stationaryphase or to permeate totally the pores of the packing. Inboth situations entropic contributions to the separationcan be minimized.

In the general case, the distribution coefficient can bewritten as in Equation (24):

Kd D as

amD e�

GRT D e�

HRT e

SR

D Kd.LC/Kd.SEC/ .24/

The elution in mixed-mode LC separations can becalculated according to Equation (25):

Ve D V0 C [Kd.LC/Kd.SEC/]Vpore .25/

This equation describes the general behavior of solutesin porous stationary phases. However, the dominantseparation mechanism in LC with entropic contributionsis the enthalpy term.

It is interesting to study the retention dependence onmolar mass in LC separations. This is especially impor-tant when applying this technique to macromolecules,where chain statistics, chemical composition and molec-ular size play an important role in chromatographicbehavior. Figure 33 shows the adsorption characteris-tics of molecules of different chain length or molar mass.The adsorption process is determined by the interactionenthalpy which itself is governed by active sites on theanalyte molecule. If molecules are small only very fewinteractive sites are present, which may differ in natureand possess different interaction energies. In such cases,retention is relatively small (Kd less than about 10). Largermolecules, especially macromolecules, are composed ofrepeating units (usually called monomers), which cantotally adsorb on the column packing. This is due tothe fact that each and every repeating unit can potentially

Retention

Complete adsorption

Log

M

Figure 33 Adsorption of macromolecules of different molarmass on interactive LC columns.

interact with the chromatographic surface. The longer thechains get, the higher the total interaction enthalpy andthe higher the retention time to elude from the column(see Figure 33).

The statistics of chain adsorption are determined bythe magnitude of the entropy loss of the adsorbedmacromolecule. If the entropy loss of the chain issmall as compared to the adsorption enthalpy gain,the molecules will completely attach to the surfaceand the Gaussian chain will collapse into a 2-D layer.This scenario will dominate in cases where the solutescontain functional groups with strong interactions andwhere the eluent strength is relatively poor. In caseswhere the adsorption energy is relatively small, theentropy loss due to adsorption can be larger than theenthalpy gain. The chain only attaches selectively atthe surface on the column packing forming loops. Insuch cases the macromolecules can desorb again. Theadsorption/desorption process can be controlled by thenature of the stationary phase or even more easily by thethermodynamic properties of the eluents and gradientcomposition. Recently, temperature was also used tomoderate adsorption behavior in chromatography..205/

Details of chain statistics in adsorption chromatographycan be found in the book by Glockner..206/

4.3.2 Liquid Chromatography at the Critical Point ofAdsorption

In LC and SEC chromatography modes either theenthalpy or the entropy dominate the separation. Severalyears ago Russian scientists found that homopolymersof different molar mass show exactly identical retentionbehavior on TLC plates.207,208/ and on silica columns,.209/

if a special eluent mixture was used for that macro-molecule. They found that under ‘‘critical’’ conditions thesorbent did not ‘‘see’’ the polymeric nature of the chain.The separation was dependent only on the enthalpicinteraction of the sample-sorbent pair (so-called ‘‘criticalchromatography’’ or ‘‘liquid chromatography at criticalconditions’’..210 – 212/

The LC/CC mode relates to a chromatographic situa-tion where the entropic and enthalpic interactions of themacromolecule and the column packing compensate eachother, as shown in Equation (26):

G D H � TS D 0 .26/

Therefore, we can derive Equation (27):

Kd D e�GRT D 1 .27/

Rewriting this result for retention in elution volumeterms, we directly get the experimentally observed resultthat the chromatographic peak position is independent


of the molar mass of the analyte and equal to theaccessible volume of the stationary phase, which is statedin Equation (28):

Ve D V0 CKd.LC/CC/Vpore D V0 C Vpore D Vt .28/

The Gibbs free energy of the macromolecule remainsconstant when it penetrates the pores of the station-ary phase (G D 0). The distribution coefficient Kd isunity, regardless of the size of the macromolecules, andall macromolecules of equal chemical structure elutefrom the chromatographic column in one peak. The term‘‘chromatographic invisibility’’ is used to refer to this phe-nomenon. This means that the chromatographic behavioris not directed by the size, but by the heterogeneities(chemical structure, branching point, endgroup, etc.) inthe macromolecular chains..207 – 210/

In general, as the Gibbs free energy is influencedby the length of the polymer chain and its chemicalstructure, contributions Gi for the polymer chain and Gj

for the heterogeneity may be introduced, as stated inEquation (29):

G D∑

niGi C∑

njGj .29/

For a perfectly uniform homopolymer chain the freeenergy change is determined by the contribution of therepeating units of the polymer chain (Equation 30):

G D∑

niGi .30/

At the critical point of adsorption of the polymerchain of a complex polymer, however, the contributionGi becomes zero and chromatographic behavior is exclu-sively directed by imperfections in the macromolecularchain (Equation 31):

G D∑

njGj .31/

This chromatographic effect can be employed todetermine imperfections in the polymer chain selec-tively and without any contribution by the repeat-ing units themselves. LC/CC has been successfullyused for the determination of the FTD of telechelicsand macromonomers,.213 – 217/ for the analysis of blockcopolymers,.218 – 220/ macrocyclic polymers,.221/ and poly-mer blends..222 – 224/

Thus, LC/CC represents a chromatographic separationtechnique yielding fractions which are homogeneous inone property (e.g. chemical composition) but polydis-perse in a different property (e.g. molar mass). Thesefractions can readily be analysed by SEC, which forchemically homogeneous fractions provides true MMDswithout interference of CCD or FTD. Therefore, thecombination of LC/CC and SEC in a 2-D chromatography

experiment can be regarded as ‘‘orthogonal’’ chromatog-raphy in the strict sense provided that LC/CC is used asthe first dimension separation mode. Consequently, forfunctional homopolymers being distributed in functional-ity and molar mass, coupling LC/CC with SEC can yieldcombined information on FTD and MMD. Such propertyinformation is important, e.g. for the quality control ofamphiphilic polyalkylene oxides.

There is another area where the 2-D combination ofLC/CC and SEC is extremely useful and can give resultsno other technique can provide in a single experiment.The 2-D separation of segmented copolymers (such asblock- or graft- or comb-shaped copolymers) allows thecomplete molecular characterization of the copolymerwith regard to individual segment molar masses andcomposition. It has been demonstrated that with sucha set-up the polydispersities of copolymer segments canbe determined independently..225/

The thermodynamics of segmented copolymers isbased on the idea that free energy change GAB ofa segmented copolymer molecule, AnBm, is the sumof the contributions of segments A and segments B,GA and GB, respectively, which can be expressed byEquation (32):

G D∑

nAGA C∑

nBGB C cAB .32/

where cAB is the Flory–Huggins parameter describingthe interactions between segments A and B. It has beendemonstrated for a number of block copolymers.27,34/

that no specific interactions between the heterosegmentsA and B (cAB D 0) can be measured by chromatography.Using this assumption the change in the Gibbs freeenergy is solely dependent on the energy contributions ofsegments A and B, as is shown in Equation (33):

G D∑

nAGA C∑

nBGB .33/

Applying experimental conditions, which correspondto the critical point of homopolymer A, the A segment inthe segmented copolymer will become chromatographi-cally invisible, as expressed in Equation (34):

GA D 0 .34/

Consequently, the retention of the segmented copoly-mer will be determined solely by the chromatographicproperties of segment B. This is shown in Equation (35):

GAB D∑

nBGB .35/

This also means that the distributions coefficient for thissystem, KAB

d can be reduced to the very simple term whichis used for homogeneous molecules (Equation 36):

KABd D KB

d .36/


Repetition of such an experiment using critical condi-tions for segment B allows the determination of molecularparameters for the other segment A in the copolymer. Thesame equations derived above apply, just the parametersfor segments A and B are exchanged. Critical conditionsfor segment B mean that Equation (37) holds:

GB D 0 .37/

and the copolymer shows only the chromatographicbehavior of segment A (Equation 38):

GAB D∑

nAGA .38/

The retention is then given by Equation (39):

KABd D KA

d .39/

The characterization of ABA triblock copolymers canbe done in a similar way to the analysis of diblockcopolymers. The two possible cases for this type ofinvestigation are:

(a) analysis with respect to the center block B using thecritical conditions of the outer blocks A

(b) the analysis of the terminal A segments using thecritical conditions of the center block B.

It is particularly useful to carry out experiments atthe critical point of A. The separation occurs then withrespect to the chain length of B, yielding fractions whichare monodisperse with respect to B and polydispersewith respect to A and A0. These fractions can be analyzedselectively with respect to the outer blocks A and A0 inseparate experiments.

4.3.3 Temperature-rising Elution Fractionation

This is not, strictly speaking, a chromatographic techniquebut it uses the same equipment and leads to detector traceswhich resemble chromatograms. This method is widelyused for the short-chain branching characterization ofpolyolefins in the petrochemical industry.

Many polyolefins are in fact copolymers; the sec-ond comonomer introduces short-chain branches intothe macromolecule. The copolymer properties dependstrongly on the average composition and its sequencelength distribution. In the case of polyethylene copoly-mers, changes in composition change the ease andtemperature at which the polymer chains can crystallize.This property allows for the determination of compositiondistribution by measuring TREF. The TREF techniquesrely on the fact that the redissolution temperature ofa precipitated sample depends strongly on the numberof short-chain branches. A chromatographic pump willtransport the redissolved species through the column

Solvent

Degasser

Pump

Pulse dampener

Detector

Temperaturecontroller

Elution temperature

Con

cent

ratio

n

TR

EF

col

umn

Figure 34 Schematic layout of a TREF instrument withthermostated TREF column.

loaded with precipitated polymer into a concentrationdetector, which in turn will measure the concentrationof the fraction redissolved at a given temperature, T, asshown in Figure 34.

4.4 Separation Techniques for the Second Dimension

The LC methods described in section 4.3 can also be usedin the subsequent separation stage in a multidimensionalchromatography set-up. However, as pointed out earlier(cf. section 4.2), it is advantageous to use gradient LC orLC/CC in the first separation dimension. On the otherhand, SEC is preferentially used as the second methodto retrieve molar mass information. In theory, TREFcan also be used in a later separation step; however, forpractical reasons, this is not advisable.

There are a number of chromatographic separationmethods which can only be used in the last stage of amultidimensional experiment.

4.4.1 Size-exclusion Chromatography

SEC is described in the section of techniques for thesecond dimension, because of its primary benefits there(cf. section 4.2.2). However, it can also be used withlower efficiency and more biased results in the firstseparation dimension. This technique was developed forthe separation and characterization of large molecules.It is also called Gel Filtration Chromatography fornatural and biopolymers and known as GPC for syntheticpolymers.

The principles are the same in both cases and rely on thefact that the macromolecule can only partially penetratethe porous packing, depending on its molecular size (notmolar mass). The molecular size of a macromoleculein solution, or more accurately its hydrodynamic volume,


will depend on its chain length, the nature of the repeatingunits, chemical composition, molecular topology andthe thermodynamic quality of the solvent. The samedependencies exist for the distribution coefficient in SEC,which can lead to the co-elution in SEC of species havingidentical hydrodynamic radius, but different composition,molecular architecture, and so forth. Large molecules canonly access pores larger than the hydrodynamic radiusof the molecule and will elute from the column early. Ifmacromolecules are larger than the biggest pores, theywill be totally excluded from the pore volume and will notbe separated into fractions of different molar mass. Onthe other hand, the smallest molecules might be able topenetrate into all pores in the packing. They also will notbe separated and will all co-elute at the total permeationvolume. This chromatographic behavior is illustrated inFigure 35.

Similarly to the thermodynamic treatment of LC, reten-tion in SEC can be described by basic thermodynamicentities and can be determined by measuring the con-centration of the molecule in the stationary and mobilephases, as expressed in Equation (40):

Kd.SEC/ D as

amD e

SR .40/

In ideal SEC separations the retention in the columnis only governed by the entropy loss when the macro-molecule enters the pore of the packing. No enthalpicinteraction should be present in order to allow for accu-rate molar mass determinations.

The retention in ideal SEC experiments is given byEquation (41):

Ve D V0 CKd.SEC/Vpore .41/

Retention

V0 Vt

Log

M

Com

plet

e ex

clus

ion

Tot

al p

enet

ratio

n

Figure 35 Size-exclusion behavior of macromolecules of dif-ferent size on a porous column packing.

This equation looks very similar to Equation (23). Theonly difference is the magnitude of Kd, which is always lessthan unity for SEC separations and reaches a maximumvalue of unity for molecules which enter all pores.

Ideal SEC conditions are difficult to obtain for realmacromolecules, however. As in the case of LC, there arecontributions from both entropic and enthalpic terms tothe distribution coefficient (cf. Equation 25).

In such cases, the retention for molecules with enthalpicinteractions is higher and they will elute later from thecolumn. This behavior can be described by Equation (42):

Ve D V0 C [Kd.SEC/Kd.LC/]Vpore .42/

In this equation the term Kd.LC/Vpore describes thedelayed elution from the column as compared to theideal case. This equation is mathematically identical tothe respective equation for LC. However, the dominantparameter is Kd(SEC) and the entropy change governsthe penetration of the analyte into the pores.

4.4.2 Capillary Electrophoresis

CE is a very efficient microseparation method (typicallyN > 100 000), which uses a strong electric field to createan electro-osmotic flow in which the species will migrate.The reason for that is that the surface of the silicateglass capillary contains negatively-charged functionalgroups that attract positively-charged counterions. Thepositively-charged ions migrate towards the negativeelectrode and carry solvent molecules in the samedirection. This overall solvent movement is called electro-osmotic flow. During a separation, uncharged moleculesmove at the same velocity as the electro-osmotic flow(with very little separation). Positively-charged ions movefaster and negatively-charged ions move more slowly.

CE can also be used as a first set in multidimensionalseparation, but its practical use here is very limited dueto the minute sample amounts injected.

For further information and details on the CE tech-nique, please consult the articles in this encyclopedia.

4.4.3 Supercritical Fluid Chromatography

SFC is a relatively recent chromatographic techniquewhich was commercialized in the early 1980s.

In SFC, the sample is carried through a capillaryor packed column by a supercritical fluid (typicallycarbon dioxide). The properties of the mobile phasecan be modified easily by polar additives and/or pressureprogramming, just as in gradient HPLC, to optimizeselectivity. All three basic modes of chromatography(interaction, size-exclusion and critical conditions) havebeen verified in SFC separations..226/ SFC is a very


efficient separation technique, which has most of itsapplications in low molecular weight separations.

SFC has several advantages over conventional chro-matographic techniques. SFC separations can be doneconsiderably faster than HPLC separations, because thediffusion of solutes in supercritical fluids is about 10times greater than that in liquids (and about 3 times lessthan in gases). This results in a decrease in resistance tomass transfer in the column and allows for fast high res-olution separations. Compared with GC, capillary SFCcan provide high resolution chromatography at muchlower temperatures. This allows fast analysis of thermola-bile and nonvolatile compounds. These advantages makeSFC a good choice for multidimensional chromatographyset-ups. Since SFC is a mobile phase-destroying tech-nique, it can only be used in the last separation step inmultidimensional separations.

4.4.4 Gas Chromatography

As with SFC, GC is a mobile phase-destroying techniqueand can only be used in the last stage of multidimensionalseparations. It also shares with SFC the high efficiencyand speed of separations. However, it is limited torelatively low molecular weight compounds which arevolatile without degradation and thermostable.

Most ‘‘multidimensional’’ applications reported inthe literature use the first dimension for precleaningand removal of high molar mass species and not forcomplete characterization of the samples. Examples ofsuch applications are the removal of humic acids frompesticides in soil extracts by SEC/GC and of highmolecular byproducts from mono, di, and triglycerides.

For an indepth description of the technique, pleaserefer to the Gas Chromatography in this encyclopedia.

4.5 State-of-the-art of On-line CoupledTwo-dimensional Chromatography

This section will illustrate the current state and futurepotential of 2-D chromatography by reviewing separa-tions published in the literature. Examples will be givenfor different separation techniques and combinations ofthese.

4.5.1 Two-dimensional Separations by LiquidChromatography and by Size ExclusionChromatography

Much work on chromatographic cross-fractionation wascarried out with respect to the combination of SECand gradient HPLC. In early experiments SEC wasused as the first separation step, followed by HPLC.In a number of early papers the cross-fractionation ofmodel mixtures was discussed. Investigations of this

kind demonstrated the efficiency of gradient HPLCfor separation by chemical composition. Mixtures ofrandom copolymers of styrene and acrylonitrile wereseparated by Glockner et al..198/ In the first dimensionan SEC separation was carried out using THF as theeluent and PS gel as the packing. In total, about 10fractions were collected and subjected to the seconddimension, which was gradient HPLC on a CN bonded-phase using iso-octane/THF as the mobile phase. Modelmixtures of random copolymers of styrene and 2-methoxyethyl methacrylate were separated in a similarway, the mobile phase of the HPLC mode being iso-octane/methanol in this case..227/ This procedure was alsoapplied to real-world copolymers..198/ Graft copolymersof methyl methacrylate onto EPDM were analyzed byAugenstein and Stickler;.228/ whereas Mori reported onthe fractionation of block copolymers of styrene andvinyl acetate..229/ For all these experiments the samelimitation with respect to the SEC part holds true: whenSEC is used as the first dimension, true MMDs are notobtained.

From the theoretical point of view, a better copolymerseparation set-up is prefractionation through HPLC in thefirst dimension and subsequent analysis of the fractions bySEC. HPLC was found to be rather insensitive towardsmolar mass effects and yielded very uniform fractionswith respect to chemical composition..230,231/

The major disadvantage of all early investigations onchromatographic cross-fractionation was related to thefact that both separation modes were combined with eachother either off-line or in a stop-flow mode. Regardless ofthe separation order SEC vs HPLC or HPLC vs SEC, inthe first separation step fractions were collected, isolated,and then subjected to the second separation step. Thisprocedure, of course, is very time-consuming and thereliability of the results, at least to a certain extent,depends on the skills of the operator.

A fully automated 2-D chromatographic system wasdeveloped by Kilz et al..23,188,194/ The operation of thecolumn-switching device is automatically driven by thesoftware, which at the same time organizes the datacollection from the detector.

One of the very few applications of 2-D gradientHPLC/SEC was published by Kilz et al. and described theanalysis of styrene–butadiene star polymers..188/ Four-arm star polymers based on poly(styrene-b-butadiene)were prepared by anionic polymerization to give sampleswith well-known structure and molar mass control. Ina first reaction step, a poly(styrene-b-butadiene) with areactive chain end at the butadiene was prepared. Thisprecursor reacted with a tetrafunctional terminating agentto give a mixture of linear (of molar mass M), 2-arm (2M),3-arm (3M) and 4-arm (4M) species. Four samples withvarying butadiene content (about 20%, 40%, 60%, and


80%) were prepared in this way. A mixture of thesesamples was used for the 2-D experiment. Accordingly,a complex mixture of 16 components, resulting from thecombination of four different butadiene contents andfour different molar masses (M, 2M, 3M, 4M) had tobe separated with respect to chemical composition andmolar mass.

Initially, the 16-component star block copolymer wasinvestigated by SEC. As can be seen in Figure 36, fourpeaks were obtained. They correspond to the four molarmasses of the sample consisting of species with one tofour arms. The molar masses are defined by the numberof arms and were in the ratio M–2M–3M–4M. Despitethe high resolution, the chromatogram did not give anyindication of the very complex chemical structure of thesample. Even when pure fractions with different chemicalcomposition were investigated, the retention behaviordid not show significant changes as compared to thesample mixture. In each case a tetramodal MMD wasvisible, indicating the different topological species. TheSEC separation alone did not show any difference inchemical composition of the samples, which varied from20% to 80% butadiene content.

Running the sample mixture in gradient HPLC modegave poorly resolved peaks, which might suggest differentcomposition, but gave no clear indication of differentmolar mass and topology, see Figure 37.

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

RI-

resp

onse

104 105 106

C

B

A

M (g mol−1)

Figure 36 SEC chromatograms of the 16-component star blockcopolymer mixture (A) overlayed with styrene–butadiene60 : 40 (B) star copolymer and 20 : 80 (C); peaks correspondto molar mass separation of different arm numbers.

0 100 200 300 400 500 600U

V-r

espo

nse


Figure 37 Gradient HPLC chromatogram of the 16-compo-nent star block copolymer mixture; separation is dominated bychemical composition and does not reveal the complexity of thesample.

Ve (mL) In

jectio

ns

Figure 38 3-D view of the 16-component star block copolymermixture; each trace represents an SEC chromatogram from atransfer injection.

The combination of the two methods in the 2-Dset-up dramatically increased the resolution of theseparation system and gave a clear picture of thecomplex nature of the 16-component sample. A 3-Drepresentation of the gradient HPLC/SEC separationis given in Figure 38. Each trace represents a fractiontransferred from HPLC to SEC and reflects the result


of the SEC analysis in the second dimension. Basedon the composition of the sample, a contour map withthe coordinates’ chemical composition and molar massis expected to show 16 spots, equivalent to the 16components. Each spot would represent a componentwhich is defined by a single composition and molar mass.The experimental evidence of the improved resolutionin the 2-D analysis is given in Figure 39. This contourplot was calculated from experimental data based on 28transfer injections.

The contour plot clearly revealed the broad chemi-cal heterogeneity (y-axis, chemical composition) and thewide MMD (x-axis) of the mixture. The relative concen-trations of the components were represented by colors.Sixteen major peaks were resolved with high selectiv-ity. These correspond directly to the components. Forexample, peak 1 corresponds to the component with thehighest butadiene content (80%) and the lowest molarmass (molar mass 1M) whereas peak 13 relates also to amolecule with 80% butadiene content but a molar massof 4M. Accordingly, peak 16 is due to the component

with the lowest butadiene content and a molar mass of4M, representing a 4-arm star block copolymer with astyrene–butadiene content of 80 : 20.

A certain molar mass dependence of the HPLC sepa-ration is indicated by a drift of the peaks for componentsof similar chemical composition: see peaks 1-5-9-13, forexample. This kind of behavior is normal for polymers,because pores in the HPLC stationary phase lead tosize-exclusion effects which overlap with the enthalpicinteractions at the surface of the stationary phase. Con-sequently, 2-D separations of this type will in general benot orthogonal but skewed, depending on the pore sizedistribution of the stationary phase and the nature of thesample. The quantitative amount of butadiene in eachpeak could be determined via an appropriate calibrationwith samples of known composition. The molar massescould be calculated based on a conventional molar masscalibration of the second dimension.

The mapping of ethoxylated fatty alcohols and ethyleneoxide–propylene oxide block copolymers by 2-D chro-matography was discussed by Trathnigg et al..232/ They

80

60

40

20

Sty

rene

con

tent

(%

)

104 105 106

50

150

250

400

500

600

800

1000

1100

1200

1400

1600

1800

2000

2400

Rel

ativ

e co

ncen

trat

ion

SEC molar mass (D)

1

5

913

2

3

4

711 15

16128

610 14

Figure 39 Contour plot of the 16-component styrene–butadiene star block copolymer mixture characterized by the 2-D HPLC/SECseparation.


100

60

0

0.000 5.00 10.00 15.00 20.00

Alk

−Alk

Cyc

les

Alk

−OH

HO

OC

−CO

OH

HO

−CO

OH

HO

−OH Eth

erE

ther

HDm

V


Figure 40 LC chromatogram of an aliphatic polyester sample at critical conditions; peak labels identify endgroups or functionality.

combined LAC and SEC and were able to determine theCCD and the MMD of the polyethers.

4.5.2 Two-dimensional Separations by LiquidChromatography at Critical Conditions and SizeExclusion Chromatography

The analysis of aliphatic polyesters with respect to FTDand MMD has been demonstrated..188,233/ Polyesters fromadipic acid and 1,6-hexanediol are manufactured for awide field of applications with an output of thousandsof tonnes per year. They are intermediates for themanufacture of polyurethanes, and their FTD is a majorparameter affecting the quality of the final products. Inparticular, nonreactive cyclic species are responsible forthe ‘‘fogging effect’’ in polyurethane foams.

For the separation of the polyesters with respect tofunctionality, LC/CC was used, the critical point ofadsorption of the polymer chain corresponding to aneluent composition of acetone–hexane 51 : 49 (v/v) onsilica gel. The critical chromatogram of a polyester sampletogether with the functionality fraction assignment isgiven in Figure 40. The ‘‘ether’’ peaks could be attributedto the formation of ether structures in the polyestersamples by a condensation reaction.

The MMDs of the functionality fractions could bedetermined by preparatively separating the fractions andsubjecting them to SEC. The SEC chromatograms offractions 1–9 are summarized in Figure 41. For a numberof fractions oligomer separations were obtained, whichcould be used to calibrate the SEC system.

The very complex nature of the sample could be verifiedin a 2-D experiment using LC/CC as the first dimensionto separate for functionality and SEC in the seconddimension to determine molar masses. The contour plot inFigure 42 reveals the structural complexity of the sampleincluding the functionality fractions from LC/CC andthe oligomer separations from SEC which were well-recognizable. The sample was prepared from an adipicacid-rich reaction mixture resulting in an acid number ofabout 5. The high content of dicarboxylic acid endgroupsis clearly reflected in the contour map. Quantification of

Fr.1

Fr.3

Fr.4

Fr.5Fr.6

Fr.7Fr.8

Fr.9

Fr.2

Ve (mL)

Figure 41 SEC separation of different fractions taken fromthe LC/CC separation of the aliphatic polyester sample (cf.Figure 40).

the contour plot yielded quantitative information on bothFTD and MMD.

The analysis of an octylphenoxy-terminated PEO withrespect to FTD and MMD has been demonstrated byAdrian et al..225/ A separation of the sample with respectto the terminal groups could be achieved by LC/CC onan RP-18 stationary phase and a critical eluent compo-sition of methanol–water 86 : 14 (v/v). All peaks in thechromatogram could be identified by MALDI/TOF MSas pure fractions of different functionality, proving thatthe separation followed the chemical structure of the end-groups. The combination of LC/CC with SEC in a 2-Dchromatography experiment resulted in the contour plotshown in Figure 43..225/ At the abscissa, the retention vol-ume of the SEC runs (second dimension) is given, whereasthe ordinate gives the retention volume of the LC/CC(first dimension). Relative concentrations are mapped toa color code on a log scale to make small quantities visible.


95.0

90.0

85.0

80.0

75.0

70.0

65.0

60.0

55.0

50.0

45.0

40.0

35.0

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5.0

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ativ

e co

ncen

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SEC volume (mL)

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[-O-]n

HO-OH

HO-COOH

HOOC-COOH

Alk-OH

Cycles

Alk-Alk

Figure 42 Contour map of the 2-D LC/CC and SEC separation of the aliphatic polyester with acid number of about 5; functionality(on y-axis) was calibrated by model compounds.

The contour plot (cf. Figure 43) clearly reveals fivespots corresponding to the five functionality fractions,fraction 2 being the main fraction containing the a-octylphenoxy-w-hydroxy oligomers. In addition, a,w-di(octylphenoxy) oligomer fractions and fractions havingbutylphenoxy endgroups are identified.

The 2-D experiment yielded separation with respectto functionality and molar mass, and FTD and MMDcould be determined quantitatively. For calculatingFTD, the relative concentration of each functionalityfraction must be determined. These concentrations areequivalent to the volume of each peak in the contourplot. With the appropriate software this can be doneeasily. Determination of the MMD for each fractionwas possible after calibrating chromatograph 2 with PEOcalibration standards. Calculation of the MMD could thenbe achieved in the usual way, taking one chromatogramfor each functionality fraction, preferably from the regionof the highest peak intensity.

In similar approaches other polyalkylene oxides havebeen analysed by 2-D chromatography. Murphy et al..234/

separated PEGs and Brij type surfactants according tochemical composition and molar mass by RP HPLCversus SEC.

The analysis of methacryloyl-terminated PEOs byLC/CC versus SEC was described by Kruger et al..233/

The functionality-type separation was conducted onan RP system at a critical eluent composition ofACN–water 43 : 57 (v/v). The functionality fractions,including PEG, a-methoxy-w-hydroxy, a-methoxy-w-methacryloyloxy, and a,w-di(methacryloyloxy) PEO,were identified by MALDI/TOF MS.

Finally, a technical C13,C15-alkoxy-terminated PEOwas analysed by Pasch and Trathnigg using LC/CC vsSEC..235/

4.5.3 Two-dimensional Combination of Different HighPerformance Liquid Chromatography Modes

The deformulation of alcohol ethoxylates, which arean important class of nonionic surfactants, by 2-Dchromatography has been published by Murphy et al..236/

This class of products possesses a polydispersity of


12.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

13.00 14.00 15.00

Elution volume GPC (mL)

Elu

tion

volu

me

HP

LC (

mL)

OO

n

O

HO On

HOO

n

On

Figure 43 2-D separation of octylphenoxy-terminated PEO shown as a contour plot; the labels indicate the molecular structureverified by MALDI/MS.

methylene groups and a distribution of ethylene glycolunits in the PEG segment of the nonionic surfactant.The authors established chromatographic methods forboth heterogeneities independently and then combinedthem later for the 2-D investigations. They used anRP system with a 3 µm C-18 column with 100 A porewidth to separate the alcohol alkyl chains using anisocratic mobile phase (methanol–water (95 : 5)) (seeFigure 44). The PEG segments were separated on anormal phase system comprising a 3 µm nonmodifiedsilica with 70 A pores run with a concave water–ACNgradient (see Figure 45). Both chromatograms show highresolution separations for the alcohol groups or the PEGMMD, without any indication of any other unresolvedproperty.

The combination of both techniques in a 2-D exper-iment revealed the complex nature of the surfactant.The authors applied the gradient normal phase liquidchromatography (NPLC) separation of PEG as the firstdimension with the reversed phase liquid chromatogra-phy (RPLC) as the second dimension using ELSD as ahighly sensitive means of detection. Figure 46 shows the2-D contour map which displays both the alkyl chain sep-aration on the x-axis and the ethylene oxide chain length

10

100 000

200 000

300 000

20 30

Time (min)

UV

40 50

Figure 44 RP separation of FAE non-ionic surfactant forethylene oxide content determination.

on the y-axis. The separation is not truly orthogonal,which the authors attribute to the concave gradient in theNPLC separation. This separation clearly demonstratesthe increased peak capacity of 2-D experiments. The highresolution separation in NPLC showed very high peakcapacities of about 15, whereas the RPLC experimentresulted in an extremely good separation into the four


30

Time (min)

200 000

400 000

600 000

800 000

40

UV

Figure 45 High resolution normal-phase chromatogram ofnon-ionic surfactant; separation occurs by alkyl chain lengthwithout indication of ethylene oxide polydispersity.

alkyl alcohol chains. The total peak capacity in the 2-Dexperiment is about 70, which agrees very favorably withthe theoretical value of about 60 (cf. section 4.1).

The authors were able to distinguish between differentsamples of one manufacturer and samples of different pro-ducers using this technique. They used the polydispersity

x =12 x =13 x =14

100

125

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175

2000.00 0.25 0.50

RPLC (min)N

PLC

(m

in)

0.75 1.00

H-(CH2)x-O-(CH2CH2O)12-H

x =15

Figure 46 2-D contour plot of FAE characterized in anNPLC/RPLC experiment with very high peak capacity (about70).

of the PEG segment and the presence of various Cn chainsfrom the 2-D contour plot as indicators for identifyingsample, synthetic process and manufacturer.

2500

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Rel

. con

c.

Rel. conc.

2500+2250 to 25002000 to 22501750 to 20001500 to 17501250 to 15001000 to 1250750 to 1000500 to 750250 to 5000 to 250

Figure 47 Surface plot of the 2-D TREF/SEC characterization of the ethylene–butene copolymer (Ziegler–Natta catalyst). Thetemperature axis reflects short-chain branching, the molar mass axis shows the broad MMD of the sample.


4.5.4 Temperature-rising Elution Fractionation–SizeExclusion Chromatography Two-dimensionalSeparations

This method combination has high potential for the studyof the physical properties of polyolefins and can relatethem to their molecular structure. The thermal propertiesand the performance of many polyolefin applicationsdepend on the formation of crystalline domains, whichare controlled by the type and the degree of branching ofthe macromolecule.

TREF (section 4.3.3) is a technique which is widelyused for the short-chain branching investigation ofpolyolefins. Micklitz et al. reported on the 2-D char-acterization of poly(ethylene-co-butene) using differentinitiator systems..237/

The 2-D system comprised a preparative TREF unitrunning in xylene from which samples were collected andmanually transfered into a high-temperature SEC systemrunning in 1,2,4-trichlorobenzene at 135 °C. Figure 47shows the surface plot of the TREF/SEC characteriza-tion of the ethylene–butene copolymer, which has beensynthesized from a traditional Ziegler–Natta type cat-alyst. Both the branching distribution (indicated by the

measured property elution temperature by TREF) andthe MMD (by SEC calibration) are very broad, as wouldbe expected from a Ziegler–Natta type catalyst. The aver-age molar mass of the sample was about 100 000 g mol�1.

Figure 48 shows an ethylene–butene copolymerformed from a metallocene catalyst using otherwiseidentical conditions (comonomer ratio, molar mass).Metallocene catalysts marked a new step in polyolefinsynthesis, because they allow a much improved controlof the structure of the growing macromolecule. The highdegree of structure and molar mass control with a met-allocene catalyst can be seen from this figure. The widthof the 3-D peak with respect to the elution temperatureand the molar mass axis is much smaller than that of theZiegler–Natta product. This indicates that the degree ofbranching and the insertion of new monomers into thegrowing polymeric chain can indeed be controlled in away not possible before.

4.6 Conclusions and Future Developments

Multidimensional chromatography separations are cur-rently one of the most promising and powerful methodsfor the fractionation and characterization of complex

2500

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. con

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2500+2250 to 25002000 to 22501750 to 20001500 to 17501250 to 15001000 to 1250750 to 1000500 to 750250 to 5000 to 250

Figure 48 3-D surface plot from a TREF/SEC experiment of a ethylene–butene copolymer formed by a metallocene catalyst (referto Figure 47 for details).


sample mixtures in different property coordinates. It com-bines extraordinary resolution and peak capacity withflexibility and overcomes the limitations of any givensingle chromatographic method. This is the basis forthe identification and quantification of major compoundsand byproducts, which might adversely affect productproperties if not detected in time.

Using a chromatographic separation which is selectivetowards functionality or chemical composition in thefirst dimension and SEC in the second dimension, truly‘‘orthogonal’’ separation schemes can be established.Thus, the combination of gradient HPLC versus SECyields quantitative information on CCD and MMD, whilecoupling LC/CC and SEC is useful for the analysis offunctional homopolymers and block copolymers in thecoordinates FTD/MMD and CCD/MMD respectively.Even more complex systems, such as graft copolymersand polymer blends, in which each component may bechemically heterogeneous itself, can be analyzed.

Although 2-D LC is experimentally more demandingthan other chromatographic techniques, the completecharacterization yields much more qualitative and quan-titative information about the sample, and results arepresented in an impressively simple way. The contourplot of a 2-D separation maps all obtainable informationand allows a fast and reliable comparison between twosamples. For future development, the automated compar-ison of the results of different samples can be consideredas an important step to improve process control andquality management. Currently, there is much activity toidentify the best method combinations for multidimen-sional chromatography of many applications which aredifficult to separate and quantify.

Recent improvements in the efficiency of the interactivechromatography of macromolecules and other separationmodes (e.g. SEC, CE) widen the applicability of 2-Danalyses. The next step is the on-line identification offractions using information-rich detection systems suchas FTIR and MS described in section 3 of this article. Thiswill advance the state of the art to a still higher level.

LIST OF SYMBOLS

am activity (concentration) in mobile phaseas activity (concentration) in stationary phasec concentrationf response factorG Gibbs free energyKŁ optical constant in light scatteringKd distribution coefficientM molar massMw weight-average molar massmi mass of species i

ni peak capacity of the i-th dimensionN number of theoretical platesP./ scattered light angular dependenceR universal gas constantR./ intensity of scattered light at the angle V0 exclusion volumeVe elution volumeVh hydrodynamic volumeVp volume at peak maximumVpore pore volumeVt total penetration volumew weight fractionh viscosity of a solutionl wavelength# projection angle between two separation

methodsi portion of copolymers elutedV volumex area of slicej detectorMC molar mass of copolymersA2 second virial coefficientNA Avogadro’s constant[h] intrinsic viscositycAB Flory–Huggins parameterRg radius of gyrationh0 viscosity of a solvent (viscosity of the pure

mobile phase)hsp specific viscosityP pressurea Mark–Houwink exponentMn number-average molar massK constant factor in the Mark–Houwink

equationhrel relative viscosityH interaction enthalpyT absolute temperaturedp diameter of column packingf dilution factorMv viscosity-average molar massni number of species iS entropyVinj injection volume

ABBREVIATIONS AND ACRONYMS

ACN AcetonitrileCCD Chemical Composition DistributionCE Capillary ElectrophoresisEA Ethyl AcrylateELSD Evaporative Light Scattering DetectionEPDM Ethylene–Propylene–Diene RubberESI Electrospray Ionization


FAE Fatty Alcohol EthoxylateFFF Field-flow FractionationFTD Functionality Type DistributionFTIR Fourier Transform InfraredGC Gas ChromatographyGC/MS Gas Chromatography/Mass SpectrometryGPC Gel Permeation ChromatographyGTP Group Transfer PolymerizationHPLC High-performance Liquid ChromatographyIR InfraredLAC Liquid Adsorption ChromatographyLALLS Low Angle Laser Light ScatteringLC Liquid ChromatographyLC/CC Liquid Chromatography

at the Critical Point of AdsorptionLS Light ScatteringMAD Molecular Architecture DistributionMALDI Matrix-assisted

Laser Desorption/IonizationMALLS Multiangle Laser Light ScatteringMMD Molar Mass DistributionMS Mass SpectrometryNMR Nuclear Magnetic ResonanceNPLC Normal Phase Liquid ChromatographyPDMS Polydimethyl SiloxanePEG Polyethylene GlycolPEO Polyethylene OxidePMMA Polymethyl MethacrylatePnBMA Poly-n-butyl MethacrylatePPG Poly(propylene glycol)PS PolystyreneRI Refractive IndexRP Reversed PhaseRPLC Reversed Phase Liquid ChromatographySAN Styrene–acrylonitrileSEC Size Exclusion ChromatographySFC Supercritical Fluid ChromatographyS/N Signal-to-noiseTHF TetrahydrofuranTLC Thin-layer ChromatographyTOF Time-of-flightTREF Temperature-rising Elution FractionationUV UltravioletVIS VisibleVISC On-line Viscometry2-D Two-dimensional3-D Three-dimensional

RELATED ARTICLES

Polymers and Rubbers (Volume 8)Polymers and Rubbers: Introduction

Polymers and Rubbers cont’d (Volume 9)Field Flow Fractionation in Analysis of Polymers andRubbers ž Infrared Spectroscopy in Analysis of Poly-mers and Rubbers ž Size-exclusion Chromatography ofPolymers ž Supercritical Fluid Chromatography of Poly-mers ž Temperature Rising Elution Fractionation andCrystallization Analysis Fractionation

Infrared Spectroscopy (Volume 12)Infrared Spectroscopy: Introduction ž Theory of InfraredSpectroscopy

Liquid Chromatography (Volume 13)Liquid Chromatography: Introduction ž Capillary Elec-trophoresis ž Column Theory and Resolution in LiquidChromatography ž Gradient Elution Chromatographyž Normal-phase Liquid Chromatography ž SupercriticalFluid Chromatography

Nuclear Magnetic Resonance and Electron SpinResonance Spectroscopy (Volume 13)High-performance Liquid Chromatography NuclearMagnetic Resonance

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