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Fast Protein Liquid Chromatography

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FPLC A semi-automatic microprocessor controlled machine used primarily

for the separation of macromolecules

FOUR TYPES OF LIQUID CHROMATOGRAPHY

Partition chromatography

Adsorption, or liquid-solid chromatography

Ion exchange chromatography

Size exclusion, or gel, chromatography

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1903: Russian botanist Mikhail Tswett Separated plant pigments through

column adsorption chromatography.

Packed open glass column with Calcium carbonate and alumina particles

Poured sample into column, along with pure solvent.

History

As the sample moved down the vertical column, different colored bands could be seen.

Bands correlated to the sample components.

Coined the term chromatography from the Latin word meaning “color writing”.

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Chromatography Theory Review

Several chromatographic techniques Even though each method utilizes different

techniques to separate compounds, the principles are the same.

Common to all: Stationary phase-

A solid or a liquid supported on a solid Mobile phase-

A liquid or gas 7

Column Chromatography Similar to thin layer

chromatography Stationary phase silica gel Mobile phase liquid solvent

In column chromatography, this stationary phase is packed into a vertical glass column.

Mobile phase moves down the column as a result of gravity.

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Column Chromatography

Blue compound, more polar

Adsorb more to the silica gel

Elutes slower Yellow compound ,

less polar

Spends much of its time in the mobile phase

Elutes faster

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Example of column chromatography separation:

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HPLC History

1966: Horváth

Built the first HPLC instrument and gave it its name HPLC = High Pressure Liquid Chromatography.

Improved columns and detectors

Production of small silica packing material

1970’s: HPLC became popular with an increase in technology

By 1972 particle sizes less than 10µm were introduced

This allowed for more precise and rapid separations.

As new technology continued to develop, HPLC became more efficient.

HPLC = High Performance Liquid Chromatography

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FPLC Introduction:

FPLC; improved form of column chromatography

Instead of the mobile phase moving through the column as a result of gravity, it is forced through the column under high pressure.

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FPLC is a form of high-performance

chromatography that takes advantage of

high resolution made possible by

small-diameter stationary phases.

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It was originally developed for proteins and features high loading capacity, biocompatible aqueous buffer systems, fast flow rates, and availability of stationary phases in most common chromatography modes (e.g., ion exchange, gel filtration, reversed phase, and affinity)

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FPLC- A Modification of HPLC

In 1982 Pharmacia introduced a new chromatographic method called FPLC.

FPLC is basically a “protein friendly” HPLC system.

FPLC can also be used to separate other biologically active molecules, such as nucleic acid.

Stainless steel was thought to denature proteins .

Also many ion-exchange separations of proteins involve salt gradients; thought that these conditions could results in attack of stainless steel systems.

Stainless steel components replaced with glass and plastic.

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FPLC pump delivers a solvent flow rate in the range of 1-499 ml/hr

HPLC pump, 0.6-600 ml/h

FPLC operating pressure: 0-40 bar HPLC, 1-400bar

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Since lower pressures are used in FPLC than in HPLC, a wider range of column supports are possible.

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FPLC System Components

The buffers carry the sample through the system. There are two pumps to move the buffers. Sample is injected into the valve. Then the sample passes to the column. The UV light detector measures the amount of

protein based on absorbance. The sample is divided into fractions by the fraction

collector.

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Column components

1. Packing

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Design & Operation of an FPLC Instrument

Mobile phase degassing:

Dissolved gases in the mobile phase can come out of solution and form bubbles as the pressure changes from the column entrance to the exit.

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Spraying is used to remove any dissolved gas from the mobile phase.

An inert and virtually insoluble gas, such as helium, is forced into the mobile phase solution and drives out any dissolved gas.

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Degassing may also be achieved by filtering the mobile phase under a vacuum.

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Gel de-gassing.

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Filling liquid in the bottomof the column

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Pouring gell slurry to the column.

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Settling of gels.

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Adjusting the adaptor

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Fixed adaptor

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Connecting a line to the adaptor

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Adaptor touching the upper layer of gels.

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Gel level lowers due to the solution flow into the column.

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Stopping the outlet line

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Connection of pump line to the adaptor

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Blue dextran solution flows in the column.

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Valve control

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Injection of sample

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FPLC controller.

Remote controller

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Multimeter for digital data aquisition

Multimeter

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Types of Detectors

Absorbance (UV with Filters, UV with Monochromators)

Fluorescence

Refractive-Index

Evaporative Light Scattering Detector (ELSD)

Electrochemical

Mass-Spectrometric

Photo-Diode Array41

UV detector

Detector

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Software UV signal data aquisition

Software (PROTEK 506)

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IN CONCLUSSION

the FPLC method appeared to be an appropriate method for identification and quantification of the β-thalassaemia, HbA /E, and could be a useful screening and diagnostic tool in laboratories equipped with a FPLC analyzer.

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