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Gel Permeable chromatography

Gel permeation chromatography (GPC) is a type of size exclusion chromatography (SEC), that separates analytes 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 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 (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), the size average molecular weight (Mz), or the viscosity molecular weight (Mv). Some of the companies dealing with Gel Permeable Chromatography Technique are: 1. Tosoh Bioscience GmbH (Germany) 2. Shimadzu (USA) 3. Intertek (UK) – only deals with process outsourcing and testing 4. Malvern Instruments (UK) 5. Water Associates limited (US) 6. Polymer Char 7. The Dow Chemical Company Basic GPC equipment

This diagram illustrates how the sample is injected into the mobile phase and the path the sample takes to the detector. 1. Pump Pumps the polymer in solution through the system. Different polymers produce solutions of different viscosities. To compare data from one analysis to the next, the pump must deliver the same flow rates independent of viscosity differences. In addition, some detectors are very sensitive to the solvent flow rate precision. Such constant flow must be a critical feature of the instrument.

2. Injector Introduces the polymer solution into the mobile phase. The injector must be capable of small volume injections (for molecular weight determinations) and large volume injections (if fraction collecting is desirable). The injector should not disturb the continuous mobile phase flow. It should also be capable of automatic multiple sample injection when the sample volume is large. 3. Column Set Efficiently separates sample components from one another. High efficiency columns give maximum separating capability and rapid analyses. Every column must provide reproducible information over extended periods for both analytical and fraction collecting purposes. 4. Detector Monitors the separation and responds to components as they elute from the column. Detectors must be nondestructive to eluting components if they are to be collected for further analysis. In addition, the detectors must be sensitive and have a wide linear range in order to respond to both trace amounts and large quantities of material if necessary. Since all compounds refract light, the differential refractometer (RI) is referred to as a "universal" detector. As a result it is the most widely used detector to monitor molecular weight distribution. The refractive index of polymers is constant above approximately 1000 MW. Therefore, the detector response is directly proportional to concentration. Beside information about molecular weight averages and distribution obtained with RI, the use of UV absorbance detectors may provide information about composition, while on-line light scattering detectors and viscometers provide information about polymer structure. 5. Automatic data processing equipment Automatically calculates, records, and report numerical values for Mz, Mw, Mv, Mn, and MWD. Data systems can also provide complete control of GPC systems so that large numbers of samples can be run unattended and raw data can be automatically processed. Today's GPC software offerings need to be able to provide special calculations for multi-detection processing, band broadening correction, special calibration routines and polymer branching determination, just to name a few. Many of the above mentioned companies produce industry oriented GPC equipments enhanced and equipped with the latest technology, here we'll be focusing on Malvern Viscotek HT-GPC systems, Viscotek TDA max in particular. What is HT-GPC?

HT- GPC system is a new version of GPC system wherein it is held at elevated temperature to ensure no part of the sample can re-precipitate before or during analysis leading to erroneous results. HT-GPC allows the measurement of average molecular weights and molecular weight distributions of polymers that will not dissolve in GPC solvents at conventional GPC temperatures. It is used for the characterization
of polyolefins and other synthetic polymers that are soluble only at elevated temperatures.

A word about Malvern Viscotek HT-GPC systems: Designed specifically for the characterization of polyolefins, but with the ability to handle other polymers, such as polythiophene PEEK and PVF, the Viscotek High Temperature GPC (HT-GPC) system brings advanced triple detector technology to these demanding applications. The HT-GPC operates up to 160°C to handle even the toughest polypropylene samples and is engineered in a modular format for flexibility, reliability and easy maintenance.     The system is available in four detector configurations: Conventional Calibration – using RI (refractive index) detection with column calibration Light Scattering – using RI + light scattering (LALS) detection for direct molecular weight measurement without column calibration. Universal Calibration – using RI + Viscometer detection to give true molecular weights plus branching data. Triple Detection – using RI + Viscometer + light scattering (LALS) for direct molecular weight measurement and branching data. A comparison of the GPC instruments available with Malvern:

MALVERN VISCOTEK-TDAmax

Viscotek TDAmax is a high end GPC equipment which is broadly divided into three sub units which are discussed below in detail: 1. TDA(Triple Detector Array) + UV Viscotek is using the latest tetra detector configurations, which includes a triple detector array which employs Refractive Index, Viscometer and light scattering detectors, an additional UV detector in it's tetra detector format. The detector cells and the chromatographic columns are together housed in the same thermal chamber thus giving precise and accurate temperature control over them. Now let us have a brief look of the detectors used: a) Light Scattering Detector The powerful light scattering detector provides two separate measurement points: i. ii. The unique 7 Low angle(LALS) detector, which measures the absolute molecular weight of even the largest macromolecules directly without the need of extrapolation or correction. and The 90 Right angle(RALS), for small molecules such as proteins.
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b) Viscometer detector The differential viscometer detector features the patented Digital Inert Transducer Technology(DIT), which gives a faster and a more sensitive response to the viscosity changes compared to the traditional magnetic reluctance transducers. In addition, the innovative construction in the 316 stainless steel means they are chemically robust with a few limitations on salts or pH. c) Refractive Index(RI) detector The high sensitivity RI in the TDA has been designed specifically with multi detection GPC/SEC in mind. The unique design of the purging system allows the detector to be in series between the light scattering and the viscometer, which the ideal configuration to obtain maximum signals on all the detectors in a multi detector GPC system. d) UV detectors Ultraviolet detectors are an excellent tool for compositional analysis of co-polymers or proteins. 2. The OmniSEC(GPC/SEC software) It is the software that controls the functioning of the individual units and also interprets the data obtain from all the detectors. OmniSEC provides support for:       Muti-detection(single/dual/triple/tetra) Absolute molecular weight Protein Analysis Molecular conformation/branching Copolymer analysis Intrinsic viscosity,etc.

3. GPCmax GPCmax is the leading solvent and sample delivery module extensively for GPC/SEC applications, which promises the highest reproducibility and accuracy. Some of the important components of GPCmax are:  Degasser & eluent sensor The GPCmax is equipped with an inline degasser to remove dissolved gases and allow optimum performance of the pump. Furthermore, it improves baselines on all the detectors. To avoid

damage to valuable GPCmax includes an automatic eluent sensor which will stop the flow if it detects the solvent supply is dangerously low.  Pump The dual piston pump provides an extremely low pulsation and stable eluent flow, to allow optimum performance of concentration, viscostly and light scattering detectors. The pump features a unique user programmable soft start/stop to maximize the lifetime of your columns by protecting them from pressure shocks. The pump is compatible with any solvent, even acidic buffers.  Autosampler The Autosampler provides the system with reliable, accurate and reproducible injection volumes, enabling comprehensive macro-molecular characterizations to be made with confidence. In addition, the advanced programming features via the OmniSEC software allows takes like 2 virial coefficient(A2) determinations to be completed simply and unattended.  Columns and standards A comprehensive selection of columns and standards for all types of GPC/SEC are available as part of the Visotek range. Columns for: Aqueous soluble polymers Organic soluble polymers High Temperature GPC Protein analysis Advantage of using Viscotek GPCmax:  PROTEINS It provides:       Absolute molecular weight Aggregation Protein size and density Oligomeric composition A Second virial coefficient Standards: Polystyrene(PS) Polymethylmethacrylate(PMMA) Polyethylene glycol(PEG) Pre-weighed and bulk polymers
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POLYMERS      Absolute molecular weight Molecular weight distribution Branching and structure Molecular size Copolymer composition

Now let us discuss the future trends...

The latest trend in the field of characterization of polymers is Asymmetric Flow Field Fractionation Technique. Field-flow fractionation, abbreviated FFF, is a separation technique where a field is applied to a fluid suspension or solution pumped through a long and narrow channel, perpendicular to the direction of flow, in order to cause separation of the particles present in the fluid, dependent on their differing "mobilities" under the force exerted by the field. It was invented and first reported by J. Calvin Giddings. In field-flow fractionation the field can be electrical, transverse flow through a semi-permeable membrane, gravitational, centrifugal, magnetic, thermal-gradient, etc. In all cases, the separation mechanism is born from differences in particle mobility. Advantage over GPC systems:
1.Broad Molar Mass Separation Range at high Temperatures: Compared with High Temperature GPC, the separation in High Temperature Asymmetric Flow Field-Flow Fractionation can also be performed for polymers up to molar masses of

109 Da. No size exclusion effect is observed, making High

Temperature Asymmetric Flow Field-Flow Fractionation the ideal tool for the separation of large and ultralarge polymers, macromolecules and nanoparticles. 2.GentleSeparation Conditions: Because of the open channel and the absence of any stationary phase, the separation can be performed under the absence of shear forces and stress to the macromolecule. Typical High Temperature GPC problems, such as filtering/adsorption effects, shear induced chain degradation and late elution effects, can be avoided.

High molecular weight low density polyethene: Molar mass by HTFFF(red) and HTGPC(blue):

The results of GPC is limited by the size exclusion limit of the column, while in case of FFF the high molecular weight range of the Low Density polyethene can be accessed, which gives us the real distribution of the given sample. Some companies producing the equipment with FFF techniques are: Postnova Analytics GmbH, Wyatt Technology Europe, and Dow Chemicals.