Gihan Gawish.Dr

High Performance Liquid Chromatography

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

separate, identify, and quantify compounds.

Retention time varies depending on the interactions between the stationary

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

Dr Gihan Gawish

LC vs. HPLC

Liquid chromatography

High-performance liquid

chromatography (HPLC) Use large, non-rigid support

material Particles size dp: > 150 μm,

column size dc: 10 ~ 50 mm,

column length L: 50 ~ 500 cm, flow rate F: <

1mL/min Gravity. Large H, small N Poor system efficiencies

and large plate heights

Use small, uniform, rigid support material

Particle size dp < 40 μm, usually 3-10 μm in practice

Good system efficiencies and small plate heights, narrow peaks, shorter separation times

HPLC components:

Liquid Mobile Phase

Pump

Injection Valve

Separation Column

Detector

Dr Gihan Gawish

HPLC Instrumentation (Schematic diagram)

Dr Gihan Gawish

HPLC Instrumentations

High pressure:

•Several hundred atm

• Packing: 3 ~ 10 μm

•Elaborate and expensive

1. Solvent treatment system

2. Pumping system

3. Sample injection system

4. Column

5. detector

Dr Gihan Gawish

Instruments – Solvent SystemMobile phase reservoirs:

– Several reservoirs (> 500mL)

– Degassing: remove of dissolved gas band spreading and interfering detection

• Sparging: fine bubble of gas

• vacuum pumping, distillation, heating

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

•Millipore filter under vacuum

– Isocratic elution: constant composition

– Gradient elution: different solvent systems during elution, continuous change or step wise, solvent proportion valve

Mobile phase

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

Dr Gihan Gawish

HPLC pumps

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

Dr Gihan Gawish

Instruments – Sample Injection SystemSample Injection system:

– Limit of precision of HPLC

– Sample size: 0.5 ~ 500 μL

– No interference with the pressure

– Based on a sample loop, 1 ~ 100 μL,

Reproducibility: 0.1%, P < 7000 psi

– Auto sampler: inject continuously

variable volume 1 μL – 1 mL•Controlled temperature environment for derivatization reaction

Analytical Columns

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

sampleDr Gihan Gawish

Instruments – Detectors 1

Absorption detectors:– UV-Vis: Most widely used

•Z-shape, flow-through cell (V, 1 ~ 10 μL and b, 2 ~ 10 mm)

•Photometer: Hg 254 nm and 280 nm line

•for organic, D2 or W filament + interference filter

•Spectrophotometer: more versatile

– IR: filter instrument or FTIR

•Similar cell (V, 1.5 ~ 10 μL and b, 0.2 ~ 1.0 mm)

•Limit: no suitable solvent, special optics

– Fluorescence: Hg or Xe lamp

•Fluorometer and spectrofluorometer

• Fluorescing species or fluorescent derivatives

A UV-Vis detector for HPLC

Instruments – Detectors 2Electrochemical detectors:– Amperometry, voltammetry, coulormetry and conductormetry

– A: simplicity, high sensitivity, convenience and wide-spreading application

– Thin-layer flow cell of Teflon : 50 μm thick, 1 ~ 5 μL volume

– Indictor E: Pt, Au, C

– RE and CE: down stream

– Multi-electrode: simultaneous detection or sample purity indication

Amperometric thin-layer cell for HPLC

Analyze

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.

The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered a reasonably unique identifying characteristic of a given analyte.

Dr Gihan Gawish

Analytes

organic molecules Biomolecules Ions polymers

Dr Gihan Gawish

Advantage of HPLC

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.

Dr Gihan Gawish

Types of HPLC1. Normal phase chromatography

(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.

Dr Gihan Gawish

2 .Reverse Phase chromatography

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

Dr Gihan Gawish

Practical considerations: 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.

Dr Gihan Gawish