An Introduction To Gel Permeation Chromatography And Size Exclusion .

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An Introductionto Gel PermeationChromatography and SizeExclusion ChromatographyPRIMER

ContentsStart here3A note on namesSeven things you should know about GPC/SECChapter 1 – What is chromatography?44556PolymersSize mattersHow does GPC/SEC workWho uses GPC/SEC, what for and whyCalibrationsCalculations in GPC/SECTypes of polymer distribution13Solvents and solvent containersOvensSamplesInjection and injectorsColumns and column setsPumpsDetectorsConventional GPC/SECMulti-detector GPC/SECAutomatic data processing181819Chapter 5 – FAQsAppendixRecommendations forsetting up a GPC/SEC system2021Ordering InformationAgilent solutions for GPC/SECGlossary and abbreviationsSuggestions for further reading2427283021Choosing an eluent for GPC/SECChoosing a column for GPC/SECSetting up the GPC/SEC systemWhat standards should I use?Typical polymer molecular weights66788911Chapter 3 – GPC/SEC in practice18Gum arabic, good and badFingerprinting nail varnishModifying PVC4Types of chromatographyGas chromatographyHigh performance liquid chromatographyGel permeation/size exclusion chromatographyChapter 2 – GPC/SEC overviewChapter 4 – GPC/SEC in action;real world applications3313131414141516171717212122222335 years’ expertise in GPC/SEC1990 PL aquagel-OH columnsVastly improve resolution and data qualityin aqueous GPC1981 PLgel MIXED columns,PL aquagel columnsMIXED columns improve data quality, withnovel chemistries for analysis of watersoluble polymers1984 GPC softwareDedicated software streamlinesGPC/SEC calculations1976 PLgel columns, individualstandards and standard kitsPolymer Laboratories founded to developmarket leading products for organic GPC/SEC7677787980818283841900s28586878889909192

Start hereThis guide provides some background to the most commontechniques and applications of gel permeation chromatography,also known as size exclusion chromatography (GPC/SEC), foranyone not familiar with the equipment and methods used in thisimportant analytical technique. As this is a basic introduction youdo not need to know any chemistry to get something useful fromit, though a little knowledge is definitely a good thing. If you wantto know more about the science of GPC, there is a reading list inthe appendix, and a glossary at the back of this guide to explainsome of the most common terms.Seven things you should know about GPC/SECThe guide begins with some basic information in Chapter 1about the history of chromatography and its technologies, butyou can skip this and get straight to the chapters on GPC/SEC ifyou’re in a hurry.4. GPC/SEC separates molecules on the basis of their size,hence ‘size exclusion’.1. Gel permeation chromatography/size exclusionchromatography is a type of high performance liquidchromatography (LC).2. GPC/SEC can be performed in a wide range of solvents.From non-polar organics to aqueous applications.3. GPC/SEC uses columns packed with very small, round, porousparticles to separate molecules contained in the solvent that ispassed through them.5. The first GPC/SEC columns were packed with materialsreferred to as gels, hence ‘gel permeation’.A note on names6. GPC/SEC is used to determine the molecular weightdistributions of polymers.In this booklet, the terms GPC and SEC are used to describethe same chromatography process with the different acronymsbeing used by different industries, but generally speaking whenanalysts discuss GPC and SEC they are referring to the sametype of chromatographic analysis. The International Union ofPure and Applied Chemists (IUPAC) prefer the term SEC forexperiments of this type, but GPC is still in common use.7. The particles in the columns are made from polymers thathave been cross-linked to make them insoluble, or inorganicmaterials, such as spherical silicas.2007 PLgel Olexis columnsOptimized for polyolefin analysis withhighest resolution and data quality for evenultrahigh molecular weight samples2004 PlusPore columns andEasiVial standardsNew chemistries deliver high-pore-volumematerials for increased resolution, andEasiVial standards simplify calibrationprocedures even further1999 PL-GPC 220 instrumentMarket leading high temperature GPCsystem for routine analysis of even the mostdifficult samples by multi-detector GPC93949596972009 1260 InfinityMulti Detector Suiteand PolarGel columnsThe 1260 Infinity MDSturns any LC intoa powerful multidetector GPC system,and PolarGel columnsanalyze polar samplesin any solvent system2003 PL-GPC 50instrument with lightscattering and viscometryCost-effective solution to lowtemperature polymer analysis,including multi-detector GPC/SEC1993 EasiCal standardsNew format shortens sample preparationtime and the speed of calibration98990001021900s0304052000s3062010 Polymer Labsis now AgilentTechnologies07080910

Chapter 1 – What is chromatography?The invention of chromatography, itstechniques and the place of GPC/SECwithin the chromatography familybased on their chemical or physical properties. These includecolor, viscosity, the way the molecules behave when light isshone through them, or whether they carry an electric charge.Not surprisingly, chromatography equipment and techniqueshave also been refined over the years. For example, Tsvet reliedon gravity to move his extracts through the column, but nowhigh-pressure pumps or compressed gases are often used.Chromatography is a separation method used for chemicalanalysis invented by the Russian, Mikhail Tsvet, in 1901. Tsvetground up plant leaves in solvents such as ether and ethanolthat dissolved the chlorophyll and carotenoid pigments in theleaves. He poured the resulting solution into a glass column filledwith solid calcium carbonate, using gravity to draw the solutiondown the column. Colored bands were formed as the pigmentsin the solution moved through the column at different speedsdue to greater or lesser degrees of interaction with the calciumcarbonate, revealing that the extract was made up of differentcomponents. Tsvet was able to collect these different coloredmolecules into separate containers as they dripped or, ‘eluted’,from the bottom of the column.Chromatography is now accepted as probably the most powerfuland versatile analytical technique available because it canseparate mixtures in a single step, and measure the amount ofevery component and their relative proportions.Chromatography instruments are widely used in academia andindustry for research and development of new compounds.The instruments can be complex and expensive, or simple andinexpensive, so much so that practical chromatography canbe done in school. One of the most common chromatographydemonstrations in schools is to separate plant pigments –Mikhail Tsvet would be pleased.This simple experiment demonstrated the great potential ofchromatography in separating mixtures of molecules. Today,samples can be gases, liquids or solids, in simple mixtures or incomplex blends of widely differing chemicals. The solvent canalso be a gas or liquid, depending on the type of chromatography.Types of chromatographyThere are many forms of chromatography, but two are verycommon and well known to all analytical scientists and serveto illustrate the diversity of analytical techniques. These are gaschromatography (GC) and liquid chromatography (LC). GPC/SECis a form of LC. These techniques are described in brief below.Chromatography systems employ a column, capillary or someother container that is filled with a mobile and a stationaryphase, which can be solid, liquid or gas. The stationary phaseremains in position and does not move during the analysis,whereas the mobile phase moves through the container. InTsvet’s experiment, the stationary phase was the calciumcarbonate and the mobile phase was the organic solvent.Gas chromatographyIn GC, the stationary phase is (typically) polydimethylsiloxane(PDMS) applied to the inner surface/walls of a very narrowcapillary tube. A gas such as helium is used as the mobilephase. Volatile samples, or samples volatilized by heating, areintroduced into the gas phase and flow through the column,during which the components of the sample have time to interactwith the stationary phase, mainly through physical interactionsand adsorption. As a result, molecular components of the sampleare separated based on their degree of affinity for the stationaryphase (of which there are a very wide variety which areThe separation occurs because molecules partition between thetwo phases, and the more association the molecule has with thestationary phase, the longer it takes to leave the container. Allthe different forms of chromatography that scientists use todaydescribe the use of different mobile and stationary phases. Youwill come across some examples later.Since the early days, a great deal of work has been done to findthe best method of detecting the components as they exit thecontainer holding the mobile and stationary phases, usually4

application dependent). Components exiting the column are then”detected”, typically using a flame ionization detector or a massspectrometer. GC is the preferred technique for analyzing volatilesamples that contain molecules of differing chemistries.nature of the samples and the phases employed. For example, inone of the most common types of HPLC, the sample componentsinteract with the stationary packing material based on howhydrophobic, or “greasy”, the components are. A mobile phasethat can dissolve these hydrophobic components is then passedthrough the column. Those components that are only somewhatgreasy will easily come off, while those that are more greasy willcome off the column much later.Agilent J&W offers the largest selection of the highest quality GC columnsavailable todayAgilent’s HPLC column range includes Poroshell, ZORBAX, Polaris,PLRP-S, and moreSilica gel is commonly used as a stationary phase in normalphase, adsorption HPLC. Normal phase HPLC works well withcompounds that are insoluble in water, and organic normalphase solvents are more MS ‘friendly’ than some of the typicalbuffers used in reversed phase HPLC. However, the techniquesuffers from poor reproducibility of retention times becausewater or protic organic solvents (which have a hydrogen atombound to an oxygen or nitrogen atom) change the hydration stateof the silica. This is not an issue for reversed phase HPLC, whichhas become the main HPLC technique worldwide. In reversedphase systems, the silica particles are non-polar or hydrophobic,and the mobile phase is a polar liquid. If you need a column forrobust HPLC with high sensitivity, polymeric packings provide analternative to silica based materials.Gel permeation chromatography/size exclusionchromatographyThe Agilent 7890A GC with 7693A Series Automatic Liquid SamplerAs we have seen, GPC/SEC is a type of LC and so solidstationary and liquid mobile phases are again used. However,the separation mechanism here relies solely on the size ofthe polymer molecules in solution, rather than any chemicalinteractions between particles and the stationary phase.High performance liquid chromatographyHPLC is a technique that employs a liquid mobile phase.Stationary phases are typically chemically modified inorganicsilicas or polymeric beads packed into a column. In HPLC, theseparation mechanism that partitions components of a samplebetween the two phases can take many forms, depending on the5

Chapter 2 – GPC/SEC overviewGPC/SEC instruments produceinformation on polymer molecularweight distributionswhen polymers join together is called polymerization.Take, for example, polyethylene – made up of repeating unitsof ethylene (C2H4)n, where n can be a very large number.The interesting thing about polymers is that the length of themolecular chains can be shorter or longer and the compoundwill still be recognizable as the same polymer. In practice, asample of polymer will contain a distribution of molecules ofdifferent lengths. Does this matter? Yes it does!GPC/SEC employs a stagnant liquid present in the pores ofbeads as the stationary phase, and a flowing liquid as themobile phase. The mobile phase can therefore flow betweenthe beads and also in and out of the pores in the beads. Theseparation mechanism is based on the size of the polymermolecules in solution. There are several names given todifferent types of SEC, but all are based on the same principle,that of size exclusion, hence size exclusion chromatography.Historically the porous medium was made of a gel andtherefore gel permeation chromatography was coined, a termstill prevalent in the industry today. Low pressure analysisof biological compounds is often referred to as gel filtrationchromatography (GFC). For our purposes SEC and GPC refer tothe same instrumentation and column technology.Size mattersPlastics, such as those used to make polyethylene bags,polystyrene foam cups, and polypropylene drain pipes, aremade by linking monomers together to form chains. Manyof the useful properties of plastics, such as mechanicalstrength and elasticity, come from the intertwining of theselong molecular chains. Generally, the longer the chains, themore intertwined they are, and the harder and tougher thematerial will be. So, depending on the chain lengths in asample of polyethylene, the material could be liquid, a wax ora rigid solid, with its physical state obviously having a majorimpact on how it is used. In this case, the chemistry of thesematerials is the same – they’re all polyethylene – it’s just thephysical state of the materials that differs. Furthermore, allsynthetic polymers contain a distribution of polymer chainlengths; in fact, it’s impossible to make polymers in whichall the chains are the same length. GPC/SEC is a techniquePolymersPolymers have many physical characteristics that make themattractive to industry, and to us as consumers. Beneficialphysical properties, include hardness, thermal and electricalinsulation, optical properties, and resistance to chemicals.These parameters are influenced by a polymer’s attributes,such as chemistry, molecular structure and shape, molecularweight and the presence of branching. Furthermore, theseparameters can be characterized by measuring some basicattributes. The physical attributes can either be directlyassessed by examination of the finished product or bepredicted from an understanding of the polymer molecules.So what exactly are polymers? Polymers are moleculescomposed of many repeating units joined together. The termis derived from the Greek words polloi (many) and meros(parts). There are a large number of natural polymers fromplants and animals, such as rubber, polysaccharides, starch,cellulose and glycogen. Proteins, nucleic acids and someinorganic large molecules can also be thought of as polymers.The chemical basis for the formation of polymers is the abilityof the single, or ‘monomer’, units to form long chains. Manymolecules can do this, leading to the development of manydifferent types of man made polymers. The chemical reaction6

as they travel down the column. As a result, small polymercoils that can enter many pores in the beads take a long timeto pass through the column and therefore exit the columnslowly. Conversely, large polymer coils that cannot enter thepores take less time to leave the column, and polymer coils ofintermediate size exit the column somewhere between theseexamples. Thus, the way in which the samples elute from thecolumn depends very much on the size of the pores in thebeads. Imagine you are walking with a child through the toysection of a department store. You want to get straight to thecar park, but your small companion wanders off to sample allthe delights on offer, so you will reach the exit straight awaywhile junior takes his time to get there, pausing to investigateall the toys on display. It is the same with GPC/SEC – thelarger bodies get to the exit first.that allows you to separate out the different lengths ofpolymer chain in a sample and measure their relativeabundance. Clearly, the example of the ‘liquid – wax – solid’we mentioned above is an extreme case, but you don’t needmuch of a difference in the distribution of polymer chainlengths to drastically alter its physical properties.GPC/SEC is a technique for measuring the chain lengths andother characteristics of polymers by separating them on thebasis of their size. It’s as simple as that!How does GPC/SEC workA GPC/SEC instrument consists of a pump to push thesolvent through the instrument, an injection port tointroduce the test sample onto the column, a column tohold the stationary phase, one or more detectors to detectthe components as they leave the column, and software tocontrol the different parts of the instrument and calculate anddisplay the results.The separating mechanism is shown in Figure 1. This diagramshows how different sized sample molecules can be excludedcompletely, partially, or not at all from entering the pores inthe particles, depending on the size of the pores and of thesample molecules.The polymer sample is first dissolved in a solvent. This is animportant step, because although polymer molecules canbe described as long chains of monomers linked together,they don’t exist like that in solution. Once they have beendissolved, the molecules coil up on themselves to form acoil conformation, which resembles a ball of string. Soalthough they are chains, when we analyze them byGPC/SEC they behave like tiny spheres, with the size of thesphere dependent on the molecular weight; higher molecularweight polymers coil up to form larger spheres.Polymer chainCoiled in solutionThese coiled up polymer molecules are then introduced intothe mobile phase and flow into the GPC/SEC column. Thedissolved polymer molecules move past the beads as themobile phase carries them down the column. As the polymercoils move past each bead, several things can happen. If thepolymer coils are much larger than the biggest pores in thebeads, they cannot enter the pores and so are carried straightpast by the mobile phase. If the polymer coils are a littlesmaller than the biggest pores they can enter the larger, butnot the smaller pores as they pass by, occupying some, butnot all of the available stationary phase. If the polymer coilsare smaller than the smallest pores in the beads, then theycan enter any of the pores and so can potentially occupy allof the stationary phase. As the molecules enter the column,this partitioning occurs repeatedly, with diffusion acting tobring the molecules into and back out of any pores they passPorous structureof polymer beadsKeySmaller coils can access many poresLarger coils can access few poresVery large coils access very few poresFigure 1. How GPC/SEC separates molecules ofdifferent sizes7

As the components exit the column they are detected invarious ways, and the elution behavior of the sample isdisplayed in a graph, or chromatogram. The chromatogramshows how much material exited the column at any onetime, with the higher molecular weight, larger polymer coilseluting first, followed by successively lower molecular weight(and therefore smaller) chains emerging later. The primaryseparation is according to elution volume. This is convertedto time for ease of measurement, on the assumption that youhave a constant flow rate. The time it takes for a group ofmolecules of the same size (a fraction) to emerge from thecolumn is called the retention time, because the moleculeshave been retained on the column.To avoid damage to fragile biological compounds duringchromatography, biologists and biochemists typically uselow pump pressures and columns packed with a gel, suchas polyacrylamide, dextran or agarose. The advantage of thetechnique for these scientists is that the biological activityof the compounds is not destroyed and so the fractions thatcome out of the column can be used in other experiments,though the technique is not very efficient. On the other hand,polymer chemists and engineers in industry are more likely touse high pump pressures on columns filled with cross-linkedpolystyrene or silica, as this gives higher resolution andbetter results.GPC/SEC is frequently combined with additional methodsthat further separate molecules by other characteristics, suchas their acidity, basicity, charge or affinity.The data that produced the chromatogram is then comparedto a calibration that shows the elution behavior of a seriesof polymers for which the molecular weight is known. Thisallows the molecular weight distribution of the sample tobe calculated, providing important information for polymerchemists because they can use the distribution to predicthow the polymer will perform.Now that we know how GPC/SEC works, here’s some detailon how we calculate molecular weights.CalibrationsAs we have seen, to determine the molecular weights of thecomponents of a polymer sample, a calibration with standardpolymers of known weight must be performed. Values fromthe unknown sample are then compared with the calibrationgraph to generate molecular weights and molecular weightaverages. Standards are now available in a wide range ofmolecular weights, and as kits and individual molecularweights for maximum choice. Not surprisingly, standardsneed to be of very high quality and with extremely narrowmolecular weight distributions so that the position of thetop of the peak, the peak molecular weight (Mp - seeFigure 3) can be assigned with confidence. It is the Mp valuewe use to set up the calibration. For example, Agilent’scurrent polystyrene at MW 1,000,000 g/mol has a very narrowdistribution, or polydispersity index of 1.05 (we’ll come on topolydispersity soon).One important thing to bear in mind about GPC/SEC – theseparation is based on size, not chemistry. These techniquesgive information regarding the size of polymer molecules insolution that are converted into molecular weights throughthe use of a calibration. They don’t tell us anything aboutthe chemistry of the sample, or even if the sample hascomponents of different chemistries. GPC/SEC is purely aphysical partitioning of the sample on the basis of size.Who uses GPC/SEC, what for and whyGPC/SEC has two main uses – to characterize polymers andseparate mixtures into discrete fractions, such as polymer,oligomer, monomer and any non-polymeric additives.GPC/SEC is the only technique available to characterizethe molecular weight distribution of polymers, a property allsynthetic polymers have. Furthermore, the polymer mixturecan be separated into individual components, such as polymerand plasticizer. Naturally occurring polymers such as lignins,proteins and polysaccharides are routinely investigatedusing GPC/SEC in polar organic or aqueous solvents.GPC/SEC is also excellent for separations of oligomers andsmall molecules.Figure 2 shows a calibration curve from an EasiVialpre-weighed polymer standards kit from Agilent.You can see that the molecular weight is determined from thecalibration curve by simply noting the retention time (RT) ofthe sample, and reading the molecular weight on the vertical8

,000100,00010,0001,0001001517192123252729Retention time/minRetentiontime (min)Figure 2. A calibration graph used to determine the molecular weight of a polymer from its retention timeThe commonly calculated average is actually called thenumber average molecular weight, abbreviated to Mn.Looking at the distribution in Figure 3 we can see that theMn value marks the value at which there are equal numbersof molecules on each side, at higher and lower molecularweight. The value of Mn influences the thermodynamicproperties of the molecule. There are several other waysof describing molecular weight average, including weightaverage molecular weight (Mw). Mw is defined as the valueaxis that corresponds to this time. To generate a molecularweight distribution, we cut the peak into a number of equallyspaced ‘slices’. We measure the abundance of each slicefrom the peak height or area at that point, and the molecularweight of the slice from the calibration. We then performsome summations to get our averages – more on this below.It is also possible to measure sizes directly by using someform of detector that reacts directly to the molecular weightof the components as they leave the column. These detectorsrely on the physical properties of the polymer, such as theirability to scatter light, or their viscosity.Mp MwCalculations in GPC/SECMnWeight fractionIn polymers, as we have already seen, molecular weightoccurs not as a discrete value but as a distribution. Thismeans that to accurately assess the molecular weightdistribution of a polymer, we have to count how manyparticles there are at every weight in the distribution, andfrom these counts, calculate an average figure that describesthe whole sample. Figure 3 shows the typical position ofmolecular weight average.MzMz 1Molecular weightFigure 3. The average molecular weights of a mono-modal polymer – in thiscase the distribution is nearly symmetrical9

values. This is a common mathematical method ofcompressing the length of a graph’s axis when very largenumbers are involved, and the molecular weight of polymerscan exceed 10,000,000 g/mol.at which there are equal masses of molecules on each side,at higher and lower molecular weight. Mw is large-moleculesensitive and influences the bulk properties and toughnessof the polymer. Unsurprisingly, the Mw value is alwaysgreater than the Mn value unless the polymer is completelymonodisperse. Mw affects many of the physical propertiesof polymers, and is the most often quoted molecular weightaverage. As well as Mn and Mw, there are other molecularweight averages that take increasing account of the highermolecular weight components of the sample, such asz-average molecular weight (Mz) and Mz 1. Mz is sensitive toeven larger molecules and influences viscoelasticity and meltflow behavior. The ratio of Mw to Mn is used to calculatethe polydispersity index (PDI) of a polymer, which providesan indication of the material’s range of molecular mass. Thebroader the molecular weight distribution, the larger the PDI.The molecular weight values of a polymer are important, sincethey influence properties such as brittleness, toughness, andelasticity. Slight differences in these values can cause majordifferences in the way a polymer behaves and determine itssuitability for a particular industrial use. Figure 4 illustrateshow molecular weight influences the properties of a polymer.You can see from this example that knowledge of themolecular weight distribution of a polymer allows chemiststo predict how it will behave. This information is obviouslyvaluable before moving from laboratory R&D scale to fullcommercial production because it shows whether the endproduct of a production run will be able to meet the industrialspecification required. In this way, expensive mistakes canbe avoided.However, obtaining all these numbers is not as easy as itseems. On their own, standard GPC/SEC detectors cannotcount the number of molecules that elute from the column, sothese weight averages cannot be measured directly.Nonetheless, we can measure the concentration of moleculeson a weight/volume basis, and calculate the weight averagesfrom the concentration using a concentration detector, suchas one that measures the differential refractive index.Substituting concentration units for numbers in the equationsallows us to recalculate Mn, Mw and Mz, based on theconcentration units instead. All of these averages help build apicture of the nature of a polymer and provide information onits likely behavior, as we can see in Figure 4. Molecularweights on the horizontal scale are often expressed in logFigure 4. The effects of molecular weight on the properties of polymers10

Types of polymer distributionso having a very narrow molecular weight distribution isbeneficial. In this case, the molecular weight averages arevery close to each other. The distribution is nearly normalin shape, i.e. the areas under the curve on either side of thecenter line are equal.Polymer distributions can be wide with lots of high molecularweight and low molecular weight components, or narrow withmost of the components grouped around the same molecularweight. In chromatographic terms this is measured by thedispersity of the polymer. Dispersity values are importantguides to polymer behavior. The most common synthetic polymers have a mediumwidth distribution, with an Mw/Mn ratio between 1.2and 3, because many of the synthetic procedures usedtend statistically towards these values. Monodisperse polymers consist of molecules all with thesame molecular weight where the values of Mn, Mw andMz are identical. In practical terms, the only monodispersepolymers we come across are those found in nature, suchas proteins and nucleic acids. Broad distribution polymers may also be synthetic, ornatural such as polysaccharide and starch carbohydrates.These distributions are rarely normal in shape; for example,they may have a long tail towards the lower molecularweight. The presence of the low molecular weightcompounds shifts Mn more than Mw and Mz. Narrow distribution polymers are synthetic compoundswhere we try and make all the chains as close in molecularweight as possible. The Mw/Mn polydispersity ratio is lessthan 1.2, a subjective but handy definition of a polymer witha narrow distribution. Examples include polymers used asstandards for calibrations – here we want to determine theposition of the top of the peak as accurately as we can, Sometimes synthetic polymers have multimodaldistributions and this is where it gets interesting. Oneexample is shown in Figure 5. Importantly, the values ofthe molecular weight averages do not reveal in themselvesthat the sample is multimodal – you have to look at themolecular weight distribution to see that.The molecular weight averages and the ratio of Mw to Mnare thus very useful in revealing the width of a polymerdistribution, but you need to look at the molec

There are many forms of chromatography, but two are very common and well known to all analytical scientists and serve to illustrate the diversity of analytical techniques. These are gas chromatography (GC) and liquid chromatography (LC). GPC/SEC is a form of LC. These techniques are described in brief below. Gas chromatography

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