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CHAPTER 6(HIGH PERFORMANCE LIQUID CHROMATOGRAPHY)

6.1 HPLC INTRODUCTION6.1.1 Founder of Liquid Chromatography

Mikhail TsvetBorn on May 14, 1872 Asti, ItalyInvented chromatography in 1901 while performing research on plant pigmentsColumn adsorption liquid chromatography

6.1.2 HPLC AnalysisQualitative Analysis

Identification of compound identityRequire a known standardIdentified by comparing retention time

Quantitative AnalysisIdentify the amount/concentration of the compoundRequire a standard with known amount of concentrationsIdentified by interpolating the area of unknown into a set of

standards with known concentration

Qualitative Analysis »

Comparing Retention TimeQuantitative Analysis »

Comparing Peak Area/Height

6.1.3 Basic Concept of HPLCSample are injected into a HPLC system.

Interactions happen between the samples with the mobile phase

and stationary phase (column) which results in separation of

samples which is detected through the detector and converted into

a chromatogram.

V

VV

VV

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VV

V V

V

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Elution

V

VV

V

V

V

XXX

A

A

A

A

A

A

X

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X A

AA

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A A

X

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A

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AAA

A

Injection

Interaction

6.1.4 HPLC PeaksA separation by changing the relative speed of each analyte band(Competition between the mobile phase and stationary phase).

As analyte

“Bands”

pass through Detector Flow Cell, an electrical signal is sent to the Computer Data Station (recorder) to draw the “Peak”.

Blue band is now the broadest as it exits the column since it was moving so slowly in the column, it takes a lot of mobile phase to finally sweep it all out –

this is why the latest eluting peaks are the broadest.

How Peaks Are Created?

ISOCRATIC CONDITIONS

Few, Earliest Arriving AnalyteMolecules

Fron

t

Ape

x

Bac

k

Chromatogram

Analyte “Band” flowing

Into Detector Cell

Most AnalyteMoleculesArrive (Highest Concentration)

Few, Latest Arriving AnalyteMolecules

Computer Data Station

Signal from the Analyte “Band” in the Detector Flow Cellis Translated into a “Peak”The higher the analyte concentration, the higher the peak height –better Sensitivity

Most Concentrated Point in the Analyte “Band”

Time

D E T E C T O R

MobilePhase

MobilePhase

Mobile Phase

Base Line

Mobile Phase

Base Line0

How an Analyte

Becomes a Peak?

6.1.5 Band Spreading

Narrow Band –Narrow Peak More Concentrated –

Increased Peak Height/Sensitivity More Resolution Capability

Broad Band –

Broad PeakLess Concentration –Less SensitivityLess Resolution Capability

Broader “Band”More “Band Spreading”Broader Peak

Narrower “Band”Less “Band Spreading”Narrower, Taller Peak

MobilePhase

MobilePhase

MobilePhase

Less Concentrated

0 0

MobilePhase

Better separationMore concentrated “Bands”Higher Sensitivity

In this region, both analytes

(blue and red) are not separated [a partial co-elution –

shown as a “purple”

band]

System withMORE Band Spreading

System with LESS Band Spreading

Narrower, Sharper Bands create Narrower Sharper Peaks, which provide a better separation.This results in better Resolution between the 2 peaks and greater peak heights for better Sensitivity

6.2 TYPES OF HPLCHPLC : is a type of chromatography that employs a liquid mobile

phase and a very finely divided stationary phase.

The types of HPLC are often classified by separation mechanism or

by the type of stationary phase :

Partition / Liquid-liquid chromatography

Adsorption / liquid solid chromatography

Ion exchange

Size-exclusion

Affinity chromatography

Chiral chromatography

The selection separation procedure is governed by what the analytes

are.

6.3 APPARATUSIn HPLC solvent at high pressure is forced through a column to obtain a separationThe instrument is made of a solvent system, an injection valve, a column, and a detector.

HPLC system have :1.

Solvent reservoir 7. Sample Loop 2.

Degasser 8. Guard Column3.

Gradient Valve 9. Column4.

Mixer 10. Detector5.

High Pressure Pump 11. Acquisition Computer 6.

Switching Valve 12. Waste Reservoir

6.3.1 (a) Mobile Phase Reservoir and Solvent Treatment Systems

Solvent reservoir is made from glass and it contains 500 mL or more of a solvent volume.However, dissolved gas and dust from liquid can form bubbles in column »

band spreading. Thus, Degassers is needed for a sparging

process.Sparging : the removal of an unwanted dissolved gas by aeration with an inert gas.Elution can be performed isocratically or with a gradient :

Isocratic elution is performed with a constant solvent (pure or constant mixture).Gradient elution is the solvent mixture is altered during the run if one solvent is not satisfactory.

Isocratic elution

Mixture of acetonitrile

and aqueous phase buffer solution (KH2

PO4

)Separation of aromatic compounds, including alcohols and ketones.

Gradient elutionIncreasing ratio of acetonitrile

during the analysis behaves like temperature programming in GC

6.3.1 (b) Pumping SystemThe requirement of pump :

Ability to generate pressures up to 6000 psi

Pulse-free output

From rate ranging 0.1 to 10 mL/min

Flow reproducibilities of 0.5% relative or better

Resistance to corrosion by a

variety of solvent

There are 3 types of pumps

Syringe-type pumps

Reciprocating pump

Pnuematic or constant pressure pump

A reciprocating pump for HPLC

6.3.1 (c) Sample Injection SystemThe most widely used method of sample introduction in liquid chromatography is based on a sampling loop.These devices are an integral part of some liquid chromatography equipment.

Interchangeable loops are available to provide a choice of sample sizes ranging from 5 to 500 μL.Many HPLC instruments have autosamplerwith an automatic injector.

6.3.1 (d) Analytical ColumnsHPLC column :

made from stainless steel10 – 30 cm long2 – 5 mm internal diameterParticles size for column packing is 3 – 10 μm and provide 40000 –

60000 plates/m

Microcolumn :3 – 7.5 cm long1 – 4.6 mm internal diameterParticles size for column packing is 3 – 5 μm and provide 100000 plates/mHave disadvantage of speed and minimal solvent consumption.

The most packing for liquid chromatography is prepared from silica

particles

which are synthesized by agglomerating submicron silica

particles under conditions that lead to larger particles with highly

uniform diameters.

The resulting particles are coated with thin organic films, which are

chemically or physically bonded to the surface.

Guard Column Positioned ahead of the analytical column to increase the life of the analytical column by removing particulate matter and contaminants from solvents.It also serves to saturate the mobile phase with the stationary phase so that losses of stationary phase from the analytical column are minimized.Composition is similar to analytical column except particle size is larger to minimize pressure drop.

Column ThermostatsBetter chromatograms are obtained by maintaining column temperatures constant to a few tenths of a degree celcius.Equipped with heaters to control column temperatures.Columns may be fitted with water jackets from a constant-temperature bath to give precise temperature control.

6.3.1 (e) DetectorsDetectors for HPLC must have low dead volume to minimize extra-column band broadening.The detector :

small and compatible with liquid flownot highly sensitiveuniversal detector system

There are 2 types of detector :Bulk property detector : measure property of mobile phase (refractive index, dielectric constant, density).

Solute property detector : measure property of solute

present in mobile phase (UV absorbance, fluorescence, IR

absorbance).

The most widely used detectors for liquid chromatography are

based on absorption of ultraviolet or visible radiation.

Both photometer and spectrophotometer specifically designed for

use with chromatographic column are commercially available.

Source :

single line (arc or hollow cathode lamp)

continuum (Xe, D2 lamp)

Detector :

photodiode/ photomultiplier tube

photodiode array

UV-Vis Detection for HPLC

Performance of LC Detectors

6.4 HIGH PERFORMANCE PARTITION CHROMATOGRAPHY

The most widely used and popular type of HPLC → the stationary phase is a second liquid that is immiscible with the liquid

mobile phase.

Sample molecules equilibrate (PARTITION) between liquid stationary phase and mobile phase. Retention depends on a sample molecule's escaping tendency into the mobile phase versus its solubility in the stationary phase.

It can be divided by 2 types, liquid-liquid and liquid-phase-bounded chromatography.i. Liquid-liquid partition chromatography »

the stationary phase is a solvent that is held in place by absorption on the surface of packing material.

ii. Liquid-bounded-phase chromatography »

the stationary phase is an organic species that is attached to the surface of the packing particles by chemical bonds.

6.4.1 Bonded-Phase PackingsMost of the bonded-phase packings are prepared by rxn of an organochlororsilane

with the ―OH groups formed on the surface of silica particles (3, 5 or 10 μm)

by hydrolysis in hot, dilute HCL to produce organosiloxane.

Advantage : not required periodic recoating of stationary phase due to thee phase will be dissolved away by mobile phase and very practical for gradient elution.

6.4.2

Normal and Reversed-Phase PackingsBase on the relative polarities of the mobile and stationary phase :

Normal-Phase HPLC → nonpolar (solvent)/ polar (column)Reversed-Phase HPLC → polar (solvent)/ nonpolar (column)

In Normal-Phase Chromatography → the least polar component is eluted first »

↑

the polarity of the mobile phase, ↓

the elution time.In Reversed-Phase Chromatography → the most polar components elutes first, ↑

the polarity of the mobile phase, ↑

the elution time.

6.4.3 Choice of Mobile and Stationary PhasesThe partition chromatography requires a proper balance of intermolecular force among analyte, mobile phase and stationary phase.This intermolecular force are describes qualitatively in terms of the relative polarity posses

by each components. The order of polarities of common mobile phase solvents » water> acetonitrile> methanol> ethanol > tetrahydrofuron

> propanol

> cyclohexane

> hexane.

For stationary phase choice : choose column with similar polarity to analyte

for maximum interaction.Reversed-Phase » column (nonpolar)Normal-Phase » column (polar)

For mobile phase choice : Polar strong solvent interacts most with polar analyte

(solute) –

elute faster but less resolution.

6.4.4 Applications

Field Typical Mixture Separated

Pharmaceuticals Antibiotics, sedatives, steroids

Biochemicals Amino acids, proteins, carbohydrates, lipids

Food Products Antioxidants, additives

Industrial Chemicals Dyes, propellants, surfactants

Pollutants Pesticides, herbicides, phenols

Forensic Chemistry Drugs, poisons, blood alcohols

6.5 HIGH PERFORMANCE ION-EXCHANGE CHROMATOGRAPHY

The interactions between the chromatographic medium and theproteins in the mixture are based primarily on ionic charge.Ion exchangers are resins often coupled on cross-linkedpolysaccharides that can exchange ions with water solutions.

Most common types of ion-exchangers:DEAE (diethylaminoethyl)-cellulose; an anion exchange resin used primarily for neutral and acidic proteinsCM (carboxymethyl)-cellulose; a cation exchanger used primarily for the separation of neutral and basic proteins.

Elution from the column:by altering the pH of the elution bufferby increasing the ionic strength of the elution buffer

Two types of ion chromatography in use :Suppressor basedSingle Column

6.5.1 Ion Chromatography Based on SuppressionIn suppression-based ion chromatography, the ion exchange column is followed by a suppressor column or by a suppressormembranes that converts an ionic eluent

into a non-ionic species that does not interfere with the conductometric

detection of analyteions.

6.5.2 Single Column Ion ChromatographyIn single-column ion-exchange chromatography, analyte ions are separated on a low-capacity ion exchanger by means of low-ionic strength eluent

that does not interfere with the conductometricdetection of analyte

ions

6.6 HIGH PERFORMANCE SIZE-EXCLUSION CHROMATOGRAPHY

It is based on molecular size.Used for large MW compounds protein and polymers.Separation mechanism is sieving not portioning.Stationary phase » porous silica or polymer particles (Polystyrene, polyacrylamide)(5 –

10 μm)

6.6.1 Column PackingWell-defined pore sizes (40 – 2500 Å)

Large molecules excluded from pores – not retained, first eluted (exclusion limit –

term of MW).Intermediate molecules – retained, intermediate elution times.Small molecules permeate into pores – strongly retained last eluted (permeation limit – term of MW).

6.6.2 ApplicationsSeparation of sugars in caneSeparation of proteins/ peptideDetermination of molecular mass of large polymers or natural products