Basic Principles of HPLC

Column ChromatographyTswett experiment

The flow through the column is by gravity

Column Chromatography

Thin Layer Chromatography

Flow is created by capillary action as the mobile phase diffuses into the dry layer & moves up to the glass plate.

• 1970 : HPLC acronym was coined by late Prof. Csaba Horvath during 1970 Pittcon

High Pressure Liquid Chromatography

indicating high pressure was use to generate flow

HPLC

What is Liquid Chromatography?

• It is a separation technique.• It makes use of stationary phase and a mobile

phase.

The components of the sample mixture are carried through the stationary phase by the flow of the mobile phase.

• In LC, the mobile phase is the liquid.• Separation is based on the differences in migration

rates among the sample components.

Methods of Separation

• adsorption

• Partitioning

• Solubility

• Vapor pressure

• Molecular size

• Ionic charge density

Partitioning

• Separation is based on the analyte’s relative solubility between two liquid phases

Stationary PhaseMobile Phase

Solvent Bonded Phase

Main Parts of HPLC

• Solvent Reservoir

• Solvent Pump

• Injector Port ( Autosampler)

• Column

• Detector

• Data Processor ( Software)

HPLC Solvent Reservoir

Sinker frits help keep the inlet line submerged and provide a final line of defense against particulate contamination.

Sinker frits

Degassing Methods

• Sonication – recommended for premixed mobile phase, often in conjunction with vacuum degassing.

• Vacuum – the mobile phase immediately begins to re-equilibrate with air.

• Helium Sparge ( helium is insoluble in most mobile phase.

Vacuum Filtration

Helium Sparge

Typical HPLC : Waters Alliance

How does HPLC works

HPLC System

HPLC Pump

Single –piston reciprocating pump

HPLC Pump

• Delivers/force the mobile phase to through the column under pressure

• Maintain a constant flow of mobile phase through the HPLC regardless of the pressure ( back pressure) caused by the flow resistance of the packed column.

HPLC Pump Head Assembly

Types of Pumping System

• High Pressure Mixing ( 2 pump gradient) The pump outputs are blended on the down stream or high pressure side of the flow path.

• Low Pressure Mixing ( one pump gradient) The mobile phase composition is

controlled by valve on the upstream or low pressure side of the pump.

High Pressure Mixing

2 pump system blends the output of 2 high pressure pumps to control the mobile phase composition.

Low Pressure Mixing

One pump system

Types of Chromatographic Separation (based on solvent delivery)

• Isocratic flow: The mobile phase composition remains constant throughout the procedure.

• Gradient Flow : The composition of the mobile phase is changed during the separation process.

Isocratic LC System

High Pressure Gradient LC System

Low Pressure Gradient LC System

• Dwell time ( gradient delay volume)

This is the volume of the liquid in the system between the point where the gradient is formed(usually in the mixing chamber)and the point where it enters the column.

Columns

How a chromatographic column works

Column Ends

Column Failures

• Peak shape deterioration ( peak broadening & peak tailing) – maybe due to blockage of the inlet frit by particulates or creation of void in the inlet.

• Gradual decrease in retention times because of loss in column plate number (due to accumulation of strongly retained compounds on column packing).

• Loss of bonded phase due to chemical attacked by mobile phase or by sample.

Column Ends

In-Line filter

The job of this is to catch particulate matter that could plug the column frit or column, causing poor separartion & high pressure.

Guard Column

A guard cartridge traps chemical contamination before it can go to the column. This can also serve as filter to the mobile phase & sample that enters the analytical column.

The use of precolumn increased the lifetime of a bonded-phase analytical column used at high pH.

Caring For Your HPLC Column• Use in-line filters & guard columns whenever

possible.• At the end of the day, wash out the column with

organic solvent & leave the column stored in it.• Filter the mobile phase.• Filter all samples before injecting.• Avoid large & sudden pressure fluctuations.• Keep track of peak shape & pressure problem &

fix the problem.• Seal the columns with plugs and store properly.

Column Efficiency

• Separation & band broadening• Theoretical Plates

Theoretical Plates, N

This is a measure of column efficiency, the higher the number of plates, N, efficiency increases.

How peaks are created

Resolution

Partitioning Process

• All chromatographic separations are based upon differences in the extent to which the solutes are partitioned between the mobile phase and stationary phase.

Xm Xs

Identifying Compounds by Retention Time

Identification (by Retention Time)

Quantification (by Peak Area Response)

Injector

HPLC Autosampler (Autoinjector)

How injector port works

DetectorsBased on Bulk property of eluting solution• Conductivity - senses all ions whether from solute or

mobile phase. for ion-exchange chromatography• Refractive Index

Based on specific property of the analyte• UV-VIS Detector follows Beer- Lamberts Law ( A= Ebc) Photodide Arrays take spectra of all wavelengths

simultaneously• Flourescence Detector monitors both excitation & emission wavelengths

• Electrochemical

Amperometric

Coulometric

• Mass Spectrometry

Quadrople

Fourier Transformed

Ion Trap

Time of Flight

• Electrochemical

Measures the current generated when solute is oxidized or reduced.

• Refractive Index

Sense the differences in refractive index between the eluent with solute and pure mobile phase.

(The presence of the solute in the mobile phase changes the refractive index of the eluting solution).

often use for analyte that does not contain chromophore.

PDA Detector

HPLC - Modes

• Normal Phase.- Polar stationary phase and non-polar solvent.

• Reverse Phase.- Non-polar stationary phase and a polar solvent.

Normal Phase Chromatography

Reversed Phase Chromatography

HPLC Separation Modes(based on Compound Characteristics)

• Polarity

• Electrical Charge

• Molecular Size

Compound/Analyte Chromatographic Polarity Spectrum

Characteristics of Separation based on Polarity

Mobile Phase Chromatographic Polarity Spectrum

Stationary Phase Particle Chromatographic Polarity Spectrum

Separation based on Charge(Ion Exchange Chromatography)

Separation based on Size(Size exclusion Chromatography)

•Separation is based on size of molecules rather than charge or polarity

•Filtered through controlled-porosity hydrophilic dextran polymer

( gel permeation chromatography)

Typical Chromatogram

Two factors contributing to good separation

• Difference in elution times: The larger the difference, the better the separation

• Width of the peaks : the broader the peaks, the poorer the separation

Resolutiondescribes the quality of the separation between 2 peaks

• By definition, a Rs > 1.5 is considered baseline resolution.

Tailing Factor

Peak Tailing

Any increase in peak tailing is a symptom of a problem that should be fixed.

Asymmetry Factor

In most cases, Asymmetry factor & Tailing factor will be roughly the same ( 1.0 & 1.5 for new column)

Calibration Curve

Retention Times

-time between the start of elution(sample introduction), and the emergence of peak maximum.

Applications of HPLC

• Pharmaceutical /Nutraceutical Industry

• Cosmetic Industry

• Petroleum Industry

• Food & Beverage Industry

• Environmental Science

• Medical, Clinical & Science Research

• Toy Manufacturing Companies