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Fast Protein Liquid Chromatography 2

FPLC A semi-automatic microprocessor controlled machine used primarily for the separation of macromolecules A semi-automatic microprocessor controlled machine used primarily for the separation of macromolecules 3

FOUR TYPES OF LIQUID CHROMATOGRAPHY Partition chromatography Partition chromatography Adsorption, or liquid-solid chromatography Adsorption, or liquid-solid chromatography Ion exchange chromatography Ion exchange chromatography Size exclusion, or gel, chromatography Size exclusion, or gel, chromatography 4

1903: Russian botanist Mikhail Tswett Separated plant pigments through column adsorption chromatography. Separated plant pigments through column adsorption chromatography. Packed open glass column with Calcium carbonate and alumina particles Packed open glass column with Calcium carbonate and alumina particles Poured sample into column, along with pure solvent. Poured sample into column, along with pure solvent. 5 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”. 6

Chromatography Theory Review Several chromatographic techniques Several chromatographic techniques Even though each method utilizes different techniques to separate compounds, the principles are the same. Even though each method utilizes different techniques to separate compounds, the principles are the same. Common to all: Common to all: Stationary phase- Stationary phase- A solid or a liquid supported on a solid A solid or a liquid supported on a solid Mobile phase- Mobile phase- A liquid or gas A liquid or gas 7

Column Chromatography Similar to thin layer chromatography Similar to thin layer chromatography Stationary phase silica gel Stationary phase silica gel Mobile phase liquid solvent Mobile phase liquid solvent In column chromatography, this stationary phase is packed into a vertical glass column. In column chromatography, this stationary phase is packed into a vertical glass column. Mobile phase moves down the column as a result of gravity. Mobile phase moves down the column as a result of gravity. 8

Column Chromatography Blue compound, more polar Blue compound, more polar Adsorb more to the silica gel Adsorb more to the silica gel Elutes slower Elutes slower Yellow compound, less polar Yellow compound, less polar Spends much of its time in the mobile phase Spends much of its time in the mobile phase Elutes faster Elutes faster 9 Example of column chromatography separation: Example of column chromatography separation:

HPLC History 1966: Horváth Built the first HPLC instrument and gave it its name Built the first HPLC instrument and gave it its name HPLC = High Pressure Liquid Chromatography. HPLC = High Pressure Liquid Chromatography. Improved columns and detectors Improved columns and detectors Production of small silica packing material Production of small silica packing material 10

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. This allowed for more precise and rapid separations. As new technology continued to develop, HPLC became more efficient. As new technology continued to develop, HPLC became more efficient. HPLC = High Performance Liquid Chromatography 11

FPLC Introduction: FPLC; improved form of column chromatography 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. Instead of the mobile phase moving through the column as a result of gravity, it is forced through the column under high pressure. 12

FPLC is a form of high-performance chromatography that takes advantage of high resolution made possible by small-diameter stationary phases. 13

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) 14

FPLC- A Modification of HPLC In 1982 Pharmacia introduced a new chromatographic method called FPLC. In 1982 Pharmacia introduced a new chromatographic method called FPLC. FPLC is basically a “protein friendly” HPLC system. FPLC is basically a “protein friendly” HPLC system. FPLC can also be used to separate other biologically active molecules, such as nucleic acid. FPLC can also be used to separate other biologically active molecules, such as nucleic acid. 15

Stainless steel was thought to denature proteins. 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. 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. Stainless steel components replaced with glass and plastic. 16

FPLC pump delivers a solvent flow rate in the range of ml/hr FPLC pump delivers a solvent flow rate in the range of ml/hr HPLC pump, ml/h HPLC pump, ml/h FPLC operating pressure: 0-40 bar FPLC operating pressure: 0-40 bar HPLC, 1-400bar HPLC, 1-400bar 17

Since lower pressures are used in FPLC than in HPLC, a wider range of column supports are possible. Since lower pressures are used in FPLC than in HPLC, a wider range of column supports are possible. 18

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. 19

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Column Column components 1. Packing 21

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. 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. 22

Spraying is used to remove any dissolved gas from the mobile phase. 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. An inert and virtually insoluble gas, such as helium, is forced into the mobile phase solution and drives out any dissolved gas. 23

Degassing may also be achieved by filtering the mobile phase under a vacuum. Degassing may also be achieved by filtering the mobile phase under a vacuum. 24

Gel degassing. 25

Filling liquid in the bottom of the column 26

Pouring gell slurry to the column. 27

Settling of gels. 28

Adjusting the adaptor 29

Fixed adaptor 30

Connecting a line to the adaptor 31

Adaptor touching the upper layer of gels. 32

Gel level lowers due to the solution flow into the column. 33

Stopping the outlet line 34

Connection of pump line to the adaptor 35

Blue dextran solution flows in the column. 36

Valve control 37

Injection of sample 38

FPLC controller. Remote controller 39

Multimeter for digital data aquisition Multimeter 40

Types of Detectors Absorbance (UV with Filters, UV with Monochromators) Absorbance (UV with Filters, UV with Monochromators) Fluorescence Fluorescence Refractive-Index Refractive-Index Evaporative Light Scattering Detector (ELSD) Evaporative Light Scattering Detector (ELSD) Electrochemical Electrochemical Mass-Spectrometric Mass-Spectrometric Photo-Diode Array Photo-Diode Array 41

UV detectorDetector 42

Software UV signal data aquisition Software (PROTEK 506) 43

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. 44

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