FFF-MALS—A New Tool for the Characterisation of Polymers and Particles

Polymer International 43 (1997) 373È379

New Tool for theFFF–MALS—ACharacterisation of Polymers and

Particles*

R. J. White

Optokem Instruments Ltd, Pistyll Farm, Nercwys, Flintshire, CH7 4EW, UK

(Received 22 October 1996 ; revised version received 13 February 1997 ; accepted 22 February 1997)

Abstract : As industrial applications for polymers and biopolymers have ex-panded in recent years, the limitations of traditional characterisation techniqueshave become apparent. Size exclusion chromatography is widely used to deter-mine the molecular weight distribution of a polymer, but su†ers from a numberof drawbacks when used with ultra-high molecular weight (MW) polymers orpolyelectrolytes. As a result, interest in the use of Ðeld Ñow fractionation (FFF)for the separation of macromolecules has grown.

FFF relies on a combination of Ðeld-driven and di†usive transport mech-anisms to separate polymers in the range 103È1015 g mol~1 and/or particles of5 nmÈ100 km, allowing MW and size distributions for virtually all macro-molecules to be determined without the need for Ðltration. The open channelgeometry minimises shear e†ects making it possible to separate fragile, high MWpolymers, while the absence of a stationary phase means adsorption e†ects canbe minimised. As a result, samples can be run in a mobile phase suitable for thepolymer rather than selecting conditions to minimise column interactions whichleads to the risk of aggregate formation in polyelectrolytes.

While it is possible to calculate the MW of a polymer eluting from an FFFchannel using calibration techniques similar to those for size exclusion chroma-tography, the same problems are encountered as well. When analysing ultra-highMW polymers and/or gels, these are compounded by the need for calibrationstandards with a MW in excess of 106 g mol~1. The addition of multi-angle lightscattering (MALS) to FFF has allowed absolute MW and size distributions to beobtained without the need for calibration, standards or assumptions. Complexmixtures of polymer, microgel, and macrogel can all be studied in a single run.By combining all of the information derived from FFFÈMALS, molecular con-formation, density and branching levels can also be determined.

This paper outlines the principles of operation of FFFÈMALS, before movingon to discuss recent applications of the technique for the analysis of macro-molecules and sub-micrometre particles.

Polym. Int. 43, 373È379 (1997)No. of Figures : 9 No. of Tables : 0 No. of References : 10

Key words : Ðeld Ñow fractionation, multi-angle light scattering, absolute molec-ular weight, size distribution, separation, characterisation, polymers, bio-polymers, particles.

* Presented at “The Cambridge Polymer Conference : Partnership in PolymersÏ, Cambridge, UK, 30 SeptemberÈ2 October 1996.

3731997 SCI. Polymer International 0959-8103/97/$17.50 Printed in Great Britain(

374 R. J. W hite

INTRODUCTION

Anyone currently involved in the characterisation ofmacromolecules using traditional chromatographictechniques must be aware of the limitations of suchmethods for the determination of molecular weight dis-tributions (MWDs). Many modern engineering poly-mers have high molecular weight (MW) tails in excess of107 g mol~1, above the exclusion limit of most sizeexclusion chromatography columns, while otherscontain signiÐcant levels of gel that will a†ect end-useproperties. Filtration is often used to remove this ultra-high MW material but results in the determination ofan erroneously low MW material, making it difficult torelate the properties of a polymer batch back to its realMWD.

Many natural and synthetic polymers, particularlythose used in the paints, food, and pharmaceuticalindustries, exhibit polyelectrolyte behaviour and inter-act with the stationary phase during separation in a tra-ditional SEC column. When this occurs it is impossibleto predict from classic SEC theory what the MW is at agiven elution volume. Even when absolute detectionmethods such as light scattering are used, adsorptioncan result in the polymer slice taken at a given elutionvolume exhibiting polydispersity, when an adsorbedand non-adsorbed molecule of di†erent MW are co-eluting. As light scattering determines the absoluteweight average MW, of each volume slice, thenM1 w ,uses SEC algorithms to construct an MWD assumingeach slice is monodisperse, the distributions obtainedwill tend to be narrower than is actually the case.

To overcome these e†ects, bu†ers can be added to themobile phase to prevent interaction occurring. This initself can cause problems, particularly when analysingbiopolymers such as proteins. Changes in bu†er fromthat used to dissolve the native polymer to one compat-ible with the column packing can cause aggregation tooccur, again disrupting the “realÏ MWD. This will thenlead to problems in relating observed end-use propertiesto the measured MW.

Other large molecules, such as elastomers and highMW polyoleÐns, are fragile and easily degraded by theelongational shear Ðelds found in high resolution SECcolumns. This degradation will cause a downward shiftin the apparent MWD from that present in the unsep-arated material. Shear e†ects in the column will alsocause problems for polymers with an extended confor-mation, e.g. rod-like polysaccharides. As these materialspass down the column, the Ñow acts to orientate therods preferentially down the length of the column, pre-venting true permeation through the stationary phasefrom occurring, and resulting in poor, if any, MWresolution.

As polymers have come to be used in increasinglydemanding applications, the need for accurate charac-terisation has become more acute. A key advance in

SEC was the elimination of column calibration usingÐrst low-angle then, more recently, multi-angle lightscattering which has enabled absolute MW informationto be obtained. However, these detection techniquescannot overcome problems associated with columninteraction causing incomplete separation, exclusion ofultra-high MW molecules on packed columns,Ðltration-induced modiÐcation of the real MWD, ormethod-induced aggregation/gel formation.

To overcome these limitations, many researchersaround the world have switched to an alternativepolymer separation technique known as Ðeld Ñow frac-tionation (FFF). Originally developed in the late 1960sby Prof. Calvin Giddings at the University of Utah,1FFF uses an open channel for separation rather than apacked column, making it possible to separate macro-molecules and particles ranging in size from 5 nm (or103 g mol~1) to 100 km. The geometry of the channelensures that shear forces are minimised, allowing fragilemolecules to be separated without degradation, whilethe absence of a stationary phase avoids the possibilityof interactions between channel and sample. As themechanism of separation is based primarily on di†usivetransport, non-spherical conformations can also be suc-cessfully handled. Virtually any solvent can be used as amobile phase (pH 2È12), making it possible to separateproteins in a native bu†er when screening for aggre-gates, or to dissolve polyamide in formic acid for aroom temperature determination of gel content.

As there is no high MW exclusive limit, FFF is alsocapable of separating emulsions, colloids and particlesuspensions ranging in size from 2 nm to 100 km. Evencomplex mixtures of soluble polymers, gels and sus-pended particles can be characterised in a single run.

While FFF can be used to separate complex macro-molecules across an enormous size range, problems areencountered when size and/or MWDs need to be calcu-lated. Elution order is based on the hydrodynamicvolume, with the smallest molecule coming o† thechannel Ðrst. To determine MW it is necessary to cali-brate the channel using similar techniques to thosedeveloped for classical SEC. Size can be calculateddirectly from elution volume as long as the maximumparticle size falls below 1km and a constant Ðeldstrength is used during the separation. If not, a latexstandard calibration must be undertaken with all of theattendant problems this brings. Gradient Ðeld pro-gramming is an essential tool in the separation ofcomplex samples by FFF, as without such techniquesrun times of the order of 3È4 h are required rather than10È30 min as is the case with gradient FFF. If materialis held on the channel for too long, some sample lossthrough adhesion to the channel wall may occur.

An alternative approach pioneered by Wyatt Tech-nology has been to couple the FFF separation with amulti-angle light scattering (MALS) photometer capableof measuring both MW and size in real time as the

POLYMER INTERNATIONAL VOL. 43, NO. 4, 1997

FFFÈMAL SÈa new tool 375

sample elutes from the channel.2 Problems associatedwith non-ideality of separation, gradient Ðelds, MWcalibration and branching e†ects can be overcomeimmediately, providing a Ñexible and versatile tool foruse in macromolecular and particle characterisation.

FIELD FLOW FRACTIONATION (FFF)

FFF uses a combination of hydrodynamic and Ðeld-driven mechanisms to e†ect a separation. The channelhas a high aspect ratio (thickness 100È200 km, width20 mm) to provide a highly parabolic Ñow proÐle assolvent Ñows through it (Fig. 1).

To separate the sample, a Ðeld is applied across thechannel at 90¡ to the Ñow. When a sample is injectedthe solvent Ñow is switched o†, retaining it at the headof the channel. The applied Ðeld forces the macro-molecules and/or particles to migrate towards thebottom of the channel (the accumulation wall), whiledi†usive transport acts to oppose this motion. At equi-librium, a series of zones are produced with the smallestparticles protruding nearest to the centre of the channel(highest di†usion coefficient) while the largest are keptclosely conÐned at the accumulation wall. When thechannel Ñow is then reapplied, the small particles are ina faster Ñow region than the large ones and are sweptdown the channel ahead of them, e†ecting a size-basedseparation. They then elute from the channel and aredetected using normal chromatographic detectors, suchas UV, to produce a chromatogram where elutionvolume is proportional to hydrodynamic radius.

A variety of Ðelds has been used in commercial FFFsystems, including solvent cross-Ñow, thermal, sedimen-tation and electrical. Each has unique beneÐts for spe-ciÐc applications, and careful choice of Ðeld allows

Fig. 1. Principles of Ðeld Ñow fractionation separation.

parameters other than size to be determined, e.g. sedi-mentation FFF (SedFFF) can be used to measure par-ticle density as well as size, while thermal FFF (ThFFF)has been used to study compositional di†erences acrossthe MWD of a copolymer. With electrical FFF (EFFF),zeta potential can be determined.

MULTI-ANGLE LIGHT SCATTERING (MALS)

Commercial MALS detectors use an array of 18 detec-tors positioned around the Ñow cell to measure theangular dependence of scattering from polymers and/orparticles eluting from the channel in the range 5È171¡(Fig. 2). The intensity of scatter can be directly relatedto the of polymer, and the root mean square radius,M1 w

according to eqns (1) and (2) :3Rg ,

K*cR(h)

\ 1[M1 w P(h)]

] 2A2 c (1)

where

K* \ 4n2n02(dn/dc)2j04 NA

(2)

See Appendix for deÐnitions of terms. P(h) describes theangular dependence of scattering, and at low angles itsvariation depends only on the mean square radiusSRg2T.

To determine the MW for a given elution slice, thescattering is plotted as a function of angle, then extrapo-lated back to zero angle. The intercept value is thenused to determine the absolute of each mono-M1 wdisperse slice, while the slope of the line gives an inde-pendent measure of as shown in Fig. 3.Rg ,

The data for each slice are combined to produce aseries of distribution plots for the sample as a whole,providing absolute particle size distributions which do

Fig. 2. Scattering geometry of MALS Ñow cell.


376 R. J. W hite

Fig. 3. Debye plot from MALS software.

not ignore the presence of particles with a diameter lessthan 100 nm, and absolute MWDs that do not varywith separation conditions.

By combining size and MWD information, a range ofother parameters can be derived, including molecularconformation and a range of branching parameters. Byuse of secondary detectors, UV compositional varia-tions can also be studied, while particle density can bederived using a combination of MALS and classic FFFsize distributions.

APPLICATIONS OF FFF–MALS

Although the combination of FFF with MALS is a rela-tively recent innovation, it has already been used tocharacterise many di†erent types of material that haveproved problematical using traditional techniques.These have included natural and synthetic elastomers

which are a complex mixture of soluble polymer micro-gel and macrogel, protein-based pharmaceutical formu-lations where aggregate content is dependent on thebu†er used during separation, food and pharmaceuticalgrade polysaccharides, cationic and anionic poly-electrolytes, latex particle dispersions, and biologicallyactive colloids of importance in environmental andpharmaceutical research.4h6 Some examples from thislist are presented below.

Elastomers

Two samples from nominally identical batches of poly-butadiene showed unexpected di†erences in their rela-tive viscosities.7 Analysis using traditional SECÈMALSrevealed very little di†erence in their MWD (Fig. 4),while ThFFF (Fig. 5) showed that the low viscositysample had shifted to lower MW, accounting for thedi†erence in processability. Chromatographic condi-

Fig. 4. Di†erential MWD for polybutadiene by SECÈMALS.



Fig. 5. Di†erential MWD for polybutadiene by ThFFFÈMALS.

tions for the SEC were selected to minimise column-induced shear, but it was thought likely that the MWDplots obtained were inÑuenced by degradation duringseparation. ConÐrmation of this would appear to begiven when the (peak molecular weight) of each dis-Mptribution is compared. It can be seen that by SECÈMALS, was of the order of 5 ] 105 g mol~1 whileMpusing ThFFF this increased to in excess of1 ] 106 g mol~1.

Natural rubber is a highly complex material contain-ing a mixture of proteinaceous debris, soluble linear iso-prene, branched isoprene, insoluble microgel, andinsoluble macrogel. Attempts to separate this polymerusing SEC have met with limited success as it is neces-sary to Ðlter out all of the insoluble material beforeinjection, which alters the distribution of molecularspecies present. Secondly, ultra-high MW isoprene is avery fragile molecule that tends to degrade as it passesdown the column, again altering the MWD, as dis-cussed above.

To fully characterise natural rubber, recent studieshave looked at using ThFFFÈMALS to avoid sheardegradation and allow full characterisation of the gel

phases present. Gentle sonication was used to disruptvery large gel particles, but no attempt was made toÐlter the sample prior to injection. Figure 6 shows theresultant di†erential MWD plot for natural rubber incyclohexane, showing that it contains molecules/gelswith a MW of 1010 g mol~1, more than three orders ofmagnitude greater than indicated by SEC. Indeed, ascan be seen from Fig. 7, more than 40% of this samplehad a MW in excess of 107 g mol~1 (typical exclusionlimit for SEC columns).

Proteins

While the MW of proteins has been determined using arange of di†erent chromatographic techniques, e.g.reverse phase liquid chromatographyÈMALS, SECÈMALS and ion exchangeÈMALS, they all have a funda-mental Ñaw associated with their application to thestudy of protein aggregation. Because the mobile phaseused during separation has to be chosen to be compat-ible with the column stationary phase as well as theprotein molecule, it is not possible to be certain that the

Fig. 6. Di†erential MWD for natural rubber separated using ThFFF.


378 R. J. W hite

Fig. 7. Cumulative MWD for natural rubber separated using ThFFF.

aggregates found were present in the original formula-tion, or were formed during the separation through theaction of the bu†er system used to prevent columninteraction. This uncertainty makes the accurate charac-

terisation necessary for safety reasons difficult toachieve.

By applying Ñow FFFÈMALS to the problem it isnot necessary to select bu†ers that suppress interactions

Fig. 8. MW vs. elution volume for BSA showing aggregate peak.

Fig. 9. FFFÈMALS size distribution for Duke standard PS latex of nominal size 100 nm.



with the column packing as there is no stationary phasein the channel. As a result, aggregation phenomena canbe studied in the same mobile phase as the protein for-mulation was produced in, and a true characterisationcan be undertaken.

Figure 8 gives an example of the type of separationobtained for proteins using FFFÈMALS.8 The presenceof both dimer and trimer aggregate forms of the BSAmolecule can be clearly identiÐed from the MW vs.elution volume plot provided from MALS.

Particles

By combining Ñow FFF with MALS, it is possible tostudy particle size distributions that vary by just a fewper cent. The Duke standards shown in Fig. 9 representsix batches of a latex with a nominal size of 100 nm.9Using traditional techniques such as photon correlationspectroscopy (PCS) and classic FFF, these samples areseen to be identical. However, combining FFF withMALS made it possible to split the batches into twodiscrete size distributions with a peak size that di†ers by3 nm.

These applications, while broad, represent only thetip of the iceberg and the coming years will establishthis hybrid technique as a key element in the analysis ofcomplex macromolecular materials as well as sub-micrometre particle dispersions, emulsions, colloids,liposomes, etc.10

CONCLUSIONS

This paper has demonstrated the wide applicability ofFFFÈMALS to the analysis of polymers, biopolymersand particles, and has highlighted the synergistic bene-Ðts of combining two powerful techniques that devel-oped in isolation but are both targeted at thecharacterisation of macromolecules and particles.

REFERENCES

1 Giddings, J. C., Science, 260 (1993) 1456.2 Roesner, D. & Kulicke, W.-M., J. Chromatogr., 687 (1994) 249.3 Wyatt, P., Anal. Chim. Acta, 272 (1993) 1.4 Wittgren, B. & Wahlund, K.-G., poster presented at Mol Mass

1996, 1È4 April, Windermere, UK.5 Wyatt Technology Corporation, Aramide-T hermal FFF Applica-

tion Note, 1996.6 Wyatt Technology Corporation, L iposome Characterization Appli-

cation Note, 1996.7 Foulton, S., Thorpe, W. & White, R., Eur. Rubber J., pp 30È31,

Oct. 1996.8 Roessner, D., Characterization of Proteins by Means of Multi-

Angle L aser L ight Scattering Coupled with Flow Field-Flow Frac-tionation. Wyatt Technology Corporation, March 1995.

9 Shortt, D. W., Studies of V arious Duke and NIST PolystyreneMicro-spheres by MAL L S/FFF. Wyatt Technology Corporation,October 1995.

10 Shortt, D. W., Roessner, D. & Wyatt, P. J., Am. L ab., 21È28 Nov.1996.

APPENDIX

The equation describing the relationship between lightscattering intensity and the absolute weight averagemolar mass of a polymer molecule can be deÐned(M1 w)as :

K*cR(h)

\ 1[M1 w P(h)]

] 2A2 c (1)

where

K* \ 4n2n02(dn/dc)2j04 NA

(2)

c \ polymer concentrationR(h) \ excess Rayleigh scattering factor (light scat-

tered by molecule at an angle (h))M1 w \ weight average molar massP(h) \ scattering function (\1 for molecules smaller

than j0/20)A2 \ second virial coefficient (polymer/solvent con-

stant, normally assumed to be zero except forpolyelectrolytes

n0 \ refractive index of the solventdn/dc \ speciÐc refractive index increment (polymer/

solvent constant)j0 \ vacuum wavelength of the incident light

(690 nm for the MiniDAWN)NA \ AvogadroÏs number

To measure the molar mass of a polymer eluting from aSEC column, measurements of the scattered light aretaken in narrow time slices, typically 1 s, at a series ofangles (three for the MiniDAWN), while a refractiveindex (RI) detector is placed in line to measure the con-centration of the eluting polymer. The light scattering(LS) data are then extrapolated back to h \ 0, where itcan be shown from eqn (1) that :

K*cR(0)

\ 1M1 w

(3)

From SEC it is assumed that molecules separate on thebasis of size and the LS/RI detector combination mea-sures the of these separated molecules as they elute.M1 wBecause the measurement is made over a narrow timeinterval, it is further assumed that, within each slice, thepolymer is monodisperse, i.e.

M1 n\ M1 w \ M1 z\ M (4)

This is used in conjunction with the concentration mea-surements from the RI to plot molar mass distributionsfor a separated polymer. Molar mass averages can becalculated from standard SEC algorithms, i.e.

M1 \;i

NiM

in

;i

NiM

in~1 (5)

n \ 1 for n \ 2 for and n \ 3 forM1 n , M1 w M1 z .


Documents

FFF-MALS—A New Tool for the Characterisation of Polymers and Particles