High Performance Liquid Chromatography

Instrumentation

solventsolvent

pumppump

injectorinjector

columncolumn

detectordetector

HPLC Instrument

Schematic of HPLC Instrument

Mobile phase Quality

High purity

Reasonable cost (and disposal)

Boiling point 20-50 °C above column temperature

Low viscosity

Low reactivity

Immiscible with stationary phase

Compatible with detector

Safety – limited flammability and toxicity

Mobile phase selection

k’ of 2-5 for two or three component mixturek’ of 0.5-20 for multicomponent mixtureMatch analyte polarity to stationary phase polarityMobile phase of different polarity

Normal Phase:

nonpolar solvent, polar stationary phase least polar component elutes first increasing mobile phase polarity decreases elution time

Reversed Phase:

polar solvent (water, MeOH, ACN), nonpolar stationary phase most polar component elutes first increasing mobile phase polarity increases elution time most widely used

Solvent Treatment Systems

For Dissolved gases (Irreproducible flow rates and band broadening)

Degassers

i. Vacuum system and distillating system.ii. Sparging system

(inert gas insoluble in mobile phase)

For Dust and particulate matter(Damage the pumping or injection system and clog the column)

Filtering the mobile phase through millipore filters.

Solvent Reservoir and Elution

Elution :

Isocratic : Elution with a single or mixed solvents of constant composition.

Gradient Elution: Elution of mixed solvents, with different polarities with composition varied with time.

HPLC instruments equipped with proportioning valves to introduce solvents from different reservoirs.

The ratio is preprogrammed before elution.

Pumping Systems

Why pressure?

The typical particle sizes in HPLC is 3-10 μm. In order to achieve flow rates of 0.5 to 5 mL/min, for a 10-30 cm column, pressures of 1000 to 6000 psi are required.

Requirements for HPLC pumping system

pressures to 6000 psipulse free outputcontrol flow rate from 0.1 to 10 mL/minresistance to corrosion by solvents

Different types of pumping Systems

Displacement syringe pump Pulse free Small capacity (250ml)

Only for isocratic elution.

Reciprocating pumps Small internal volume High output pressure Adaptable for gradient elution Large capacity Constant flow rate

Pump used in most commercial design.

Reciprocating pumps

www.lcresources.com/resources/getstart/2b01.htm

Disadvantage: Causes pressure pulses which leads to

i. poor quatitative analysis

ii. detection problems.

Solution

i. Dual pump system

ii. Pulse dampers ( act as shock absorbers)

Injection ( Sampling Valves)

Six-port sample injection system is used. It takes

i. Small amount of sample (≤ 500µl of sample)

ii. In a pressurized system

Injection ( Sampling Valves)

http://www.restek.com/info_sixport.asp

Guard Column ( Pre-column)

Prevents the contamination of the expensive analytical columns with fine particles that can eventually clog the mobile phase flow.

Porous stainless steel column(0.5 -2µm)

Composition same as analytical column.

Particle size is large to minimize pressure drops.

When contaminated, discarded and replaced by new one.

Analytical Column

Generally made of stainless steel or teflon components

10-30 cm long x 4-10 mm internal diameter

Packing usually 5 or 10 µm diameter

Analytical Column Packaging

Pellicular

Spherical, nonporous, glass or polymer beads 30-40- µm diameter Thin porous layer of silica, alumina, or ion-exchange resin deposited on surface

Porous

Most common 3-10- µm diameter Silica (most common), alumina, or ion-exchange resin Thin organic film bonded to surface

solventsolvent((mobile phasemobile phase))

andandsamplesample

wax coated beadswax coated beads

HPLC ColumnHPLC Column

to detectorto detector

Normal vs Reverse phase

nonpolarnonpolarstationary phasestationary phase

3 mmicroscopic view of beadmicroscopic view of bead

Reverse phase(C4, C8 or C18): Non-polar stationary phase

Normal phase: Polar stationary phase

Detectors

Ideal Characteristic of a detector

Adequate sensitivity

Good stability and reproducibility

Linear response to solutes

Short response time

Response to all solutes in a mixture

Non-destructive

Temperature stability

Types of Detectors

Bulk property Detectors (Mobile

phase property)Refractive index

Density

Dielectric constant

Electrochemical

Solute property DetectorsUV-Visible

Fluorescence

Diode array

Mass spectrometry

Diode array 3D plot