Facing the Challenge of

A PRIMER IN 2-DIMENSIONAL LIQUID CHROMATOGRAPHY (2D-LC)FOR

DETERMINING THE MOLECULAR WEIGHT AND COMPOSITIONALDISTRIBUTIONS OF COMPLEX POLYMERS

Analyzing Complex Polymers

POLYMER ST ANDARDS SERVICE

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Advances in polymer synthesis techniqueshave resulted in a proliferation of new com-plex polymers (block copolymers, graftcopolymers, blends etc.) with predeterminedchemical composition, functionality and archi-tecture. The task of characterizing thesepolymeric materials has now become a chal-lenge, since no single analytical techniqueprovides adequate information regarding allthe different distributions. Analyticalchemists have thus opted for hyphenatedtechniques such as two-dimensional liquidchromatography (2D-LC) in which two liquidchromatography methods are combined toachieve selective separations according tothe various distributions.

The increased use of hyphenated techniqueshas prompted Polymer Standards Service(PSS) to publish this primer on the imple-mentation of a 2D-LC technique. PSS hascommercially pioneered the development ofa unique software package that has thecapability of performing 2D-LC analysis.WinGPC Unity allows data acquisition andcontrol of fraction transfer between two liquidchromatographic systems.

This primer, "Facing the Challenge ofAnalyzing Complex Polymers" explains thebasic concepts, the reasons to use 2D-LC,how it works, what you need, and what PSSoffers in the way of equipment and supplies."Facing the Challenge for Analyzing ComplexPolymers" is divided into four main sections:

1. Basic Concepts Review

Complex polymers

High Performance LiquidChromatography (HPLC), and the kind ofinformation it yields

Gel Permeation Cromatography(GPC),and the kind of information it pro-vides

2. Why use hyphenated techniques

When it is useful to combine GPC andHPLC into one technique

Why use HPLC for 1st- DimensionSeparation

3. How 2D-LC works

Schematic diagram of a 2D-LC system

How to collect and process data

What the processed data look like

Minimum Requirements for a 2D-LCSystem

4. 2D-LC Sources and Resources

Reference publications about 2D-LCAnalysis of Complex Polymers

Where to purchase a 2D-LC System

Who is PSS

1

INTRODUCTION

COMPLEX POLYMERS

Complex polymershave more than onedistribution. They aredistributed accordingto MOLAR MASS,CHEMICAL COMPO-SITION, FUNCTION-ALITY, and ARCHI-TECTURE. A copolymer, forexample, has a molar mass distribution(MMD) owing to the various chain lengths.It has a chemical composition distribution(CCD), reflecting the comonomer units thatare present in varying amounts. It presentsmolecular architecture distributions (MAD),which are associated with the polymer struc-ture: linear, star-shaped, comb-like, graftedor branched polymer. Finally, functionalitytype distributions (FTD) occur when polymerchains have differing end-groups.

HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY (HPLC) AND THE KINDOF INFORMATION IT YIELDS

High Performance Liquid Chromatography,(HPLC), has been the technique of choice formany years to both identify and quantifychemical compounds that are usually solid,or liquids that have boiling points too high toanalyze by gas chromatography. The material to be analyzed is dissolved in asuitable solvent and injected onto a columnthat is usually silica-based, and most oftencontains a bonded phase that can interact(via partitioning or adsorption) with the com-ponents. Depending on the chemistry ofthese interactions between the eluting com-ponents and the bonded phase, the resultingchromatogram will show a clear separation ofthe component mixture. The materials that

have little affinity or exhibit little interactionwith the column will elute first, followed bythe other materials that interact more strong-ly with the column (usually adsorption), andelute later. The stronger this interaction, thelater the elution occurs.Commonly, the mobile phase (or eluent) isnot a single solvent that does not changecomposition throughout the analysis. Usually,a second (or more) solvent is brought in tochange the polarity of the eluent and forcelater eluting species that interact stronglywith the column to elute more quickly toshorten analysis time (gradient mode). Theflow rate may also be changed during thecourse of the separation (flow programming),which will also help to optimize the analysistime and still maintain good peak capacityand resolution. An ultraviolet, (UV) or evapo-rative light scattering detector (ELSD) is usu-ally used.

GEL PERMEATION CHROMATOGRAPHY (GPC)AND THE KIND OF INFORMATION IT PROVIDES

Gel permeation chromatography, (GPC), alsoreferred to as size exclusion chromatography(SEC), has been the technique of choice formore than 30 years to obtain the molecularweight distribution of macromolecules. Othertechniques, such as osmometry, end-groupanalysis, mass spectrometry, etc., have also

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BASIC CONCEPT REVIEW

Architecture

Linear, branched,grafted, cyclic, star

Molar Mass

classical chain lengthdistribution

Chemical Composition

comonomers present in different proportions

Functionality

Chains have same ordifferent end-groups

Architecture

Linear, branched,grafted, cyclic, star

ArchitectureArchitecture

Linear, branched,grafted, cyclic, star

Molar Mass

classical chain lengthdistribution

Chemical Composition

comonomers present in different proportions

Functionality

Chains have same ordifferent end-groups

been used for low molecular weight materi-als, but for polymers with molecular weightsexceeding ~50,000, the GPC or SEC tech-nique is still the best choice. The GPC or SEC technique involves theseparation of the polymeric material accord-ing to its hydrodynamic volume, which in turngives information regarding the various molarmass averages (Mn, Mp, Mw and Mz) andpolydispersity, (Mw/Mn), of the polymer.

The molar mass averages are simply statisti-cal moments of the molar mass distribution,and can be used to make correlations withimportant physical properties and processingparameters. For example, Mn, the numberaverage molecular weight, is the first statisti-cal moment, and can be used to predict apolymer's flow properties and brittleness. Thesecond statistical moment, Mw, (the weightaverage molecular weight), is directly relatedto the strength properties of a polymer, suchas modulus, tensile strength and impactresistance. Mz is the third statistical momentof the distribution, and is used to make pre-dictions about a polymer's flexibility and elon-gation, important parameters for elastomers.In SEC the polymer sample passes througha column, which is packed with porousbeads. As the polymer chains pass throughthe column, the longer polymer chains areexcluded from many of the pores, and travelbetween the beads and reach the columnexit more quickly. The short polymer chainsare capable of passing, (permeating) through

the pores of the beads and experience alonger residence time in the column and as aconsequence elute later from the column.With SEC there is usually at least twocolumns connected in series to provideenough pore volume to do the analysis accu-rately. After exiting the last column, the sep-arated polymer passes into a concentrationdetector, usually a differential refractometer.SEC is a relative method; that is, the columnset has to be calibrated with standards ofknown molar masses. The standards arereferred to as "narrow standards", whichmeans that the polydispersities (Mw/Mn), are<1.20. The calibration curve is constructedusing the Mp values, plotting log molar massversus elution volume. . The molecularweight averages obtained are relative to thecalibration molecule used. The major advantage of the technique is itscapability of analyzing polymers over a largemolar mass range; from 102 to >107 g/mol.The SEC technique works well whenhomopolymers are analyzed since for thesethe hydrodynamic volume is directly relatedto the molar mass.

The analysis of copolymers is more complex.Since the hydrodynamic volume in the caseof copolymers is related to both the molarmass and chemical composition, thus SECalone is insufficient for providing chemicalcomposition information within the copoly-mer.

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SEC SEPARATION OF STAR COPOLYMER HPLC GRADIENT

P. Kilz, Chromatographia 2003, 58, July (No.1/2)

Complex polymer topologies, polymer blendsand multi-component formulations require adifferent approach to perform a propermolecular characterization than simple poly-mers. In 2D chromatography different sepa-ration techniques are used to avoid co-elu-tion of species and to measure molar massand chemical composition in a truly inde-pendent way.A comprehensible advantage of coupling twochromatographic techniques over performingtwo isolated methods is following: Even if thesingle runs show good separations, forexample one according to composition andone according to molar mass, then it is stillnot possible to correlate e.g. one specialcomposition to a certain molar mass. In fact,there are numerous possible combinationswhich give the same one-dimensional chro-matograms. This is also depicted below,where schematic contour plots with differentcompounds show the same projections to theX- and Y-axes. Those contour plots can beeasily produced when both separation tech-niques (e.g. HPLC and GPC) are coupled.Besides this easy visualization, the benefitsof 2D chromatography versus one dimen-sional separation techniques can be quanti-fied as well:

It is obvious that n independent molecularproperties require n-dimensional methods foraccurate (independent) characterization of allparameters. Additionally, the separation effi-ciency of any single separation method islimited by the efficiency and selectivity of thisseparation mode, i.e. the plate count N of thecolumn and the phase system selected.Adding more columns will not overcome theneed to identify more components in a com-plex sample, due to the limitation of peakcapacities, n. The corresponding peakcapacity in an n-dimensional separation issubstantially higher due to the fact that eachdimension contributes to the total peakcapacity as a factor and not as an additiveterm for single dimension methods:

where ntotal represents the total peak capaci-ty, ni the peak capacity in dimension i andσi is the "angle" between two dimensions; fororthogonal separations this angle will be 90°and the peak capacity will be maximized.

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WHY USE HYPHENATED TECHNIQUES

CONTOURPLOTS OF DIFFERENT POLYMER BLENDS WITH THE SAME CHROMATOGRAPHIC BEHAVIOUR

The angle between dimensions is deter-mined by the independence of the methods;a 90 degree angle is obtained by two meth-ods, which are completely independent ofeach other and will e.g. separate two proper-ties solely on a single parameter withoutinfluencing themselves.

WHEN IT IS USEFUL TO COMBINE GPC ANDHPLC INTO ONE TECHNIQUE

It is useful to combine techniques when asingle technique does not provide you withthe entire picture. See the following example.

Two thermoplastic elastomers from differentvendors were analyzed using SEC. The

chromatograms and the SEC results don'tshow any differences between the two rawmaterials, but one of them gives a poor finalproduct when used.

A 2D-LC separation performed on the ther-moplastic elastomers show that they also dif-fer in their chemical composition distribution,which influences the integrity of the finalproduct.

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Vendor A Vendor B

Mn[kD] 99 90

Mw[kD] 109 103

Mw/Mn 1.08 1.14

Mp[kD] 108 104

by product 0.8% 1.7%

VENDOR A SHOWS NARROW CCD VENDOR B SHOWS TETRAMODAL CCD

WHY USE HPLC FOR 1ST- DIMENSIONSEPARATION

HPLC is one of the most widely used analyti-cal techniques owing to its capability of ana-lyzing a diverse range of compounds accord-ing to chemical composition. Its popularitycan be attributed to the large variety of com-mercially available columns having stationaryphases with varying polarities, the choice ofselecting a combination(s) of mobile phasesfor achieving the desired separation and theavailability of literature collected over thepast decades.

HPLC is considered as the method of choicefor separating polymers according to chemi-cal composition; however, the techniquedoes not provide information regarding molarmass averages and polydispersities for poly-mers.

ILLUSTRATION:

The SEC trace of a block copolymer shows asingle peak with tailing at the low molar massend. In other words there's polymeric materi-al that's not being separated.

By using HPLC at critical conditions, twopeaks that are attributed to the block copoly-mer and homopolymers are clearly resolved.

Using HPLC in the first method of separationallows greater flexibility for manipulating theliquid chromatographic conditions, i.e. HPLC

can be conducted at isocratic or gradientconditions or by using liquid chromatographyat critical conditions (LCCC).

The various LC conditions are an advantagesince this enhances probability of achievinggood separation of complex polymersaccording to chemical composition and/orfunctionality.

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A typical 2D-LC system consists of a HighPerformance Liquid Chromatography HPLCsystem (1st dimension) with its appropriatecolumns and mobile phases, and a (SEC)system (2nd dimension). These are coupledtogether via an automatic transfer valve,which allows the transfer of fractions fromthe 1st dimension to the 2nd dimension.In some cases, workers have used HPLC inboth dimensions, such as reverse phase inthe 1st dimension, and normal phase (oreven ion chromatography), in the 2nd dimen-sion. There is no restriction to just utilizingHPLC/GPC for the two dimensions, althoughthat is the technique most commonly used.

In 2D-LC analysis a polymeric sample isinjected into the HPLC system and passesthrough the HPLC column where it is sepa-rated according to its chemical compositioni.e. homopolymers, copolymers and additivesare separated depending on the degree oftheir interaction with the HPLC column. Asthe effluent leaves the HPLC column it is col-lected into storage loops (e.g. 100 µl or 200µl) for a pre-determined collection time andthen injected automaticallyinto the SEC system. Thefractions then passthrough the SEC columnwhere the polymeric mate-rial is separated accordingto its hydrodynamic vol-ume (molar mass). As thepolymeric material leavesthe SEC column it isdetected with a sensitivedetector such as a Ultra-Violet (UV) and/or anEvaporative LightScattering (ELSD) detec-tor. Since this is an onlinetechnique, minimum oper-ator time is required. For successful operation

of the 2D-chromatographic system it isimportant that fractions (after being separat-ed by the HPLC column) pass through theSEC system quickly before the next fractionis injected in order to avoid the overlappingof peaks of two chromatograms in the 2nddimension. This requires that the flow rate inthe 2nd dimension be extremely fast in com-parison to the1st dimension flow. Since oneconventional SEC column requires an analy-sis time of about 14 minutes (at a flow of1ml/min) the analysis time for a single 2D-analysis can be as long as ~8 hours.However, with the advent of High-SpeedSEC columns, flow rates of 5ml/min allowfractions to pass through the 2nd dimensionwithin 2 minutes, resulting in much faster 2D-LC analysis. A 2D run can be completed inabout one hour. The High-speed columnshave lower back-pressures and use theequivalent amount of mobile phase as theconventional SEC columns. The 2nd dimension shall be calibrated usingappropriate standards of which narrowly dis-perse polystyrene or poly (methyl methacry-late) are commonly used.

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HOW 2D-LC WORKS

WHICH CORRELATION OF THE DIFFERENT COMPONENTS CAN BE MADE TO THE SINGLEDIMENSIONS OF HPLC (CHEMICAL COMPOSITION: CC) AND SEC (MOLAR MASS: MM) ?

MIXTURE OF 3 COMPONTENTSMIXTURE OF 2COMPONENTS

SCHEMATIC DIAGRAM OF A 2D-LC SYSTEM

HOW TO COLLECT AND PROCESS DATA

Polymer Standards Service has developed aunique software package that has a separatemodule especially for 2D-LC analysis. Thiseasy-to-use software package, WinGPCUnity, allows data acquisition and control offraction transfer between two liquid chro-matographic systems. In addition, the rawdata is presented as 2-dimensional contouror 3-dimensional surface plots, which uponintegration yield molecular weight averages(Mn, Mp and Mw) and quantification ofspecies present in complex samples. Othersalient features in the software are:

1 Data acquisition from a variety of LC detectors

2 HPLC and SEC performed in a single run

3 Requires minimum operator intervention which saves time

4 Step-by-step 2D-Wizard guides you through the instrumentation set-up procedure

5 Saving and re-calling integration grids for future analysis

6 Calibration of both X- and Y-axes according to molecular weight, % composition etc.

7 360º angle rotation of 2D-contour and 3D-surface plots

8 The SEC and HPLC traces are projected along their respective axes

9 Overlaying of 2D-contour plots from different analyses for comparison

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Degasser

Pump

Degasser

Pump

Injector

HPLCcolumn

Detector

DataProcessing

DataProcessing

Waste

SEC Column

1. Dimension:HPLC/LC-CC

2. Dimension:GPC / SEC

WHAT THE PROCESSED DATA LOOKLIKE

The contours represent the variousspecies present in the copolymersample. The SEC is plotted alongthe x-axis and represents themolar mass distribution for thecopolymer sample, but does notprovide information about thechemical composition of the sam-ple. The HPLC chromatogram(representing chemical composi-tion distribution) is plotted alongthe Y-axis and shows four chemi-cally different species present inthe sample. Each contour is inte-grated to yield the molar massaverages for each species; thepolydispersity (PDI) for eachspecies is calculated from the ratioMw/Mn. The relative amounts ofeach species, in terms of area orvolume, are also listed.

MINIMUM REQUIREMENTS FOR A2D-LC SYSTEM

HPLC pump (isocratic or gradient)Manual injector or autosamplerSEC/GPC pump (isocratic)HPLC columns (PSS)PSS high speed SEC/GPCcolumns (PSS)Detector(s) (UV, ELSD or both) Universal Dater Center UDC810,A/D converters, fiber optic cables,8-port transfer valve with 2 storageloops and transfer valve actuatorWinGPC Unity software with 2Dchromatography module, interface

(All equipment provided by PSSwhen not available)

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REFERENCE PUBLICATIONS ABOUT 2D-LCANALYSIS OF COMPLEX POLYMERS

1 Balke, S.T.; Patel, R.D.; J. Polym. Sci. Polym.Lett 18 (1980) 453-456.2 Augenstein, M.; Stickler, M.; Makromol. Chem.191 (2) (1990) 415-428.3 Mori, S. J. Chromatogr. A 503 (1990) 411-419.4 Glöckner, G., Gradient HPLC of Copolymersand Chromatographic Cross-Fractionation,Springer: Berlin Heidelberg, 1991.5 Kilz, P; Krüger, R.P.; Much, H.; Schulz, G.;Polym. Mater. Sci. Eng. 69 (1993) 114.6 Kilz, P.; Krüger, R.P.; Much, H.; Schulz, G.;Advan. Chem. Ser. 247 (1995) 223-241.7 Schunk, T.C.; Long, T.E.; J. Chromatogr. A 692(1995) 221-232.8 Pasch, H.; Trathnigg, B.; HPLC of Polymers;Springer: Heidelberg, 1997.9 Adrian, J.; Braun, D.; Pasch, H.; LC-GC Int. 11(1998) 32.10 Adrian, J.; Braun, D.; Pasch, H.; Angew.Makromol. Chem. 267 (1999) 82–88.11 Schure, M.R.; Anal. Chem. 71 (1999) 1645-1657.12 Kilz, P.; Pasch, H.; Encyclopedia of AnalyticalChemistry, (R.A. Meyers, ed.) Wiley, Chichester,2000.13 Siewing, A.; Schierholz, J.; Braun, D.; GoetzHellmann, G.; Pasch, H. Macromol. Chem. Phys.202 (2001) 2890-2894.14 Pasch, H.; Mequanint, K.; Adrian, J. e-Polymers 005 (2002).15 Biela, T.; Duda, A.; Penczek,S.; Rode, K.;Pasch, H. J. Polym Sci, Part A: Polym Chem.40(16) (2002) 2884-2887.16 Graef, S.M.; van Zyl, A.J.P.; Sanderson, R.D.;Klumperman, B.; Pasch, H. Journal of AppliedPolymer Science 88 (2003) 2530-2538.17 Siewing, A.; Lahn, B.; Braun, D.; Pasch, H.; J.Polym. Sci. Polym. Chem. 41 (2003) 3143.18 Heinz, L.C.; Siewing, A.; Pasch, H.; e-poly-mers 065 (2003).

19 Kilz, P. Chromatographia 58 (2003) 1-12 .20 Li, M.; Jahed, N. M.; Min, K.; Matyjaszewski,K. Macromolecules 37 (2004) 2434. 21 Lutz, J.-F.; Jahed, N.; Matyjaszewski, K. J.Polym. Sci., Part A: Polym. Chem. 42 (2004)1939.22 Pasch, H.; Adler, M.; Rittig, F.; Becker, S.Macromol. Rapid Commun. 26 (2005) 438–444.23 Jiang, X.; van der Horst, A.; Lima, V.;Schoenmakers, P.J. J. Chromatogr. A 1079 (2005)51-61.24 Adler, M.; Rittig, F.; Becker, S.; Pasch, HMacromol. Chem. Phys. 206 (2005) 2269-2277.25 Pasch, H.; Adler, M.; Knecht, D.; Rittig. F;Lange, R. Macromol. Symp. 231 (2006) 166-177.

WHERE TO PURCHASE A 2D-LC SYSTEM

PSS can provide you with all of the neces-sary components, including software, the col-umn chemistry, valving and transfer loops, do

the installation, and show you how to operatethe 2D-LC system. You need to provide theLC instrument for the 1st dimension, (injectorand gradient pump), and a pump for the 2nddimension (GPC separation), as well as adetector, (usually an ELSD, but a UV detec-tor is also used on occasion.).

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SOURCES AND RESOURCES

Ordering Information WinGPC Unity

Software 400-0073

Module for 2DChromatography 400-1008

Universal Data CenterUDC810 401-0018

Upgrade 1, to 2 GPC 400-0301

8-Port transfer Valve 409-9012

Optional ReportDesigner 400-1001

WHO IS PSS

Polymer Standards Service GmbH (PSS) iscertified DIN EN ISO 9001 to produce highquality Reference Polymers, GPC Columnsand Software for the characterization of poly-mers by their molecular weight and theirstructural characteristics. PSS employs thelatest findings in polymer science for the syn-thesis and characterization of polymers,copolymers and biopolymers.

PSS operates a manufacturing facilityequipped with a complete state-of-the-art characterization laboratory at the headquar-ters in Mainz (Germany), fully supporting

customers working under stringent require-ments, i.e. GLP, DIN, ISO 9000x certifica-tions.

Polymer Standards Service-USA Inc. (PSS-USA, Inc.) is the sales and service partner ofPolymer Standards Service GmbH (PSS) inNorth America for the US, Canada and LatinAmerica customers since 1994. PSS partners with other product manufactur-ers such as Agilent, Brookhaven, WGE,among others, to integrate the products nec-essary to place at your service the mostcomprehensive Gel PermeationChromatography solution. Contact usthrough phone, fax or e-mail

PSS IS ONE OF THE WORLD’S LEADING COMPANIES OFFERINGSOLUTIONS FOR POLYMER CHARACTERIZATION

Reference Polymer StandardsGPC/SEC StandardsCertified reference materialsMALDI KitsViscosity and light scattering validation kitsReadyCal kitsDeuterated polymersTailor made polymers

GPC/SEC ColumnsFor organic and aqueous eluentsFor high and low molecular weight synthethic and bio-polymersVarious dimensionsFor HighSpeed polymer and bio-polymer analysis

SoftwareWinGPC UnityPorocheck- for pore size analysis

Analytical ServicesMolar mass determinationStructuretnalysisMethod development and transferComplete product de-formulation

GPC Schools and SupportGPC and software trainingGPC in-house trainingUser meetingsNet community & applications

GPC InstrumentsComplete systems and componentsLight scattering detectorsViscosity detectorFTIR/MALDI interfacedn/dc Instrumentation

Polymer Standards Service GmbHPOB 3368

D-55023 MainzPhone: +49(0)6131962390

Fax: +49(0)61319623911e-mail: info@polymer.de

Polymer Standards Service-USA Inc43 Jefferson Blvd

Warwick, RI 02888, USAPhone: 401-780-8884

Fax: 401-780-8824email: info@pssgpcshop.com

Contact your local distributor

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