ADVANCED PHARMACEUTICAL ANALYSIS

Gel Permeation Chromatography (GPC): The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company who investigated the technique in 1964 . Gel permeation chromatography (GPC) is a chromatographic method in which molecules in solution are separated by their size. The stationary phase consists of beads of porous polymeric material. Organic solvent is used as mobile phase. It is usually applied to large molecules or macromolecular complexes such as polymers.

How GPC Works: GPC separates based on the size 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.

Fig: 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.
1|Page Md. Rezaul Alam Razib 22nd Batch (Pharmacy)

Mrs. Taslima Akter :: Lecturer :: Dept. of Pharmacy :: Jahangirnagar University

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. 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. Applications: Gels: 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. There should be absence of ionizing groups in the gel forming agents. Another desirable property of the Gel forming agent is low affinity for the analytes to be separated. Commercial gels like Sephadex, Bio-Gel (cross-linked polyacrylamide), agarose gel and Styragel are often used based on different separation requirements. Polymer characterization Molecular weight Viscosity Aggregation Branching Folding Polydispersity index Copolymer composition Polymer purification

Md. Rezaul Alam Razib 22nd Batch (Pharmacy)

2|Page

Mrs. Taslima Akter :: Lecturer :: Dept. of Pharmacy :: Jahangirnagar University

Eluent: The eluent (mobile phase) should be a good solvent for the polymer should permit high detector response from the polymer should wet the packing surface Eluent The most common eluents for polymers that dissolve at room temperature GPC are tetrahydrofuran (THF), o-dichlorobenzene and trichlorobenzene at 130150 C for crystalline polyalkines and m-cresol and o-chlorophenol at 90 C for crystalline condensation polymers such as polyamides and polyesters. Sample preparation: 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. Pump: There are two types of pumps available for uniform delivery of relatively small liquid volumes for GPC: piston or peristaltic pumps. Designed not to produce any pressure pulses Filters: Prevent major junk from getting into the column Column: Contain the beads of gels through which the sample is allowed to pass. Very expensive Detector: UV absorption detector Differential refractometer (DRI) or refractive index (RI) detectors Infrared (IR) absorption detectors Low angle light scattering detectors. Multi angle light scattering detectors.

Md. Rezaul Alam Razib 22nd Batch (Pharmacy)

3|Page

Mrs. Taslima Akter :: Lecturer :: Dept. of Pharmacy :: Jahangirnagar University

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 The determination of most copolymer compositions is done using UV and RI detectors, although other combinations can be used Advantages GPC has well defined separation time It can provide narrow bands. There is a lower chance for analyte loss to occur, since the analytes do not interact chemically or physically with the column. GPC has allowed for the quick and relatively easy estimation of molecular weights and distribution of polymer samples. Other methods like fractional extraction and fractional precipitation were used past for polymer analysis and these processes were quite labor intensive. Disadvantages there is a limited number of peaks that can be resolved within the short time scale of the GPC run As a technique GPC requires around at least a 10% difference in molecular weight for a reasonable resolution of peaks to occur. Another disadvantage of GPC for polymers is that filtrations must be performed before using the instrument to prevent dust and other particulates from runing the columns and interfering with the detectors. Although useful for protecting the instrument, the prefiltration of the sample has the possibility of removing higher molecular weight sample before it can be loaded on the column. Gel-Filtration Chromatography. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography.

Md. Rezaul Alam Razib 22nd Batch (Pharmacy)

4|Page