HPLC

HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY [HPLC]
HPLC
 HPLC is a High Performance liquid Chromatography.
 High Pressure Liquid Chromatography.
 High Priced Liquid Chromatography.
 It is column chromatography.
 It is Liquid Chromatography.
 It is modified from of gas chromatography, it is applicable for both Volatile as well as
Non volatile compound.
 It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
 It is having a high resolution and separation capacity.
 It is used as qualitative as well as quantitative analysis.
 High performance liquid chromatography (HPLC) is a chromatographic technique used
to separate a mixture of compounds in analytical chemistry and biochemistry with the
purpose of identifying, quantifying or purifying the individual components of the
mixture.
7
PRINCIPLE
 High Performance Liquid Chromatography [HPLC] is principle is based on
adsorption as well as partition chromatography is depending on the nature
of stationary phase, if stationary phase is solid principle is based on adsorption
chromatography and if stationary phase is liquid principle is based on partition
chromatography.
 It is important for determination of volatile and non volatile compounds.
 It is important for determination qualitative and quantitative analysis.

 It is important for determination of Retention Time (the time is required , after
sample injection maximum angle peak reaches to detector)
Common mobile phases used include any miscible combination of water with various
organic solvents (the most common are acetonitrile and methanol)
A separation in which the mobile phase composition remains constant throughout the
procedure is termed isocratic (meaning constant composition).
A separation in which the mobile phase composition is changed during the separation
process is described as a gradient elution
ADVANTAGES
 It is simple, rapid , reproducible.
 High sensitivity.
 High performance.
 Rapid process and hence time saving.
 It is having a high resolution and separation capacity.
 Accuracy and Precision.
 Stationary phase was chemically innert.
 Wide varities of stationary phase.
 Mobile phase was chemically innert.
 Less requirement of mobile phase in developing chamber.
 Early recovery of separated component.
 Easy visualization of separated components.
 It is having Good reproducibility and repeatability.
 It is analytical technique is important for validation of product, quality control
studies of product.
 It is important for qualitative and quantitative analysis.
 It is used for both analytical and preparative purpose.
TYPES OF HPLC SEPARATIONS
 Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded
stationary phase.
 Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar,

bonded stationary phase.
WHY USE HPLC

 Simultaneous analysis
 High resolution
 High sensitivity
 Good repeatability
 Moderate analysis condition
 Easy to fractionate and purify
 Not destructive
INSTRUMENTATION OF HPLC
 Solvent storage bottle

 Gradient controller and mixing unit
 De-gassing of solvents
 Pump
 Pressure gauge
 Pre-column
 Sample introduction system
 Column
 Detector
 Recorder
SELECTION OF DETECTORS
Detectors Type of compounds can be detected
UV-Vis & Compounds with chromophores, such as aromatic rings or
Photo diode array multiple alternating double bonds.
detector
Radio freqency Fluorescent compounds, usually with fused rings or highly
detector RF conjugated planar system.
Conductivity Charged compounds, such as inorganic ions and organic

detector CDD acid.
Electron capture
detector ECD For easily oxidized compounds like quinones or amines.
Refractive index (detects the change in turbidity)

Mass spectrometry (HPLC-MS)
COMPARISION BETWEEN GC AND HPLC
SR.NO. GC HPLC
1 Gas Chromatography High Performance Liquid

Chromatography
2 Less resolution High resolution
3 Limited Flexibility Extreme Flexibility
4 Determination of Volatile Determination of Volatile and Non
compounds Volatile Compounds
APPLICATION
 Drug Discovery
 Clinical Analysis
 Proteomics
 Forensic Chemistry
 Drug Metabolism study
 Environmental chemistry
 Diagnostic studies
 Cosmetic analysis
 Structural Determination
 Pharmaceutical Applications
 Clinical Applications
 Biochemical Genetics
 qualitative and quantitative analysis
 Therapeutic Drug Monitoring

HPLC

Uploaded by

Copyright:

Available Formats

HPLC

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

HPLC

Uploaded by

Copyright:

Available Formats

HIGH PERFORMANCE LIQUID

 It is important for determination qualitative and quantitative analysis.

 Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar,

WHY USE HPLC

 Moderate analysis condition

 Easy to fractionate and purify

 Solvent storage bottle

 Sample introduction system

Conductivity Charged compounds, such as inorganic ions and organic

Refractive index (detects the change in turbidity)

1 Gas Chromatography High Performance Liquid

You might also like