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Gel permeation Chromatography

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Table of Contents

Abstract .................................................................................................................................................. 3

Introduction ............................................................................................................................................ 4

Mechanism ............................................................................................................................................. 5

Working, Apparatus and Methods ......................................................................................................... 6

Data Analysis / Calculations ................................................................................................................... 9

Advantages ........................................................................................................................................... 10

Disadvantages ...................................................................................................................................... 11

Applications ......................................................................................................................................... 11

References ............................................................................................................................................ 13
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Abstract

The basic purpose of this research is to study in depth about chromatography, to be more

specific gel permeation chromatography. The paper discusses at length the working mechanisms

and the apparatus involved in this specific type of chromatography.

Followed by the working mechanisms and the basic calculations required for analysis.

advantages that make gel permeation chromatography unique and its disadvantages are also

discussed.

Towards the end of the paper some of the novel applications are also discussed at length.
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Gel permeation Chromatography

Introduction:

A separation method developed by a Russian, Mikhail Tsvet, in 1901, used for chemical

analysis is called chromatography. Mikhail dissolved some ground up leaves in in ethanol and

ether that dissolved chlorophyll from leaves. The solution that was formed as a result was poured

into a pre-filled glass column with solid CaCO3. Different strands of colors were formed depending

upon the bonding ability of pigments with calcium carbonate, the strands were formed as the

pigments moved at different speeds. Isolation of different colored molecules into separate

containers as they were collected at the bottom of the container.

Although this was a pretty simple experiment, yet it proved its worth is the separation

mixtures of molecules. Secondly, this technique was found independent of the phase of the

mixture. Depending upon the type of the mixture chromatography is divided into different

categories. Basically, the structure consists of a capillary or certain other column that is loaded

with a moving and a stationary stage, at rest, that can either be gas, solid or liquid . The stationary

phase stays static at its place whereas mobile phase moves freely. In the Mikhail’s experiment,

CaCO3 was the stationary and mobile phase was ether and ethanol. The separation is based on the

bonding ability of mixture molecules with one of the two phases. (Gaylor & James, 1978, p. 30)

Chromatography can be divided into the following types:

1) Gas Chromatography.

2) High Performance Liquid Chromatography.

3) Gel Permeation Chromatography.

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The major focus of this paper is gel permeation chromatography. So, it is discussed at length in

the sections yet to come.

Gel permeation chromatography basically belongs to a category of size exclusion

chromatography. The basic mechanism is the separation based on the size of the particles. The

nature of solvent in usually organic. Polymers are generally analyzed by this technique. This

technique was first investigated by J. C. Moore in 1964 and was also commercialized in the same

year by Waters Corporation.

In the isolation process of polymers, it is necessary to the molecular weight and dispersity

(Đ). Gel permeation chromatography allows the determination of Đ as well as molecular weight.

Mechanism:

The separation process in Gel permeation chromatography is built on the radius of gyration

of the particles that are to be separated. Porous beads packed in column are used for separation.

The tinier the volume of particle the easier it can enter the pores and longer they can stay there.

The longer the time spent by these particles in the pores and thus will elute last. Paraphrasing it,

the larger the size of the particles the less the time they will spend in the pores and the faster they

will be eluted.

“Another feature that should be focused is the size of particle. If the particle in focus is too

large it will not get into the pores and if the particles are too small, they may be preserved

completely. Unpreserved particles are eluted with free vol that is usually at the outer periphery of

the particles (Vo), while the particles that are submerged fully are eluted with the solvent’s volume
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that are kept in the pores (Vi). The overall volume can be depicted by the resulting equation, in

which (Vg) is the gel’s volume and Vt is the total volume.”

As it can be depicted from the above discussion, there is a specific range that can be separated by

the specific column, thus the packing having pores of specific size should be selected according to

the weight of the molecules that we are studying and size of the particles that are to be separated.

Thus, the selection of pore size is key in doing gel permeation chromatography. One of the

suggestions is that if there is a range of particles sizes in the polymer then multiple columns are to

be used to completely analyze the system.

Working, Apparatus and Methods:

Gel permeation chromatography is almost similar to liquid chromatography. Just like in

liquid chromatography samples in gel permeation chromatography are also first dissolved in

solvent but in this case the nature of solvent is specifically organic and after the filtration process

it is injected into the column. The separation process basically takes place inside the column. A

pump is used to make sure that the eluent is supplied in the column in constant manner. A detector

is a must as most of the naked eye is not enough to see the analytes. In some cases, multiple

detectors are used to obtained results of high quality. It generally increases the accuracy of the

process.

The material required and the apparatus is discussed below:

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1) Gel:

Gel serves the purpose of stationary phase in gel permeation chromatography. Pore

size of the gel is the most crucial part, and it must be controlled very carefully so

that it can be applied to a given separation. It is generally advised that the agents

that are responsible for the formation of gel are non-ionic. Some of the novel

examples are

 HW-20

 HW-40

 “Agarose gel”

 LH-20

 PLgel

 Styragel

2) Column:

Gel is used as the medium that is to be used to fill the column and is used for gel

permeation chromatography with a microporous packing material.

3) Eluent:

The Eluent which is the mobile phase of Gel Permeation Chromatography is

generally a solvent for the polymer whose analysis is to be performed. It should

have a good solvation ability, should allow high detector response coming from the

polymer and should wet the packing surface. Some of the most common eluents

widely available are

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 “Tetrahydrofuran (THF)”

 “O-dichlorobenzene”

 “Trichlorobenzene”

 “m-cresol”

 “o-chlorophenol”

4) Pump:

Two types of pump are generally available for continuous and consistent delivery

of fairly minute liquid quantities that are given below:

 Piston pumps

 Peristaltic pumps

5) Detector:

In GPC, the detector is used to determine the concentration of polymer un

the eluent solver. Although, there is a wide range of detectors are available

in the market, but they can be generalized in two major categories:

 “Concentration sensitive detectors, i.e., UV absorption, Differential

refractometer (DRI) or refractive index (RI) detectors.”

 “Molecular weight sensitive detectors, i.e., Low angle light

scattering detectors (LALLS) and multiangle light scattering

detectors (MALLS).”

The chromatogram that is generated as a result is shown in the figure below

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The results showed that the most sensitive detectors were UV photometer

and the most commonly found were differential refractometer (DRI)

Data Analysis / Calculations:

Gel permeation chromatography (GPC) is one of the highly used procedure for the analysis

of polymeric samples for the determination of their molecular weights and weight distributions.

The graph that shows the distributions is called a chromatogram. One of the samples is:

(Fig 1)
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“Benoit did some researched and found that the “hydrodynamic volume” (Vη), which is

the product of intrinsic or dynamic viscosity [η] of the polymer and M. If K and α, which are Mark

Houwink Constants, are known then a graph between “log (η) *M” and elution volume/time for a

specific type of solvent can be drawn. This would the provide a generalized graph which could be

easily applied for any particular polymer in that solvent. If the retaining volume is calculated by

any means, then a graph between log of molecular weight and retention time/volume. As the

adjustment graph is obtained, the chromatogram of whichever polymer in the identical solvent

can be obtained thus the molecular weights and its weight distribution can be established easily. A

sample calibration curve is shown in the figure below.”

(Fig 2)

Advantages:

Gel permeation chromatography is widely adopted because it has a large number of

advantages. Some of them are discussed below:

1) Unlike other techniques GPC offers well defined separation time.

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2) GPC is quite famous for its narrow bands.

3) Lower analyte losses due to minimal chemical or physical interaction with the column.

4) Comparatively, GPC offers a more convenient and less hectic method of determination of

molecular weights of polymers.

5) It is very easy to investigate the properties of any polymer using GPC.

6) The processes used in the past were quite hectic and time taking. The procedures were

laborious but using GPC it has been made possible to quickly determine the molecular

weights and distributions of different polymer samples.

Disadvantages:

As there are two sides of every coin, similarly there are some disadvantages of the gel

permeable chromatography. Some of them are given below:

1) Limited number of peaks resolution within short period of time.

2) Limitation of difference of molecular weight, as GPC requires at least 10% variation in

molecular weight for a good peak resolution.

3) Before the use of instruments, performing of filtration is a must to avoid contamination due

to dust and other particles that could ruin the columns and detectors.

Applications:

Due to immense range of applications gel permeation chromatography is widely adopted

around the globe. Some of its novel applications are:

1) Determination of “relative molecular weight of samples of the polymer in focus”.

2) Molecular weight distribution analysis.

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3) Measurement of “molecular volume” and “shape function” defined by dynamic viscosity.

4) Accuracy of almost ± 5% can be achieved in the determination molecular weight

distribution.
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References

Gaylor, V. F., & James, H. L. (1978). Gel permeation chromatography (steric exclusion

chromatography). Analytical Chemistry, 50(5), 29–36.

https://doi.org/10.1021/ac50028a004

Malhotra, O. P., & Kumar, A. (1989). Application of gel filtration for fractionation and

molecular weight determination of proteins. Biochemical Education, 17(3), 148–150.

https://doi.org/10.1016/0307-4412(89)90100-3

Wikipedia contributors. (2020, November 9). Gel permeation chromatography. Wikipedia.

https://en.wikipedia.org/wiki/Gel_permeation_chromatography

Wikipedia contributors. (2020a, March 3). Mark–Houwink equation. Wikipedia.

https://en.wikipedia.org/wiki/Mark%E2%80%93Houwink_equation

Size Exclusion Chromatography. (n.d.). SHIMADZU. Retrieved December 7, 2020, from

https://www.shimadzu.com/an/service-support/technical-support/analysis-

basics/basic/55/55intro.html