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