Chromatography

Chapter 4-5

1Dr Gihan Gawish

Dr Gihan Gawish2

Dr Gihan Gawish

(HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry to:

separate, identify, and quantify compounds.

3

HPLC

HPLC Instrumentation (Schematic diagram)

Dr Gihan Gawish

4

Liquid Mobile Phase

Pump

Injection Valve

Separation Column

Detector

Dr Gihan Gawish

Solvent Reservoirs can be single solvent or multi-solvents

The choice of solvents, additives and gradient depend on: the nature of the stationary phase and the analyte

5

HPLC- Mobil phase

Dr Gihan Gawish

Requirements for HPLC• apply high pressure to force liquid through

the beads faster• pressures to 6000 psi• control flow rate from 0.1 to 10 mL/min

Types of HPLC pumps Reciprocating pumps: most commercial

systems arebased on this design. Syringe pumps

6

HPLC- Pumps

Dr Gihan Gawish

Generally stainless steel and teflon components.

The stationary phase packings are microporous silica 2-10 μm in diameter.

Unmodified silica is very polar.

Some systems use Precolumns to remove impurities from solvent or

sample

8

HPLC- Analytical Columns

UV Single wavelength (filter) Variable wavelength (monochromator) Multiple wavelengths (PDA)

Fluorescence Electrochemical Mass Spectrometric

HPLC- Detectors

Dr Gihan Gawish

The sample to be analyzed is introduced in small volume to the stream of

mobile phase.

The analyte's motion through the column is slowed by specific chemical

or physical interactions with the stationary phase as it traverses the length

of the column.

The amount of retardation depends on the nature of the analyte,

stationary phase and mobile phase composition.

12

HPLC- AnalysisAnimation

Retention time varies depending on the interactions between the stationary

phase, the molecules being analyzed, and the solvent (s) used.

Retention time

Dr Gihan Gawish

13

Time at which a specific analyte elutes (comes out of the end of the

column) is called the retention time

Retention time under particular conditions is considered a

reasonably unique identifying characteristic of a given analyte.

Dr Gihan Gawish

organic molecules Biomolecules Ions polymers

14

HPLC- Analyte

Dr Gihan Gawish

HPLC results in high resolution (sharp peaks), and rapid separation (minutes to 1 hour).

HPLC can be analytical or preparative.

HPLC can be used for all types of chromatography: size exclusion, ion exchange, reversed phase, and affinity chromatography.

15

HPLC- Advantage

Types of HPLC1. Normal phase chromatography

Dr Gihan Gawish

(NP-HPLC), this method separates analytes based on polarity

NP-HPLC uses a polar stationary phase and a non-polar mobile phase

Adsorption strengths increase with increased analyte polarity, and the interaction between the polar analyte and the polar stationary phase (relative to the mobile phase) increases the elution time.

16

Types of HPLC 2. Reverse Phase Chromatography

Dr Gihan Gawish

Reversed phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase.

One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group

With these stationary phases, retention time is longer for molecules which are more non-polar

17

Practical considerations

Dr Gihan Gawish

Not all proteins can withstand the pressure of HPLC

All materials must be of the highest quality.

Solvents must be degassed to eliminate formation of

bubbles.

18