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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).
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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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.
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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