Gel permeation chromatography (GPC) is a type of
size-exclusion chromatography
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules ...
(SEC), that separates
analyte
An analyte, component (in clinical chemistry), or chemical species is a substance or chemical constituent that is of interest in an analytical procedure. The purest substances are referred to as analytes, such as 24 karat gold, NaCl, water, etc. ...
s on the basis of size, typically in organic solvents. The technique is often used for the analysis of
polymers
A polymer (; Greek '' poly-'', "many" + ''-mer'', "part")
is a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits. Due to their broad spectrum of properties, both synthetic an ...
. As a technique, SEC was first developed in 1955 by Lathe and Ruthven.
[Lathe, G.H.; Ruthven, C.R.J. The Separation of Substance and '1956', ''62'', 665–674. ] The term ''gel permeation chromatography'' can be traced back to J.C. Moore of the
Dow Chemical Company
The Dow Chemical Company, officially Dow Inc., is an American multinational chemical corporation headquartered in Midland, Michigan, United States. The company is among the three largest chemical producers in the world.
Dow manufactures plastic ...
who investigated the technique in 1964. The proprietary column technology was licensed to
Waters Corporation
Waters Corporation is a publicly traded Analytical Laboratory instrument and software company headquartered in Milford, Massachusetts. The company employs more than 7,800 people, with manufacturing facilities located in Milford, Taunton, Massachu ...
, who subsequently commercialized this technology in 1964.
[Moore, J.C. Gel permeation chromatography. I. A new method for molecular weight distribution of high polymers. ''J. Polym. Sci.'', 1964, ''2'', 835-84]
GPC systems and consumables are now also available from a number of manufacturers. It is often necessary to separate polymers, both to analyze them as well as to purify the desired product.
When characterizing polymers, it is important to consider the
dispersity
In chemistry, the dispersity is a measure of the heterogeneity of sizes of molecules or particles in a mixture. A collection of objects is called uniform if the objects have the same size, shape, or mass. A sample of objects that have an inconsi ...
(''Đ'') as well the
molecular weight
A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...
. Polymers can be characterized by a variety of definitions for molecular weight including the number average molecular weight (M
n), the weight average molecular weight (M
w) (see
molar mass distribution The molar mass distribution (or molecular weight distribution) describes the relationship between the number of moles of each polymer species (Ni) and the molar mass (Mi) of that species. In linear polymers, the individual polymer chains rarely have ...
), the size average molecular weight (M
z), or the viscosity molecular weight (M
v). GPC allows for the determination of ''Đ'' as well as M
v and, based on other data, the M
n, M
w, and M
z can be determined.
How it works
GPC separates based on the size or
hydrodynamic volume
The Stokes radius or Stokes–Einstein radius of a solute is the radius of a hard sphere that diffuses at the same rate as that solute. Named after George Gabriel Stokes, it is closely related to solute mobility, factoring in not only size but also ...
(
radius of gyration ''Radius of gyration'' or gyradius of a body about the axis of rotation is defined as the radial distance to a point which would have a moment of inertia the same as the body's actual distribution of mass, if the total mass of the body were concentr ...
) of the analytes. This differs from other separation techniques which depend upon chemical or physical interactions to separate analytes.
[Skoog, D.A. ''Principles of Instrumental Analysis'', 6th ed.; Thompson Brooks/Cole: Belmont, California, 2006, Chapter 28.] Separation occurs via the use of porous beads packed in a column (see
stationary phase (chemistry)
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...
). The smaller analytes can enter the pores more easily and therefore spend more time in these pores, increasing their retention time. These smaller molecules spend more time in the column and therefore will elute last. Conversely, larger analytes spend little if any time in the pores and are eluted quickly. All columns have a range of molecular weights that can be separated. If an analyte is too large, it will not be retained; conversely, if the analyte is too small, it may be retained completely. Analytes that are not retained are eluted with the free volume outside of the particles (V
o), while analytes that are completely retained are eluted with volume of solvent held in the pores (V
i). The total volume can be considered by the following equation, where V
g is the volume of the polymer gel and V
t is the total volume:
As can be inferred, there is a limited range of molecular weights that can be separated by each column, and therefore the size of the pores for the packing should be chosen according to the range of molecular weight of analytes to be separated. For polymer separations the pore sizes should be on the order of the polymers being analyzed. If a sample has a broad molecular weight range it may be necessary to use several GPC columns in tandem to resolve the sample fully.
Application
GPC is often used to determine the relative molecular weight of polymer samples as well as the distribution of molecular weights. What GPC truly measures is the
molecular volume and shape function as defined by the
intrinsic viscosity Intrinsic viscosity \left \eta \right/math> is a measure of a solute's contribution to the viscosity \eta of a solution. It should not be confused with inherent viscosity, which is the ratio of the natural logarithm of the relative viscosity to th ...
. If comparable standards are used, this relative data can be used to determine molecular weights within ± 5% accuracy.
Polystyrene
Polystyrene (PS) is a synthetic polymer made from monomers of the aromatic hydrocarbon styrene. Polystyrene can be solid or foamed. General-purpose polystyrene is clear, hard, and brittle. It is an inexpensive resin per unit weight. It is a ...
standards with
dispersities of less than 1.2 are typically used to calibrate the GPC.
[Sandler, S.R.; Karo, W.; Bonesteel, J.; Pearce, E.M. ''Polymer Synthesis and Characterization: A Laboratory Manual''; Academic Press: San Diego, 1998.] Unfortunately, polystyrene tends to be a very linear polymer and therefore as a standard it is only useful to compare it to other polymers that are known to be linear and of relatively the same size.
Material and methods
Instrumentation
Gel permeation chromatography is conducted almost exclusively in
chromatography
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...
columns. The experimental design is not much different from other techniques of
liquid chromatography
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...
. Samples are dissolved in an appropriate solvent, in the case of GPC these tend to be organic solvents and after filtering the solution it is injected onto a column. The separation of multi-component mixture takes place in the column. The constant supply of fresh eluent to the column is accomplished by the use of a pump. Since most analytes are not visible to the naked eye a detector is needed. Often multiple detectors are used to gain additional information about the polymer sample. The availability of a detector makes the fractionation convenient and accurate.
Gel
Gel
A gel is a semi-solid that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state, although the liquid phase may still dif ...
s are used as stationary phase for GPC. The pore size of a gel must be carefully controlled in order to be able to apply the gel to a given separation. Other desirable properties of the gel forming agent are the absence of ionizing groups and, in a given solvent, low affinity for the substances to be separated. Commercial gels like PLgel & Styragel (cross-linked polystyrene-divinylbenzene), LH-20 (hydroxypropylated
Sephadex Sephadex is a cross-linked dextran gel used for gel filtration. It was launched by Pharmacia in 1959, after development work by Jerker Porath and Per Flodin. The name is derived from separation Pharmacia dextran. It is normally manufactured in a bea ...
), Bio-Gel (
cross-linked
In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
polyacrylamide
Polyacrylamide (abbreviated as PAM) is a polymer with the formula (-CH2CHCONH2-). It has a linear-chain structure. PAM is highly water-absorbent, forming a soft gel when hydrated. In 2008, an estimated 750,000,000 kg were produced, mainly fo ...
), HW-20 & HW-40 (hydroxylated
methacrylic polymer),
agarose gel
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the ...
and are often used based on different separation requirements.
[Helmut, D. ''Gel Chromatography, Gel Filtration, Gel Permeation, Molecular Sieves: A Laboratory Handbook''; Springer-Verlag, 1969.]
Column
The column used for GPC is filled with a microporous packing material. The column is filled with the gel.
Eluent
The
eluent
In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions.
In a liquid chromatography experiment, for exa ...
(mobile phase) should be a good solvent for the polymer, should permit high detector response from the polymer and should wet the packing surface. The most common eluents for polymers that dissolve at room temperature GPC are
tetrahydrofuran
Tetrahydrofuran (THF), or oxolane, is an organic compound with the formula (CH2)4O. The compound is classified as heterocyclic compound, specifically a cyclic ether. It is a colorless, water-miscible organic liquid with low viscosity. It is ma ...
(THF),
''o''-dichlorobenzene and
trichlorobenzene
Trichlorobenzene (TCB) may refer to any of three isomeric chlorinated derivatives of benzene with the molecular formula C6H3Cl3. Trichlorobenzenes are man-made chemical compounds that occur in three different forms. Even though the forms have the ...
at 130–150 °C for crystalline
polyalkynes and
''m''-cresol and
''o''-chlorophenol at 90 °C for crystalline condensation polymers such as
polyamide
A polyamide is a polymer with repeating units linked by amide bonds.
Polyamides occur both naturally and artificially. Examples of naturally occurring polyamides are proteins, such as wool and silk. Artificially made polyamides can be made through ...
s and
polyester
Polyester is a category of polymers that contain the ester functional group in every repeat unit of their main chain. As a specific material, it most commonly refers to a type called polyethylene terephthalate (PET). Polyesters include natural ...
s.
Pump
There are two types of pumps available for uniform delivery of relatively small liquid volumes for GPC: piston or peristaltic pumps.
Detector
In GPC, the concentration by weight of polymer in the eluting solvent may be monitored continuously with a detector. There are many detector types available and they can be divided into two main categories. The first is concentration sensitive detectors which includes UV absorption,
differential refractometer (DRI) or refractive index (RI) detectors, infrared (IR) absorption and density detectors. The second category is molecular weight sensitive detectors, which include
low angle light scattering detectors (LALLS) and multi angle light scattering (MALLS).
[Trathnigg, B. Determination of MWD and Chemical Composition of Polymers by Chromatographic Techniques. ''Prog. Polym. Sci.'' 1995, ''20'', 615-65]
The resulting chromatogram is therefore a weight distribution of the polymer as a function of retention volume. The most sensitive detector is the differential UV photometer and the most common detector is the differential refractometer (DRI). When characterizing copolymer, it is necessary to have two detectors in series.
For accurate determinations of copolymer composition at least two of those detectors should be concentration detectors.
The determination of most copolymer compositions is done using UV and RI detectors, although other combinations can be used.
[Pasch, H. Hyphenated Techniques in Liquid Chromatography of Polymers. ''Adv. Polym. Sci.'' 2000, ''150'', 1-6]
Data analysis
Gel permeation chromatography (GPC) has become the most widely used technique for analyzing polymer samples in order to determine their molecular weights and weight distributions. Examples of GPC chromatograms of polystyrene samples with their molecular weights and
dispersities are shown on the left.
Benoit and co-workers proposed that the hydrodynamic volume, V
η, which is proportional to the product of
and M, where
is the intrinsic viscosity of the polymer in the SEC eluent, may be used as the universal calibration parameter. If the Mark–Houwink–Sakurada constants K and α are known (see
Mark–Houwink equation The Mark–Houwink equation, also known as the Mark–Houwink–Sakurada equation or the Kuhn–Mark–Houwink–Sakurada equation or the Landau–Kuhn–Mark–Houwink–Sakurada equation or the Mark-Chrystian equation gives a relation between intr ...
), a plot of log
versus elution volume (or elution time) for a particular solvent, column and instrument provides a universal calibration curve which can be used for any polymer in that solvent. By determining the retention volumes (or times) of monodisperse polymer standards (e.g. solutions of monodispersed polystyrene in THF), a calibration curve can be obtained by plotting the logarithm of the molecular weight versus the retention time or volume. Once the calibration curve is obtained, the gel permeation chromatogram of any other polymer can be obtained in the same solvent and the molecular weights (usually M
n and M
w) and the complete molecular weight distribution for the polymer can be determined. A typical calibration curve is shown to the right and the molecular weight from an unknown sample can be obtained from the calibration curve.
Advantages
As a separation technique, GPC has many advantages. First of all, it has a well-defined separation time due to the fact that there is a final elution volume for all unretained analytes. Additionally, GPC can provide narrow bands, although this aspect of GPC is more difficult for polymer samples that have broad ranges of molecular weights present. Finally, since the analytes do not interact chemically or physically with the column, there is a lower chance for analyte loss to occur.
For investigating the properties of polymer samples in particular, GPC can be very advantageous. GPC provides a more convenient method of determining the molecular weights of polymers. In fact most samples can be thoroughly analyzed in an hour or less.
[Cowie, J.M.G.; Arrighi, V. ''Polymers: Chemistry and Physics of Modern Materials'', 3rd ed. CRC Press, 2008.] Other methods used in the past were fractional extraction and fractional precipitation. As these processes were quite labor-intensive molecular weights and mass distributions typically were not analyzed.
[Odian G. ''Principles of Polymerization'', 3rd ed.; Wiley Interscience Publication, 1991.] Therefore, GPC has allowed for the quick and relatively easy estimation of molecular weights and distribution for polymer samples
Disadvantages
There are disadvantages to GPC, however. First, there is a limited number of peaks that can be resolved within the short time scale of the GPC run. Also, as a technique GPC requires around at least a 10% difference in molecular weight for a reasonable resolution of peaks to occur.
In regards to polymers, the molecular masses of most of the chains will be too close for the GPC separation to show anything more than broad peaks. Another disadvantage of GPC for polymers is that filtrations must be performed before using the instrument to prevent dust and other particulates from ruining the columns and interfering with the detectors. Although useful for protecting the instrument, there is the possibility of the pre-filtration of the sample removing higher molecular weight sample before it can be loaded on the column. Another possibility to overcome these issues is the separation by
field-flow fractionation (FFF).
Orthogonal methods
Field-flow fractionation (FFF) can be considered as an alternative to GPC, especially when particles or high molar mass polymers cause clogging of the column, shear degradation is an issue or agglomeration takes place but cannot be made visible. FFF is separation in an open flow channel without having a static phase involved so no interactions occur. With one field-flow fractionation version,
thermal field-flow fractionation, separation of polymers having the same size but different chemical compositions is possible.
[Thermal Field-Flow Fractionation: Ultra-Broad Polymer Separation , http://www.chemeurope.com/en/products/77045/thermal-field-flow-fractionation-ultra-broad-polymer-separation.html ]
References
{{reflist
Biochemical separation processes
Chromatography
Polymers
Polyolefins
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