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8/12/2019 FPLC Talk Hndout

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Basic Principals of Chromatography

Concepts and Definitions Chromatography is the most important separation methodfor biomolecules.The outcome of a chromatographyexperiment is a CHROMATOGRAM

Column chromatography


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Concepts and Definitions Chromatography is the most important separation method

for biomolecules.The outcome of a chromatographyexperiment is a CHROMATOGRAM

Planar chromatography

Why Chromatography?


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Why Chromatography?Adaptable to wide range of compounds, because

variety of :separation principles (retentionmechanisms), and types of experimental setup(planar or column - gas and liquid phase elution).



Separated analyte is immediately available for identification or quantification Can be scaledup for preparative use.


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Separated analyte is immediately available for identification or quantification Can be scaledup for preparative use.

WHY NOT? Some instrumentation expensive andnot easily portable Often need preliminary bench

"work-up" of sample to avoid column contaminationExamples of analytical applications Drug analysis -therapeutic monitoring or abuse detectionVitamins, hormones, specific peptides and proteinsEnvironmental pollution, pesticide residues etc

Basic ChromatographicPrinciples

All chromatographic systems contain: Astationary phase A mobilephaseSample molecules (mixture for separation)

- Movement of molecules determined bythe balance between two forces :

Impelling force of mobile phase carrieswith it molecules for which it has affinity -favoured by solubility (LC), volatility (GC)


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Factors which influence

chromatography Theoretical Plates the number of

equilibrations that a compound makeswith the stationary phase.

Chromatography columns areconsidered to consist of a number ofadjacent plates or zones where thereis enough space for a compound toachieve complete equilibriumbetween the mobile and stationaryphase. Each zone is called atheoretical plate and the length of thecolumn the plate height. The moretheoretical plates the better theresolution of protein. Consider threecolumns each with a different number of theoretical plates

Relative distribution on the column

Factors which influencechromatography

Peak resolution- due to how youelute the protein. Step vs gradient(Shallow vs steep).

peak broadening - Due to pulsingor size of matrix. Small beads =sharp peaks (T Plates) but highpressure.


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Factors which influencechromatography

Column velocity A slow flow rate will

increase the time a proteinis in the mobile phase anddiffusion occurs resulting inpeak broadening.

A high flow rate willdecrease the interaction ofthe protein with thestationary phase and

decrease the number ofpossible equilibration,resulting in a reduction oftheoretical plates possible.

A Van Deemter plot is a plot of plateheight vs. average linear velocityof mobile phase. Such plots are ofconsiderable use in determiningthe optimum mobile phase flowrate

Retention Mechanisms

Overview of five commonretentionmechanisms


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Partition Chromatography Stationary phase = sorbed solvent held on the

surface, or within the grains or fibers of an inert solidsupporting matrix

Sample molecules equilibrate (PARTITION) between liquid stationary phaseand mobile phase. (Mobile phase is liquid in LC and HPLC systems andgaseous in GLC systems). Retention depends on a sample molecule'sescaping tendency into the mobile phase versus its solubility in thestationary phase.

K D =

Quantitatively given by the PARTITIONCOEFFICIENT, KD, the ratio of solubilities in thetwo phases

Solute in mobile phaseSolute in stationary

phase


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Examples of Partition

Chromatography Systems Liquid chromatography systems based on partitioning include

paper chromatography , and thin-layer chromatography(TLC) on polar matrixes such as silica gel and alumina.

The mobile phase is always a solvent mixture- onecomponent is polar (eg H2O) --> taken up by polar matrix toform the stationary phase. Sample molecules soluble in thissolvent will be retarded - this component of solvent is theretarder . - one component is non-polar (eg butanol) --->travels around stationary phase.

Sample molecules soluble in this solvent don't spend time instationary phase -this component of mixture is the mover . -sometimes a third component to maintain miscibility andadjust pH ( eg acetic acid).

Adsorption

Chromatography Solute in liquid (or gas) phase interacts with

adsorption sites on solid surface (finelydivided particles for maximum surface area).

Suitable solids include HYDROXYAPATITE(Ca3(PO4)2.Ca(OH)2), ALUMINA (Al2O3),MAGNESIUM CARBONATE . Polar groups onsolid form dipolar interactions ( eg hydrogenbonds) with sample dissolved (usually) in organic

solvent. Elute by increasing polarity of the solvent(eg if using acetonitrile CH3CN, add methanol(CH3OH)) --> competing bonds with adsorptionsites. Gradient elution useful (also for ion-exchange and partition chromatography)


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Ion-Exchange Chromatography Retention by attraction between groups on stationary phase

with opposite charge to sample molecules. Stationary phase= insoluble, but solvent permeable polymer matrix ( eg cellulose) chemically modified to introduce ionizable groups(eg -COOH).

Elute by Change of pH toneutralise charged group on either solute or stationary phase.Increase [salts] (especiallypolyvalent) in eluant buffer -->Displace by competing ions. pHor salt gradient to enhanceseparation. Ion-exchange mediaare classified according towhether the attached ionizablegroup is strongly or weaklyacidic or basic --> determinesthe usable H ran e

Affinity Chromatography Retention due to biospecific interaction using a ligand

molecule chemically coupled to a dextran or cellulosematrix - Hence may be able to isolate analyte fromcomplex mixture.

Elution can be bydisplacement with ligandmolecules in free solution.But analyte then eluted ascomplex with ligand.Better to elute by changeof pH to weakenbinding.Chemistry of ligand coupling to matrixusing cyanogen bromide.


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Affinity Chromatography

HPLCHigh Performance (Pressure) Liquid Chromatography


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HPLC vs. FPLC Components and Uses


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Std Routing Gradient pump inletvalves may be used to

select a differentbuffer, to addsanitation or storagebuffer at the end of arun, or to load sampledirectly through thegradient pump. Anyvalve may beattached to gradientpump inlets A or B.Two examples areshown. Use the

Manual screen valvecontrol to switchbetween valve portsand to prime eachvalve position using asyringe beforerunning a method

Sequential Binding and Elution A sample is loadedonto one column,

nonbinding proteinsare washed away,and then a fractionof eluted proteins isdiverted to asecond column.This column iswashed, eluted,and fractionsdiverted to a thirdcolumn, which inturn is washed and

eluted. Gradientpump inlet valvesare used if differentbuffers are neededfor equilibrationand elution of eachcolumn.

S tep 1With valve 1 in

Purge position andvalves 2 and 3 inLoad position, thegradient pump maybe primed with thebuffers from thegradient pump inletvalves.


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Step 1BAt a predetermined timeor volume, valve 4moves to position 2 anda portion of the columneluate is captured in theloop/DynaLoop attachedto valve 2.

Step 2Valve 1 moves to

Purge position to putvalve 2 and column 2into the flow path.Valves 3 and 4 changeposition to connectcolumn 2 to thedetectors and fractioncollector. With theappropriate bufferselected from thegradient pump inletvalves, the samplestored in theloop/Dynaloop is

injected onto column 2when valve 2 moves toInject position.

Step 3 (not shown)Valve 2 reverts toLoad position andcolumn 2 is eluted.

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