High Performance Liquid

Chromatography

Instrumentation
HPLC Instrument

solvent

pump

injector

column

detector
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 mixture
k’ of 0.5-20 for multicomponent mixture
Match analyte polarity to stationary phase polarity
Mobile 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 psi

pulse free output

control flow rate from 0.1 to 10 mL/min

resistance 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

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)

www.lcresources.com/resources/getstart/2b01.htm
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
HPLC Column
solvent
(mobile phase) to detector
and
sample
wax coated beads
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
Normal vs Reverse phase
Normal phase: Polar stationary phase

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

nonpolar
stationary phase

microscopic view of bead

3 m
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

Diode array 3D plot

Solute property Detectors
UV-Visible

Fluorescence

Diode array
Mass spectrometry