TECHNICAL REVIEW:

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Prepared by: Shariffah Huzaimah Al-Junid

10 october 2008

INTRODUCTION

� HPLC terms:

� High performance liquid chromatography

� High pressure liquid chromatography

� High price liquid chromatography

� Important analytical tool for separating and � Important analytical tool for separating and

quantifying components in complex liquid mixture

� Common method use for analysis of:

� Biological compound

� Pharmaceuticals

� Environmental toxicant

BASIC PRINCIPLES OF

CHROMATOGRAPHY

� Definition- separation process (distribute sample

mixture between two phases in chromatographic bed)

� Stationary phase

� Column packing material

� Stronger interaction with stationary phase than mobile phase – elute less quickly (longer retention time)

� Mobile phase

� Liquid media – continuously flow through the column and carry the analytes

� Mixture of solvents

CHROMATOGRAPHY

PLANAR

PAPER

THIN LAYER

GAS CHROMATOGRAPHY ADSORPTION

COLUMN

GAS CHROMATOGRAPHY

LIQUID CHROMATOGRAPHY

ADSORPTION

ION EXCHANGE

SIZE EXCLUSIONSOLID PHASE CHROMATOGRAPHY

HPLC INSTRUMENT

Quaternary Pump

Solvent Degasser

Solvent Reservoirs

Column Compartment

Autosampler

Detector

Quaternary Pump

• To hold the solvents used to make up the mobile phase

SOLVENT RESERVOIR

• To prevent bubbles in the mobile phaseDEGASSER

• Quaternary pump that mixes the solvents

• Pumps them through column and detector

PUMP

• To hold column and control column temperature using thermostats

COLUMN COMPARTMENT

• Draws prescribed volumes from sample vials and injects them onto the columnAUTOSAMPLER

• Detector that monitors the entire spectrum of the column effluent at regular intervals

DETECTOR

Flow chart….

MOBILE PHASE

PUMP INJECTION

COLUMNDETECTORRECORDER

DATA SYSTEM

INSTRUMENT- SOLVENT

RESERVOIR

� Several reservoirs suited to pump type

� Degassing : remove of dissolved gas

� Sparging line bubble or gas

� Vacuum pumping

� Dust removal – interference with detection, column clogging, damage pumping system

� Millipore filter under vacuum

� Solvent containers – enclosed to protects users from toxic solvent vapors (chloroform, aromatic hydrocarbons and etc.)

Cont..

Isocratic Elution Gradient ElutionGradient ElutionGradient ElutionGradient Elution

� Eluent composition constant during the

whole analysis

� Eluent composition

changed during the

analysis

� increase separation � increase separation

efficiency

� decrease the retention

time

� Peak shape is improved (Less tailing)

Isocratic Elution (B : Acetonitrile) Gradient Elution

� Column : 0.46 * 25cm Hypersil ODS

� Flowrate : 1.0 mL/min

� Eluent : Aqueous Buffer (pH 3.5) and Acetonitrile

(1) benzyl alcohol, (2) Phenol, (3) 3’, 4’- dimethoxy-toluene, (4) benzoin

(5) ethyl benzoate, (6) toluene, (7) 2, 6 -dimethoxytoluene, (8) o-methoxybiphenyl

INSTRUMENT - PUMP

� Requirement

� High pressure up to 6000psi – force solvents

through stationary phase beds

� Pulse free, prevents remixing of solutes

� Flow rate range: 0.1 to 10ml/min� Flow rate range: 0.1 to 10ml/min

� Integrated degassing system – vacuum degasser

� Resistant to corrosion

� acidic, basic and strong eluent in mobile phase

Cont..

� Reciprocation pumps (most use) � advantages: small internal volume, high output pressures,

constant flow rates

� disadvantages: produce a pulsed flow, cause baseline noise

� Displacement pumps

advantages: output is pulse free � advantages: output is pulse free

� disadvantages: limited solvent capacity (<250 mL) inconvenience when change solvents

� Pneumatic pumps

� advantages: inexpensive, output is pulse free

� disadvantages: limited solvent capacity, not amenable to gradient elution and limit pressures <2000 psi

INSTRUMENT - COLUMN

� Stainless steel

� Resistant to the high pressure

� Inert to chemical corrosion

� Criteria for a column

� Length: 10, 15 and 25cm� Length: 10, 15 and 25cm

� Particle diameter: 3, 5 or 10um

� Internal diameter: 4 or 4.6um

� Column packing

� silica, alumina, a polystyrene-divinyl-benzene

synthetic or an ion-exchange resin

Cont..

� Silica � Particle shape, surface properties, and pore structure

help to get a good separation

� Inert to most compounds

� High surface activity

� Used to separate a wide variety of chemical compounds� Used to separate a wide variety of chemical compounds

� Guard column� Anterior to the separating

� Prevent column contamination

� Filter or remove:� Particulate matter

� Compounds and ions

� Protect analytical column

Cont..Cont..Cont..Cont..

� Flow rate

� Depends on column internal diameter

� Also depends on type of analysis

Internal diameter Standard Flow RateInternal diameter (mm)

Standard Flow Rate(µl/min)

4.6 1000

2.1 200

1.0 50

0.30 4

0.15 1

INSTRUMENT - AUTOSAMPLER

� Sample injection:

� With syringe and septum injector

� With a loop valve

� With an automated injection system (autosampler)

Autosampler

INSTRUMENT - DETECTOR

� Most commonly use detectors:� UV Detector

� Refractive Index Detector

� Fluorescence Detector

� Others detectors� Others detectors

� Electrochemical Detector

� Light Scattering Detector

� Conductivity Detector

� Photoconductivity Detector

� Infrared Detector

� Radioactivity Detector

� Mass Spectrometer

UV detector

� General detector

� Useful for wide range of analytes

� Record compounds that adsorb ultraviolet or visible light (most organic solvents)

� Unaffected by temperature fluctuations and � Unaffected by temperature fluctuations and suitable for gradient elution

� Moderate sensitivity

� Deuterium lamps (340 - 600nm) and Tungsten lamp (340 – 850nm)

Fluorescence detector

� Sensitive and selective

� Very sensitive to a few analytes which do fluoresce

and fluorescing derivatives

� Lambda 280-305 nm and emission at lambda 340-

500 nm500 nm

Refractive Index

• Mobile phase need to stay same

• Sensitive to changes in pressure and temperature

• Useless in gradient elution

• Not useful for trace analysis

• Poor sensitivity• Poor sensitivity

• Problem with baseline stability

COMPARISON OF HPLC

DETECTORS

MODES OF SEPARATION

Adsorption chromatographyAdsorption chromatographyAdsorption chromatographyAdsorption chromatography

� Normal Phase Chromatography

� Mobile phase – nonpolar solvents (n-hexane or tetrahydrofuran)

� Stationary phase – polar (silica gel-OH, NH )

Cover almost 90% of all

chromatographic applications

� Stationary phase – polar (silica gel-OH, NH2)

� Retention mechanism – dipole-dipole

� Reverse Phase Chromatography

� Mobile phase – polar solvents (Water, Methanol, Acetonitrile)

� Stationary phase – nonpolar (modified silica – C8, C18, Phenyl)

� Retention mechanism – hydrophobic

�

�

� Ion exchange chromatography�Mobile phase – aqueous buffer

� pH and ionic strength use to control elution time

�Stationary phase – ionically charge surface� opposite charge to sample ions

�Retention mechanism – ionic interaction

� Size exclusion chromatography�Stationary phase – material control pore size �Sample screened or filtered according to size� Larger molecule rapidly washed� Smaller molecules penetrate inside porous

packing material

PREPARATION OF EQUIPMENT TO

SAMPLE

SELECTION OF MOBILE PHASE

PREPARATION OF INTERPRETATION

OF RESULT SAMPLE INJECTION

PREPARATION OF MOBILE PHASE

SAMPLE SOLUTION AND SAMPLE VOLUME

OF RESULT

SELECTION OF MOBILE PHASE

� Factors to consider : must interact with a suitable

stationary phase – separate mixture as fast and

efficiently.

� Purity – HPLC grade

� UV transparency – detector compatibility� UV transparency – detector compatibility

�Refractive index

� Solubility

� Viscosity

�Chemical inertness with sample compounds

�Corrosive resistance

�Toxicity

� Price

Increasing Increasing polarity

PREPARATION OF MOBILE PHASE

� Solvent and reagent - HPLC grade

� Mixed mobile phase or buffer

� Volume contraction effects – mixing water-miscible

solvents

� pH adjustment, addition of non-ionic additives� pH adjustment, addition of non-ionic additives

� Filter immediately before use

� Pore size 0.45um to 0.8um

� Types of filter:

� Nylon – hydrophilic (high content of

water/aqueous)

� PTFE – hydrophobic, chemically resistant

� Regenerated cellulose – hydrophilic, (aqueous

solution, sample filtration)

Cont..

� Degassed

� Polar solvents (dissolve high amount of air)

� Sonicate for 5 to 10 minutes

� Fresh prepared every analysis

� Good practice to prepare only as much will be � Good practice to prepare only as much will be

within short time

SAMPLE PREPARATION AND

SAMPLE VOLUME

SAMPLE PREPARATIONSAMPLE PREPARATIONSAMPLE PREPARATIONSAMPLE PREPARATION SAMPLE VOLUMESAMPLE VOLUMESAMPLE VOLUMESAMPLE VOLUME

� To extract target

compound

� Dilute in mobile phase

� Volume of solvent

required for dissolution

may vary

� Filter to ensure that it

contain no solid

� Types of filtration

� Filtration by membrane

� Solid phase extraction with disposable cartridges

� Protein precipitation

� Desalting

� Avoid band broadening

Signals – peaksSignals – peaks

INTERPRETATION OF RESULT

Whole entity - chromatogramWhole entity - chromatogram

f

W0.05

RETENTION TIME

� Time for analyte to reach detector� Retention time (tR) – the period between sample injection and recording of peak maximum

� Ideal tracer� Dead time (tM) / breakthrough time– retention time of unretained solute (time that require by the mobile phase to pass unretained solute (time that require by the mobile phase to pass through the column)

tR

Height and area under curve –

amount of sample

ADVANTAGES AND DISADVANTAGESADVANTAGES AND DISADVANTAGESADVANTAGES AND DISADVANTAGESADVANTAGES AND DISADVANTAGES

ADVANTAGESADVANTAGESADVANTAGESADVANTAGES DISADVANTAGESDISADVANTAGESDISADVANTAGESDISADVANTAGES

� High speed

� High resolution

� High sensitivity

� Cost

� Complexity

� Low sensitivity for some � High sensitivity

� Reproducibility of ±1%

� Accuracy

� Automation

� Low sensitivity for some

compounds

THANK YOUTHANK YOU