High performance liquid chromatography (HPLC) is suited for separating and identifying molecules like amino acids, carbohydrates, lipids, nucleic acids, proteins, pigments, and steroids. It operates at ambient temperature, which allows analysis of non-volatile and thermally unstable samples. HPLC uses stationary supports with small particle sizes and large surface areas, along with high pressure on solvent flow, which provides high resolution, speed of analysis, reproducibility, and ability to automate instrumentation and data analysis. Common stationary phases include silica gel or alumina particles that are spherical and 3-10 micrometers in diameter with pore sizes of 60-300 angstroms. Liquid stationary phases are also used where the phase is chemically bonded

1. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Written by:
Naimatullah
BS (Hons) Chemistry
Univeristy of education
Lahore

2. HIGH PERFORMANCE LIQUID
CHAROMATOGRAPHY
Introduction:
• it is suited for separation and identification of
amino acids ,carbohydrates ,lipids ,nucleic acid
,protein , pigments , steroids and pharmaceuticals.
• Gas chromatography can not be used in case of non
volatile and thermally unstable sample but it is
operated at ambient temperature and does not
have limitation.

3. conti
• Due to versatility of HPLC , all chromatographic
modes are possible.
• It is known as automated liquid chromatography.
• It has following two major advances;
1. The development of stationary supports with very
small particle size and large surface area.
2. The improvement of elution rates by applying high
pressure to the solvent flow.

4. ADVANTAGES
• Resolution and speed of analysis far exceed the
classical method.
• HPLC column can be reused without repacking or
regeneration.
• Reproducibility is greatly improve because the
parameters affecting the efficiency of the
separation can be closely controlled.
• Instrumentation and data analysis are easily
automated.
• It is adaptable to large scale ,preparative procedure.

5. STATIONARY PHASE
• Original HPLC micro particles were irregular shaped.
• It may be silica gel or alumina particles.
• Modification result in spherical shape particles for
pack column.
• High purity particles and 5-10 um in diameter.
• 3 um used for high speed chromatography.t
• Pores size range is about 60-100 augsterin.
• 300 of Pore size is used for biomolecules.
• Mostly used in partition but adsorption
chromatography is useful in some cases.

6. conti
In case of liquid chromatography;
• Solid support coated with liquid stationary phase.
• Liquid stationary phase chemically bonded with solid
supports.
• Most commonly used particles are microporous
particles.
• Majority of surface area is within the surface.
• Mobile phase moves around the particles and solute
diffuse.
• Stagnant mobile phase within the pores to interact with
the stationary phase and then diffuse out into the
moving mobile phase .

7. conti
• Use of small particles minimize the path length of
the diffusion and hence band boarding.
• Silica tend to dissolve at PH 8.
• Crosslinked polymer particles are used for the
separation of bases.
• Silica particles have silinol group used for chemical
bonding of stationary phase by silination reaction.
• About half of the silinol group are chemically
bonded.
• Remaining at end capped with trimethyl silyne to
render them inert.

8. conti
• The most common non polar phases are C8 and
C18.
• C8 is intermediate non polar.
• C18 is highly non polar.