Hydrophobic interaction chromatography

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Hydrophobic interaction chromatography(HIC)
Hydrophobicinteractionchromatography(HIC) separatesmoleculesbasedontheir hydrophobicity.HIC
isa useful separationtechnique forpurifyingproteinswhile maintainingbiological activitydue tothe
use of conditionsandmatricesthatoperate underlessdenaturingconditions.
Alternative of HIC
1. Gel filtrationchromatography
2. Ionexchange chromatography
3. Reverse phase chromatography
Why we usedHIC
Differentbasisof separation
• Weakerinteractions
 Lessstructural damage
 Maintainhighactivity
Principle
 Separationof substancesisbasedontheirvaryingstrengthof interactionwithhydrophobic
groupsattachedto an unchargedgel matrix
• Hydrophobicgroupsonproteinsare sufficientlyexposedtobind tothe hydrophobicgroupson
the matrix
How is this achieved?
• The principle forproteinadsorptiontoHICmediaiscomplementarytoionexchange andsize
exclusionchromatography.Samplemoleculescontaininghydrophobicandhydrophilicregions
are appliedtoanHIC columnina high-saltbuffer.
• The salt inthe bufferreducesthe solvationof sample solutes.Assolvationdecreases,
hydrophobicregionsthatbecome exposedare adsorbedbythe media.

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• The more hydrophobicthe molecule,the lesssaltis neededtopromote binding.
Factors affecting HIC
• Ligand
A protein'sadsorptionbehaviorisdeterminedbythe type of immobilizedligand.Ingeneral,
straightchainalkyl ligandsdemonstratehydrophobiccharacterwhile aryl ligandsshow amixed
mode behaviorwhere botharomaticandhydrophobicinteractionsare possible.The choice of
ligandtype isempiricallydetermined.
• Degree of substitution
The proteinbindingcapacityincreaseswithanincreaseddegreeof substitutionof the
immobilizedligand.Withahighlevel of ligandsubstitution,the bindingcapacityremains
constant;however,the affinityof the interactionincreases.Proteinsboundunderthese
conditionsare difficulttoelute due tomulti-pointattachment.
• Matrix
The most widelyusedsupportsare hydrophiliccarbohydrates:cross-linkedagarose and
syntheticco-polymermaterials.The selectivitybetweendifferentsupportswill notbe identical
thoughthe ligandsmaybe the same.Modifyadsorptionandelutionconditionstoachieve
similarresultswhenmovingfromone mediatoanother.
• Salt concentration
The additionof structuredsaltsto the equilibrationbufferandsample promotesligand-protein
interactionsinHIC.Asthe salt concentrationincreases,the amountof boundproteinincreases
as doesthe riskof proteinprecipitationatthe higherionicstrength.
• pH
HIC mobile phasesare typicallyinthe neutral pHrange from5–7 and bufferedwithsodiumor
potassiumphosphate.Ingeneral,the strengthof the interactionbetweenproteinsandthe
mediadecreaseswithincreasingpHasa resultof increasedcharge of the proteindue tothe
titrationof acidicgroups.Thiseffectcan varyfrom proteintoprotein.
• Temperature
• The affinityof hydrophobicinteractionsincreaseswithtemperature.Temperature alsoimpacts
proteinstructure,solubility,andthe interactionwiththe HICmatrix.Because temperature
Advantages of HIC
• Large volume of sample can be loaded
• Sampleswith high ionicstrength can be used

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• Well suitedto use before gel filtration, ion-exchange andaffinitychromatography
• Sample elutedwith lowsalt
• Purificationstepsthat generate large sample volume can be coupledwith thismethod
• Goodfor samplesafter ammonium sulfate fractionation.
• These techniquesmay require pretreatmentof samples(e.g.reducingionicstrength)
• Sample can be used in ionexchange chromatography step
Affinity Chromatography
Thisisthe most selective type of chromatographyemployed.Itutilizesthe specificinteraction
betweenone kindof solute molecule andasecondmoleculethatisimmobilizedonastationaryphase.
For example,the immobilizedmoleculemaybe an antibodytosome specificprotein.Whensolute
containingamixture of proteinsare passedbythismolecule,onlythe specificproteinisreactedtothis
antibody,bindingittothe stationaryphase.Thisproteinislaterextractedbychangingthe ionicstrength
or pH.
Affinity among different chemicals/compounds
• Antigene.g.virusprotein,bacterial proteinwithspecificantibody
• Histidine hasaffinitywithNickel/Cobalt
• Glutathione Stransferase affinitywithGlutathione
• Glycosylationisthe mostcommonpost‐translationalmodificationof proteins....Glycosylation
of some proteinshasagreat impacton theirstructuresandfunctions,andinteractionsof
protein‐linkedglycanswithcarbohydrate‐specificproteins(lectins) modu
late manyimportant biological process
Advantages of Affinity chromatography
• The technique offershighselectivity,hence highresolution,andusuallyhigh capacityforthe
proteinsof interest.

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• Purificationthatwouldotherwise be time-consuming,difficultorevenimpossibleusingother
techniquescanoftenbe easilyachievedwithaffinitychromatography.
• The technique canbe usedto separate active biomoleculesfromdenaturedorfunctionally
differentforms,toisolate pure substancespresentatlow concentrationinlarge volumesof
crude sample andalsoto remove specificcontaminants.
Chromatofocusing
is a protein-separationtechnique thatallowsresolutionof single proteinsandotherampholytesfroma
complex mixture accordingtodifferencesintheirisoelectricpoint.
• Chromatofocusingisaformof gradientelutionchromatographyperformedusing ionexchange
resincolumnandan internally developedpH gradientthattravelsthrough the column.
• Thistechnique wasdevelopedby sluytermanandhiscolleagues
• Thistechnique isabit similarto isoelectricfocusingbuthere no electricfieldisinvolvedinstead
a pH gradientismade to propagate inside anionexchange chromatography
• Separationbetweenproteinisoforms differingbyasingle aminoacid residue andbylessthan
0.02 pH unit inapparentisoelectricpoint
• that may notseparate well,orat all,usingtraditional ionexchange strategies
• Thistechnique isabit similarto isoelectricfocusingbuthere no electricfieldisinvolvedinstead
a pH gradientismade to propagate inside anionexchange chromatography
• The pI of eachproteinisthe pH at whichthe proteinhaszerosurface charge.
• ProteinswithdifferentpIscanbe separatedbybeingpassedthrougha chromatofocusing
column
Procedure
• The Chromatofocusingmediumis equilibratedwithstartbufferata pH slightlyabove the
highestpHrequired.
• The elutionbuffer(polybuffer)ispassed throughthe columnandbeginstotitrate the amines
on the mediumandthe proteins.
• Thus a gradientpH isdeveloped
• sample isappliedtothe columnbymixing itwiththe start buffer
• Proteinsinthe sample thatare at a pH above theirpI are negatively-chargedand retainednear
the top of the column
• proteinsthatare at a pH belowtheirpI begintomigrate downthe columnand bind as they
reach the zone where the pHis above theirpI.
• The proteinwithhighestPI elute firstandthe protein withlowestPIelute last

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PACKING OF COLOUMN
• Columnispackedwithmonobead matrix
• Degasthe start bufferandthe slurryto avoidair bubbleswhichcan interferewithinthe
separation
LIMITATION
• It islesssuitable forthe isolationof proteinsthatprecipitateirreversiblyat or near their
isoelectricpointbecause these proteinsare likelytoprecipitate on the columnif theyreacha
highenough concentration.