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Types of LC

Planar Chromatography

Thin Layer Chromatography (TLC) Paper Chromatography (PC)

Column Chromatography Liquid Column Chromatography High Performance Liquid

Chromatography (HPLC)

LIQUID CHROMATOGRAPHY (LC)

The principle separation can be partition,adsorption or ion exchange depending onthe stationary phase used

The stationary phase is a thin layer ofadsorbent [eg silica gel, (SiO2) or alumina(Al2O3)] coated on a plate [eg glass, plastic]

The mobile phase (aka developing solvent)is organic [eg ethanol, acetone, chloroform]

The sample is applied (spot) at one end ofthe plate

The plate is placed in a chamber containinga small amount of solvent (spot at the

bottom)

THIN LAYER CHROMATOGRAPHY (TLC)

The solvent gradually moves up the platevia capil lary action carrying thesubstances along with it at differentratesComponents are adsorbed onto thestationary phase

The separated components appear as aseries of spots at different locationsalong the plate

The spots are visualized directly,observed under UV light or sprayed witha reagent to make them visible

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Rf is the retention or retardation factor The Rfvalue for a substance is the ratio ofthe distance that the substance travels tothe distance that the solvent travels upthe plate(best ratio 0.4-0.8)

Retention Factor (Rf)

ds

dx

frontsolventofDistance

componentofDistance

fR ==

Colored compounds - direct visual detectionwith the naked eye

Other compounds - indirect methods:

UV lamp (Under UV light)

Spray with reagent- Iodine vapor (or conc. H2SO 4) for all

organic compounds brown or black- N inhyd ri n f or 1 o and 2o amines purple

or brown- Flourescamine fluorescent- 2,4-DNPH for carbonyl) orange/yellow

Qualitative Analysis(Visualizing the Chromatogram)

Mainly qualitative analysis

Determine the number of components in amixture

Determine the identity of two substances

Monitor the progress of a reaction

Determine the effectiveness of a purification

Determine the appropriate conditions for acolumn chromatographic separation

Monitor column chromatography

Judge the purity of a synthesized compound orto indicate the extent of progress of a chemicalreaction

Applications Of TLC

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PAPER CHROMATOGRAPHY

The principle is partition chromatography(Analytes are separated based on the interactionsbetween two non-miscible liquid phases according

to their relative solubility)

Stationary Phase

Water (liquid phase) is adsorbed onto thehydroxyl group of cellulose (solid support) ofthe paper

- The water is t ightly bound (by hydrogenbonding) to the cellulose structure and fillinterspaces of the paper fibers

- Cellulose can also be chemically modified(eg with carboxylmethyl, diethylaminoethyl)

Mobile Phase

A solvent (aka the developing solvent) that ispartia lly miscible in water can be applied

- Choice of the solvent will depend on thenature of the substances to be separated

- A mixture of two or three solvents isoften required

The analytes that are polar (highly watersoluble or have the strong hydrogen-bondingcapacity) move slow er along the paper

Less polar compounds wi ll travel fasterwith the solvent

Qualitative Analysis Sample application and visualization of

separated components are similar to TLC

Setup of Paper Chromatography(a) Paper hanged vertically(b) Paper rolled up horizontally

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LIQUID COLUMN CHROMATOGRAPHY

The stationary phase (solid adsorbent) isplaced in a vertical column (usually glass)

- Column length: 50 - 500 cm- Column i.d.: 1 - 50 cm- Adsorbent particle size: 15 -200 m

The mobile phase ( l iquid) is added at thetop and allowed to flow through thecolumn by gravity or external pressure

Generally used as a purification techniqueto isolate desired compounds from amixture (preparative chromatography)

The column is packed with the stationaryphase and then equil ibrated with solvent(by running solvent through it)

The sample (dissolved in a small amount ofsolvent) is loaded onto the stationary phase

The mobile phase is added and allowed toflow through the column

The separated components are collected atthe end of the column at intervals

General Procedure

HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY (HPLC)

Performance means enhanced efficiency (andresolution) by the use of small diameter (2-5 m)stationary phase particles

The liquid is pressurized (to several hundred psi)for efficient flow rates

ADVANTAGES OF HPLC

High sensitivity (use of various detectors) Ideal for ionic species and large molecules Used for analytical or preparative work (can be

applied to various quantities of compounds) Wide analytical applications (eg pesticides, organic

& inorganic materials, food components, toxins,vitamins, etc)

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Schematics of The HPLC System

(1) Solvent (mobile phase) Reservoir

Reservoir (container) for mobile phase(solvent/ eluent) is pressurized

- Use organic solvents alone in Normal PhaseChromatography (eg Hexane, Cyclohexane)

- Use organic solvent-water combination inReverse Phase Chromatography (eg methanol-water or water-acetonitrile)

Mobile phase must be fi ltered (using 0.45 mmembrane fi lters) and then degassed by:

- Sparging (remove dissolved gases out of mobilephase using a stream of inert gas)

- Vacuuming (remove bubbles using a vacuumsystem)

- Sonicating (use sonicator to remove bubbles)

Elution Profile (Elution Mode)- I so cr at ic e l ut io n - Mob il e pha se

composition (ratio of polar to non polar eluent)is constant in a chromatographic run

- Gradient e lut ion - Mobi le phasecomposition changes during a run (The

strength of an eluent is increased by varying theratio of solvents eg polar vs non polar solvents toobtain a better separation in a shorter time)

Mobile phase (aka solvents, modifier)Acetoniltrile, methylene chloride, ethanol,methanol, tetrahydrofuran, water (polar)

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ELUTION MODES

Isocratic Elution

The composition of the solvent is constantthroughout the chromatographic elution

Gradient Elution

The composition of the solvent is ch angedcontinuously or in a series of steps to:

- overcome general elution problems- enhance the separation of early and late

eluting solutes

To deliver the mobile phase across theco lumn (Up to 6000 psi @ 3 mL min-1)- W it h mi ni mu m pu ls e- Cont inuously and reproducibly (2%)

- Under isocrat ic or gradient e lut ion

A reciprocating pump with a pulsedampener is used for a smoother flow

A syr inge pump can also be used withlimited capacity

(2) HPLC Pump

To inject sample into the mobile phase

Types of sample injection techniques:- A ut o s am pl er- V al ve i nj ec to r

Auto Sampler1. Can inject a large number of samples

2. Small vials w ith a septum are placed in atray

3. Needle penetrates septum and withdrawsthe sample

4. Valve introduces the sample into the column

(3) Injector

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Valve InjectorUsed to introduce samplequantitatively onto columnwithout interrupting themobile phase flow

Sampling loop consists ofvarious loop sizes for differentvolumes

- When placed in load posit ion,sample is loaded into anexternal fixed volume loop @atmospheric pressure

- When rotated to injectposition, loop becomes part ofthe eluent flow stream and thesample is carried to the column

Stainless steel or glass tubing (P:

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Common detectors:- UV/Visible- Fluorescence

- Refractive Index (a universal detector)- diode-array

Choice of detectors is determined by:- solute type- solute concentration- detector sensitivity- linear range- compatibil ity w ith solvent- elution mode

Coupled detectors: LC-MS, LC-NMR

(5) Detectors

TYPES OF HPLC

Normal Phase Chromatography

Reverse Phase Chromatography

Ion Exchange Chromatography

Size Exclusion Chromatography

NORMAL PHASE HPLC

Non polar analytes are eluted first

Increasing mobile phase polar ity ( increaseeluent strength by increasing polar solventratio) reduces retention time

- Weak solvents increase retention, strong solvents

decrease retention Stationary Phase: POLAR- Silica- Alumina- Bonded silica (Chemically bonded with

polar functional groups eg hydroxy, cyano,amino, nitrile)

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Mobile Phase: NON POLAR

- Nonpolar solvent (eg hexane) m ixed wi tha more polar modifier (eg methylenechloride)

- So lvent strength and se lect ivi ty iscontrolled to influence solute retention

Applications

- Compounds highly soluble in organicsolvent (eg fat soluble vitamins) or thatare weakly soluble in aqueous mobilephase

- Compound c lasses (eg isomers) andhydrophil ic species (eg carbohydrates)

REVERSE PHASE HPLC

Polar analytes are eluted first

Reducing mobile phase polar ity (eluentstrength decrease) reduces retention time

Stationary Phase: NON POLAR- Silica- Bonded sil ica (eg C18-sil ica) phases chemically

bound to si l ica via surface si lanol usually anoctadecyl (C18) or octadecylsilyl

Chromatographic behavior is influenced by thetype of organic group bound to sili ca, chainlength of the organic group (C4 - C18), supportparticle size and shape

- Polymeric phases eg Cross- l inked polystyrene-divinylbenzene (PS-DVB)

Mobile Phase: POLAR- Water (mixed with) methano l, acetonitr ile or

tetrahydrofuran (THF)Solute retained due to hydrophobic interactions withthe non polar stationary phase

Additives (may be required):- Amine to deac tivate s il ano ls- Aqueous buffers to suppress ionization of

sample components- Ion-pair reagents to neutralize charged solutes

and make them more lipophilic

Applications of HPLC Reverse Phase- Proteins (includes cereal proteins)- Water soluble and fat so luble v i tamins- Ca rb oh yd ra tes (requires ion-pairing reagents)- Ant iox idants, dyes, p igments, pheno l ic f lavor

compounds

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Stationary Phase:Ion exchanger functional groups

When a cation exchanger is immersed in anaqueous solution containing the M + cations,an exchange equilibrium is established:

xRSO3-H+ + M x+ (RSO3 )xM x+ + xH+

(solid) (solution) (solid) (solution)

where, M is cation, R is part of the resin moleculecontaining one sulfonic acid group

For an anion exchange resin:

xRN(CH3)3+OH + Ax- [RN(CH3) 3+]xAx- + xOH

(solid) (solution) (solid) (solution)

where, Ax- is an anion

Mechanism of The Ion Exchanger

Mobile Phase

Solute retention is controlled by changingionic strength and pH of the mobile phase

Use aqueous solution which contain ionicsolutes (but may be mixed w ith the organicsolvent when necessary)

Applications

Simple inorganic compounds (cations andanions)

Amino acids, carbohydrates, organic acids Preparative purification of proteins and

fractionation of peptides

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Instrumentation

IEC is performed in two forms:

Suppressed Ion Chromatography

- A second column (suppressor column) is usedto remove the buffer ions so that sample ionscan be more easily detected

Nonsuppressed Ion Chromatography

- Weakly conductin g buffers of lowconcentrations are carefully selected, and theentire effluent is passed through the detector

- Ions are detected above the backgroundsignal

Schematics of an IEC Chromatograph

Size-exculsion chromatography (SEC) isalso called gel-filtration or gel-permeationchromatography (GPC)

Separation of mixture is based on the

molecular size of the components Uses porous particles to separate

molecules of different sizes

Molecules that are smaller than the poresize can enter the pores and thereforehave a longer transit t ime than largermolecules that cannot enter the pores

SIZE EXCLUSION HPLC

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Large molecules do not enter pores of thestationary phase matrix and elute as the earlypeaks in the chromatogram

Molecules that enter the pores will have anaverage residence time that depends on themolecular size and shape

Small molecules have a longer residence timein the column and elute as later peaks in thechromatogram

Stationary Phase and Mobile Phase

The stationary phase is selected based on thepore size that matches the molecular weightrange of the species to be resolved

Hydrophilic packing (surface modified silica ormetacrylate resins) is used with an aqueousmobile phase for water soluble samples

Hydrophobic packing (polystyrene divinylbenzene) is used with non-aqueous mobilephase chosen based on the sample solubility

Applications

To separate biological molecules (amino acid,virus, carbohydrate, etc)

To determine molecular weights and molecularweight distributions of polymers