Gel permeation chromatography

Gel permeation chromatography (GPC) is a type of size-exclusion

chromatography (SEC), that separates analytes on the basis of size, typically in organic
solvents. The technique is often used for the analysis of polymers. As a technique, SEC
was first developed in 1955 by Lathe and Ruthven. [1] The term gel permeation
chromatography can be traced back to J.C. Moore of the Dow Chemical Company who
investigated the technique in 1964. The proprietary column technology was licensed
to Waters Corporation, who subsequently commercialized this technology in 1964.
[2]
 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 (Đ) as well
the molecular weight. 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 (Mw) (see molar mass distribution), the size average
molecular weight (Mz), or the viscosity molecular weight (Mv). GPC allows for the
determination of Đ as well as Mv and, based on other data, the Mn, Mw, and Mz can be
determined.

How it works[edit]
GPC separates based on the size or hydrodynamic volume (radius of gyration) of the
analytes. This differs from other separation techniques which depend upon chemical or
physical interactions to separate analytes.[3] Separation occurs via the use of porous
beads packed in a column (see stationary phase (chemistry)).

Schematic of pore vs analyte size

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.

Range of molecular weights that can be separated for each packing material

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 (Vo), 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 Vg is the volume of the polymer gel and Vt is the total
volume:[3] 
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[edit]
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. If comparable standards
are used, this relative data can be used to determine molecular weights within ± 5%
accuracy. Polystyrene standards with dispersities of less than 1.2 are typically used to
calibrate the GPC.[4] 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[edit]

Instrumentation[edit]
A typical GPC instrument including: A. Autosampler, B. Column, C. Pump, D. RI detector, E. UV-vis detector

Inside of an autosampler for running several samples without user interaction, e.g. overnight

Gel permeation chromatography is conducted almost exclusively

in chromatography 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. 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[edit]
Gels 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), [5][6] LH-20
(hydroxypropylated Sephadex),[7] Bio-Gel (cross-linked polyacrylamide), HW-20 & HW-
40 (hydroxylated methacrylic polymer),[8] agarose gel and are often used based on
different separation requirements.[9]
Column[edit]
The column used for GPC is filled with a microporous packing material. The column is
filled with the gel.
Eluent[edit]
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 for polymers that dissolve at room temperature GPC
are tetrahydrofuran (THF), o-dichlorobenzene and trichlorobenzene at 130–150 °C for
crystalline polyalkynes and m-cresol and o-chlorophenol at 90 °C for crystalline
condensation polymers such as polyamides and polyesters.
Pump[edit]
There are two types of pumps available for uniform delivery of relatively small liquid
volumes for GPC: piston or peristaltic pumps.
Detector[edit]
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). [10] The resulting chromatogram is
therefore a weight distribution of the polymer as a function of retention volume.

GPC chromatogram; Vo= no retention, Vt= complete retention, A and B = partial retention

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.[4] For accurate determinations of copolymer
composition at least two of those detectors should be concentration detectors. [10] The
determination of most copolymer compositions is done using UV and RI detectors,
although other combinations can be used. [11]

Data analysis[edit]
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.

GPC Separation of Anionically Synthesized Polystyrene; Mn=3,000 g/mol, Đ=1.32

GPC Separation of Free-Radical Synthesized Polystyrene; M n=24,000 g/mol, Đ=4.96

Standardization (calibration) of a size-exclusion column

Benoit and co-workers[citation needed] 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), 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 Mw) 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[edit]
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. [3] 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. [12] 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.[13] Therefore, GPC has allowed for the quick and relatively easy estimation of
molecular weights and distribution for polymer samples

Disadvantages[edit]
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.[3] 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[edit]
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.[14]

References[edit]
1. ^ Lathe, G.H.; Ruthven, C.R.J. The Separation of Substance and '1956', 62, 665–
674. PMID 13249976
2. ^ Moore, J.C. Gel permeation chromatography. I. A new method for molecular weight
distribution of high polymers. J. Polym. Sci., 1964, 2, 835-843.[1][dead
link]
 doi:10.1002/pol.1964.100020220
3. ^ Jump up to:a b c d Skoog, D.A. Principles of Instrumental Analysis, 6th ed.; Thompson
Brooks/Cole: Belmont, California, 2006, Chapter 28.
4. ^ Jump up to:a b Sandler, S.R.; Karo, W.; Bonesteel, J.; Pearce, E.M. Polymer Synthesis and
Characterization: A Laboratory Manual; Academic Press: San Diego, 1998.
5. ^ Agilent Technologies. "AGILENT ORGANIC GPC/SEC COLUMNS"  (PDF). Retrieved  2019-
12-06.
6. ^ Waters Corporation.  "STYRAGEL COLUMN CARE AND USE MANUAL"  (PDF).
Retrieved 2019-12-06.
7. ^ GE Healthcare. "Sephadex LH-20". Retrieved  2019-12-06.
8. ^ TOSOH BIOSCIENCE. "TOYOPEARL HW-40". Retrieved 2019-12-06.
9. ^ Helmut, D. Gel Chromatography, Gel Filtration, Gel Permeation, Molecular Sieves: A
Laboratory Handbook; Springer-Verlag, 1969.
10. ^ Jump up to:a b Trathnigg, B. Determination of MWD and Chemical Composition of Polymers
by Chromatographic Techniques. Prog. Polym. Sci. 1995, 20, 615-650.[2] doi:10.1016/0079-
6700(95)00005-Z
11. ^ Pasch, H. Hyphenated Techniques in Liquid Chromatography of Polymers. Adv. Polym.
Sci. 2000, 150, 1-66.[3] doi:10.1007/3-540-48764-6
12. ^ Cowie, J.M.G.; Arrighi, V. Polymers: Chemistry and Physics of Modern Materials, 3rd ed.
CRC Press, 2008.
13. ^ Odian G. Principles of Polymerization, 3rd ed.; Wiley Interscience Publication, 1991.
14. ^ Thermal Field-Flow Fractionation: Ultra-Broad Polymer Separation
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