Basics of Chromatography_KR_C-CAMP.pdf

8/15/2019 Basics of Chromatography_KR_C-CAMP.pdf

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Basics of chromatographic Techniques

Course 1

Kannan R., Ph. D.


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Investigator(s) Year Contribution

ThomsonDempster

AstonStephensHipple, Sommer, and ThomasJohnson and NierPaul and SteinwedelBeynonBiemann, Cone, Webster, and Arsenault

Munson and FieldDoleBeckeyMacFarlane and TorgersonComisarow and MarshallYost and EnkeBarberTanaka, Karas, and HillenkampFennChowdhury, Katta, and ChaitMann and WilmGanem, Li, and HenionChait and KattaPieles, Zurcher, Schär, and MoserHenzel, Billeci, Stults, Wong, Grimley, andWatanabeSiuzdak, Bothner, Fuerstenau, and Benner

1899 –19111918

1919194619491953195319561966

1966196819691974197419781981198319841990199119911993

19931996 –2001

First mass spectrometerElectron ionization andmagnetic focusingAtomic weights using MSTime-of-flight mass analysisIon cyclotron resonanceDouble-focusing instrumentsQuadrupole analyzersHigh-resolution MSPeptide sequencing

Chemical ionizationElectrospray ionizationField desorption MS of organic moleculesPlasma desorption MSFT-ICR MSTriple quadrupole MSFast atom bombardment (FAB)Matrix-assisted laser desorption/ionizationESI on biomoleculesProtein conformational changes with ESI MSMicroESINoncovalent complexes with ESI MSOligonucleotide ladder sequancing

Protein mass mappingIntact viral analysis

Historical Developments in MS


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History

Mikhail Tswett, Russian, 1872-1919 BotanistIn 1906 Tswett used to chromatography to

separate plant pigments

He called the new technique chromatographybecause the result of the analysis was 'written in

color' along the length of the adsorbent column

Chroma means“

color”

and graphein means to“

write”

Thin layer chromatographyis used to separate thecolorful components of aplant extract


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Way and Thompson

Runge, Schoenbein, andGoeppelsroeder

Lemberg

Reed

Tswett

Karrer, Kuhn, and Strain

Holmes and Adams

Reichstein

Izmailov and Schraiber

Brown

Tiselius

1848

1850-1900

1876

1892

1903-1906

1930-1932

1935

1938

1938

1939

1940-1943

Recognized the phenomenon of ion exchange in solids.

Studied capillary analysis on paper.

Illustrated the reversibility and stoichiometry of ionexchange in aluminum silicate minerals.

First recorded column separation: tubes of kaolin used forseparation of FeCI 3 from CuSO 4.

Invented chromatography with use of pure solvent todevelop the chromatogram; devised nomenclature;used mild adsorbents to resolve chloroplast pigments.

Used activated lime, alumina and magnesia absorbents.

Synthesized synthetic organic ion exchange resins.

Introduced the liquid or flowing chromatogram, thusextending application of chromatography to colorlesssubstances.

Discussed the use of a thin layer of unbound aluminaspread on a glass plate.

First use of circular paper chromatography.

Devised frontal analysis and method of displacementdevelopment.

Historical Developments in Chromatography


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Martin and Synge

Consden, Gordon, and Martin

Boyd, Tompkins, et al

M. Lederer and Linstead

Kirchner

James and Martin

Sober and Peterson

Lathe and Ruthvan

Porath and Flodin

J. C. Moore

1941

1944

1947-1950

1948

1951

1952

1956

1956

1959

1964

Introduced column partition

chromatography.

First described paper partitionchromatography.

Ion-exchange chromatography appliedto various analytical problems.

Applied paper chromatography to

inorganic compounds.

Introduced thin-layer chromatographyas it is practiced today.

Developed gas chromatography.

Prepared first ion-exchange celluloses

Used natural and modified starchmolecular sieves for molecular weightestimation.

Introduced cross-linked dextran formolecular sieving.

Gel permeation chromatographydeveloped as a practical method.

Historical Developments in Chromatography


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Importance

Chromatography has application in every branch of thephysical and biological sciences

12 Nobel prizes were awarded between 1937 and 1972 alone forwork in which chromatography played a vital role


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The substances in a mixture are not chemically combined, sotherefore they can be separated through some physical process.

chromatography, technique for separating the components, orsolutes , of a mixture on the basis of the relative amounts of each

solute distributed between a moving fluid stream, called the mobilephase , and a contiguous stationary phase . The mobile phase maybe either a liquid or a gas, while the stationary phase is either asolid or a liquid.

Chromatography is the ability to separate molecules usingpartitioning characteristics of molecule to remain in a stationaryphase versus a mobile phase. Once a molecule is separated fromthe mixture, it can be isolated and quantified.

Chromatography

http://www.britannica.com/EBchecked/topic/553701/solutehttp://www.britannica.com/EBchecked/topic/386794/mobile-phasehttp://www.britannica.com/EBchecked/topic/386794/mobile-phasehttp://www.britannica.com/EBchecked/topic/564121/stationary-phasehttp://www.britannica.com/EBchecked/topic/564121/stationary-phasehttp://www.britannica.com/EBchecked/topic/564121/stationary-phasehttp://www.britannica.com/EBchecked/topic/564121/stationary-phasehttp://www.britannica.com/EBchecked/topic/386794/mobile-phasehttp://www.britannica.com/EBchecked/topic/386794/mobile-phasehttp://www.britannica.com/EBchecked/topic/386794/mobile-phasehttp://www.britannica.com/EBchecked/topic/553701/solute


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Chromatography

Stationary Phase

1.Thin Layer Chromatography2. Paper Chromatography3. Column Chromatography

Mobile Phase

1. Liquid chromatography2. Gas Chromatography

Classification according to the force of separation

1- Adsorption chromatography.2- Partition chromatography.3- Ion exchange chromatography.4- Gel filtration chromatography.5- Affinity chromatography.

Different Chromatographic Techniques


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Thin Layer Chromatography

TLC is a method for identifying substances and testing the purity of

compounds.

TLC is a useful technique because it is relatively quick and requiressmall quantities of material.

Separations in TLC involve distributing a mixture of two or more

substances between a stationary phase and a mobile phase .The stationary phase: is a thin layer of adsorbent (usually silica gel oralumina) coated on a plate.

The mobile phase: is a developing liquid which travels up the stationaryphase, carrying the samples with it.

Components of the samples will separate on the stationary phaseaccording to how much they adsorb on the stationary phase versushow much they dissolve in the mobile phase.


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If the spots can be seen, outline them with

a pencil.

If no spots are obvious, the most common

visualization technique is to hold the plate

under a UV lamp.

Many organic compounds can be seen

using this technique, and many

commercially made plates often contain asubstance which aids in the visualization of

compounds.

Identifying the Spots (visualization)

http://chemscape.santafe.cc.fl.us/chemscape/catofp/chromato/tlc/fluoresc.htmhttp://chemscape.santafe.cc.fl.us/chemscape/catofp/chromato/tlc/fluoresc.htm


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Reagents CompoundsIodineUV lightp-AnisaldehydeBromocresol green2,4-dinitrophenylhydrazineNinhydrinSulfanilic Acid Reagent(Diazotized), Pauly's ReagentSulfuric acidAniline phthalateAntimony trichlorideDragendorff

’

s reagent

Aromatic compoundsUnsaturated compoundsCarbohydratecarboxylic acidMainly for aldehydes and ketonesGood for aminesphenolic compounds turn orangeor yellow with this reagentsprayed on the TLCSugarCardiac glycosidesAlkaloids

Visualizing Agents


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The R f (retention factor) value for each spot

should be calculated.It is characteristic for any given compoundon the same stationary phase using thesame mobile phase for development of theplates.

Hence, known R f values can be compared tothose of unknown substances to aid in theiridentifications.

Interpreting the Data


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A method of partition chromatography using filter paper stripsas carrier or inert support.

The factor governing separation of mixtures of solutes on filter

paper is the partition between two immiscible phases.

One is usually water adsorbed on cellulose fibres in the paper

(stationary phase).

The second is the organic solvent flows past the sample on the

paper (stationary phase).

Paper Chromatography


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A method of partition chromatography using

filter paper strips as carrier or inert support.

The factor governing separation of mixturesof solutes on filter paper is the partitionbetween two immiscible phases.

One is usually water adsorbed on cellulosefibres in the paper (stationary phase).

The second is the organic solvent flows pastthe sample on the paper (stationary phase).

Partition occurs between the mobile phase

and the stationary aqueous phase bound bythe cellulose.

The isolation depends on partition coefficientof the solute.

Paper Chromatography

( )( )

c stationary K

c mobile


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Column Chromatography

Stationary phase is held in anarrow tube through which themobile phase is forced underpressure or under the effect ofgravity

Column Chromatography


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EffectFactor

Decrease of size improves separation (but very smallparticles need high pressure).

Particle size of solid stationaryphase (or of support)

Efficiency increases as ratio length / width increases.Column dimensions

Non uniform packing results in irregular movementof solutes through column & less uniform zoneformation, (i.e. band broadning or tailing).

Uniformity of packing

Increase in column temperature results in speed ofelution but does not improve separation (tailing).

Column temperature

Solvents should be of low viscosity (to give efficientresolution) & high volatility (to get rapid recovery ofthe substances).

Eluting solvent

Uniform & low flow rate gives better resolution.Solvent flow rate

Discontinuous flow disturbs resolutionContinuity of flow

Deactivation of adsorbent decreases separation.Condition of adsorbent

Substances of high concentration move slowly.Concentration of solutes

Factors affecting solutes separation in CC


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MechanismMobile phaseStationary phaseMode or type

Solutes move at different ratesaccording to the forces of attractionto the stationary phase.

Liquid or gasSolid that attractsthe solutes

AdsorptionChromatography

Solutes equilibrate between the 2phases according to their partitioncoefficients

Liquid or gasThin film of liquidformed on thesurface of a solidinert support

PartitionChromatography

Solute ions of charge opposite to thefixed ions are attracted to the resinby electrostatic forces & replace themobile counterions.

Liquidcontainingelectrolytes

Solid resin thatcarries fixed ions& mobilecouterions ofopposite chargeattached bycovalent bonds

Ion ExchangeChromatography

Molecules separate according totheir size:

1.Smaller molecules enter the poresof the gel, and need a larger volumeof eluent.2.Larger molecules pass through thecolumn at a faster rate.

LiquidPorous gel with noattractive action

on solutemolecules

Molecular ExclusionChromatography

Special kind of solute molecules

interact with those immobilized onthe stationary phase

Liquid or gasSolid on which

specific moleculesare immobilized

Affinity

Chromatography


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ProcedureTechnique

Addition of solvent mixture of fixed compositionduring the whole process.

Isocratic elution

: in which there isContinuous or linear elution

continuous change in the composition of themobile phase over a period of time (e.g. polarity,pH or ionic strength).

Gradient elution

: in which theStep wise or fractional elutionchange is not continuous i.e. a sudden change inthe composition of the mobile phase is followedby a period where the mobile phase is heldconstant.

Elution techniques


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H = Theoretical Plate HeightL = Length of the Column.

N = L / H

As HETP decreases efficiencyof the column increases.

N = L (tr/W) 2

Number of Theoretical Plates (N)


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Adsorbents:

The most common are Alumina & Silica gel in which theinteractions with solute molecules is due to OH groups present on their surface . More polar molecules are adsorbed more strongly & thus, will

elute more slowly Strength of adsorption of polar groups (solutes) on polar supportis in the following order: -C=C- < O-CH3 < -COOR < >C = O < -CHO < -NH2 < -OH < -

COOH

Olefins < Ethers < Esters < Lactones < Aldehydes < Amines <Phenols < Acids.

Adsorption Column Chromatography


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24

In partition chromatography a solidsupport with a high surface area such ascrushed firebrick or keiselguhr is coatedwith a high boiling liquid which acts asthe stationary phase. Separation occursbecause of the differences in solubility

for the analytes in the stationary andmobile phases.

The partition coefficient is defined as:

Conc n. in stationary phaseK =

Conc n. in mobile phase

coated support particle

Stationary phase Analyte

Solid

support

Partition Chromatography


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Bonded phase chromatography

In bonded phase chromatography, the molecule acting as the stationary phaseis chemically bonded to the solid support.

R can be a C 18 alkane chain or an amine (NH 2) or cyano (CN) group or someother group. The nature of R determines the types of analytes which can beseparated.

The theories of partition and adsorptionchromatography are both used to describethis mode of chromatography although it isoften classified as a partition technique.

Si OH + Cl SiR Si O SiR + HCl

diagram of phase

bonded to silica


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There are two types of partition chromatography normal phase and reversed phase,they are defined by the relative polarities of the mobile and stationary phases

For this reason, the use of silica (a polar molecule) as the stationary phase (as inadsorption chromatography) is also considered to be a normal phase separationmethod.

Because of its versatility and wide range of applicability, reversed-phasedchromatography is the most frequently used hplc method.

Types Partition Chromatography


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This type is also known as:

Size Exclusion Chromatography (SEC)

Molecular Exclusion Chromatography (MEC)Molecular Sieve Chromatography (MSC)

Gel Filtration Chromatography (GFC)

Gel Chromatography .

Gel Permeation Chromatography (GPC)


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Mobile phaseMobile phase is a liquid as water or dilute acohol

Separation mechanism Based on difference between the solutes molecular weights.

Molecules will distribute themselves outside & inside the poresaccording to their size.

Larger are excluded, medium sized enter half-way & smallestpermeate all the way.

Mobile phase


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Determination of M. wt. of peptides, proteins & polysaccharides.

Desalting of colloids e.g. desalting of albumin prepared with 2% (NH 4)2SO4.

Separation of mixture of mono- & polysaccharides.

Separation of amino acids from peptides & proteins.

Separation of proteins of different molecular weights.

Separation of mucopolysaccharides & soluble RNA.

Separation of myoglobin & haemoglobin.

Separation of alkaloids & purification of enzymes.

Applications of GPC to natural products


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Affinity Chromatography

The five steps in affinity chromatography

Activation- the ligand is bound to the chromatographic solidsupport.

Loading- the analytes to be separated are introduced into themobile phase stream.

Binding- the analytes of interest are retained due toInteraction with the ligand of the stationary phase.

Washing- unwanted analytes are eluted from the column.

Elution- the analyte(s) of interest are washed from the columnby changing the mobile phase composition.

Application:-Purification of proteins-Study of drug and hormone interactions with proteins-Immunoassays


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Stationary phases for ion-exchangeseparations are characterized by thenature and strength of the acidic or basicfunctions on their surfaces and the types ofions that they attract and retain.

Cation exchange is used to retain andseparate positively charged ions on anegative surface.

Conversely, anion exchange is used toretain and separate negatively chargedions on a positive surface.

With each type of ion exchange, there areat least two general approaches forseparation and elution.

Ion Exchange Chromatography


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Different kinds of Ion Exchange Chromatography


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HPLC


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UV-Visible Fluorescence PDA Light Scattering Mass Spectrometry

More common HPLC Detectors

Less sensitive (microgram level)

Highly sensitive


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Chromatogram of Amino acid derivatives


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LC-MS System


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Triple Stage Quadrapole (TSQ)

Q00 Q0Q1

Hyperquad

Q3

Hyperquad

Q2Collision Cell

DetectionSystem

Ion Source

Interface

Ion Transfer Tube Mass Analyzer


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LC-MS Chromatogram of Amino acid derivatives

Diff Ki d f E i


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Different Kinds of Experiments

Full Scan (MS)

MS/MS Scan ( Product Ion Scan)

Parent ion scan (Precursor scan)

Selected Reaction Monitor (SRM) scan

Multiple reaction monitor (MRM) scan

Less sensitive (lower ng)

More sensitive (fg)


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Selected Reaction Monitoring (SRM) method

Nature Methods 9, 555 –566 (2012)

Q2

Q1 Set RF Only + Ar Q3 Set

Q1 Q3 Transition

Gas Chromatography


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Gas Chromatography


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“

Without Chromatographic separationit is impossible to analyze complex

samples by using Mass spec ”

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Basics of Chromatography_KR_C-CAMP.pdf