Gel permeation chromatography
(GPC) is a type of size exclusion chromatography
Size-exclusion 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 or macromolecular complexes such as proteins and industrial polymers...
(SEC), that separates analyte
An analyte, or component , is a substance or chemical constituent that is of interest in an analytical procedure. Grammatically, it is important to note that experiments always seek to measure properties of analytes—and that analytes themselves can never be measured. For instance, one cannot...
s on the basis of size. The technique is often used for the analysis of polymers. As a technique, SEC was first developed in 1955 by Lathe and Ruthven. The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company
The Dow Chemical Company is a multinational corporation headquartered in Midland, Michigan, United States. As of 2007, it is the second largest chemical manufacturer in the world by revenue and as of February 2009, the third-largest chemical company in the world by market capitalization .Dow...
who investigated the technique in 1964. 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 polydispersity index
In physical and organic chemistry, the polydispersity index , is a measure of the distribution of molecular mass in a given polymer sample. The PDI calculated is the weight average molecular weight divided by the number average molecular weight. It indicates the distribution of individual...
(PDI) as well the molecular weight. Polymers can be characterized by a variety of definitions for molecular weight including the number average molecular weight (Mn
), the weight average molecular weight (Mw
) (see molar mass distribution
In linear polymers the individual polymer chains rarely have exactly the same degree of polymerization and molar mass, and there is always a distribution around an average value. The molar mass distribution in a polymer describes the relationship between the number of moles of each polymer species...
), the size average molecular weight (Mz
), or the viscosity molecular weight (Mv
). GPC allows for the determination of PDI as well as Mv
and based on other data, the Mn
, and Mz
can be determined.
How GPC Works
GPC separates based on the size or hydrodynamic volume (radius of gyration
Radius of gyration or gyradius is the name of several related measures of the size of an object, a surface, or an ensemble of points. It is calculated as the root mean square distance of the objects' parts from either its center of gravity or an axis....
) of the analytes. This differs from other separation techniques which depend upon chemical or physical interactions to separate analytes. Separation occurs via the use of porous beads packed in a column (see stationary phase (chemistry)). The smaller analytes can enter the pores more easily and therefore spend more time in these pores, increasing their retention time. 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 either too large or too small it will be either not retained or completely retained respectively. Analytes that are not retained are eluted with the free volume outside of the particles (Vo
), while analytes that are completely retained are eluted with volume of solvent held in the pores (Vi
). The total volume can be considered by the following equation, where Vg
is the volume of the polymer gel and Vt
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 with one another to fully resolve the sample.
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] is a measure of a solute's contribution to the viscosity \eta of a solution. Intrinsic viscosity is frequently referred to as "Inherent Viscosity" in macromolecular literature...
. If comparable standards are used, this relative data can be used to determine molecular weights within ± 5% accuracy. Polystyrene
Polystyrene ) also known as Thermocole, abbreviated following ISO Standard PS, is an aromatic polymer made from the monomer styrene, a liquid hydrocarbon that is manufactured from petroleum by the chemical industry...
standards with PDI of less than 1.2 are typically used to calibrate the GPC. 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.
Gel permeation chromatography is conducted almost exclusively in chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
columns. The experimental design is not much different from other techniques of liquid chromatography. 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. A Waters GPC instrument is shown to the left. 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.
A gel is a solid, jelly-like material 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...
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 Sephadex
Sephadex is a trademark for 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 bead form and most commonly used for...
, Bio-Gel (cross-linked polyacrylamide), agarose gel and Styragel are often used based on different separation requirements.
The eluent (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 in for polymers that dissolve at room temperature GPC are tetrahydrofuran
Tetrahydrofuran is a colorless, water-miscible organic liquid with low viscosity at standard temperature and pressure. This heterocyclic compound has the chemical formula 4O. As one of the most polar ethers with a wide liquid range, it is a useful solvent. Its main use, however, is as a precursor...
-dichlorobenzene and trichlorobenzene at 130–150 °C for crystalline polyalkines and m
-cresol and o
-chlorophenol at 90 °C for crystalline condensation polymers such as polyamides and polyesters.
There are two types of pumps available for uniform delivery of relatively small liquid volumes for GPC: piston or peristaltic pumps.
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. Molecular weight sensitive detectors include low angle light scattering detectors (LALLS), multi angle light scattering (MALLS). 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.
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 PDIs are shown on the left.
Benoit and co-workers proposed that the hydrodynamic volume, Vη
, which is proportional to the product of [η] and M, where [η] 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 gives a relation between intrinsic viscosity [\eta] and molecular weight M:[\eta]=KM^aFrom this equation the molecular weight of a polymer can be determined from data on the intrinsic viscosity and vice versa....
), a plot of log [η]M 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 Mn
) 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 of GPC
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. 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. Therefore, GPC has allowed for the quick and relatively easy estimation of molecular weights and distribution for polymer samples
Disadvantages of GPC
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, the pre-filtration of the sample has the possibility of removing higher molecular weight sample before it can be loaded on the column.