HIGH PERFORMANCE

LIQUID
CHROMATOGRAPHY

Presented by:
K.SAI TEJA
13075A1023

CONTENT :
Introduction.
HPLC System
types of HPLC
Uses of HPLC
Affects of HPLC
Advantages
Applications
conclusion
References

Introduction
HPLC is a form of liquid chromatography used to separate

compounds that are dissolved in solution. HPLC instruments

consist of a reservoir of mobile phase, a pump, an injector, a
separation column, and a detector.

Compounds are separated by injecting a sample mixture onto

the column. The different component in the mixture pass

through the column at differentiates due to differences in their
partition behavior between the mobile phase and the stationary
phase. The mobile phase must be degassed to eliminate the
formation of air bubbles.

HPLC system

FOUR TYPES OF LIQUID

CHROMATOGRAPHY

Partition chromatography

Adsorption, or liquid-solid

chromatography

Ion exchange chromatography

Size exclusion, or gel, chromatography

COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM

Solvent
Solvent Delivery System (Pump)
Injector
Sample
Column
Detectors (Diode Array)
Waste Collector
Recorder (Data Collection)

Picture of HPLC instrument

Uses of HPLC
This technique is used for chemistry and biochemistry

research analyzing complex mixtures, purifying chemical

compounds, developing processes for synthesizing
chemical compounds, isolating natural products, or
predicting physical properties. It is also used in quality
control to ensure the purity of raw materials, to control and
improve process yields, to quantify assays of final
products, or to evaluate product stability and monitor
degradation.

In addition, it is used for analyzing air and water

pollutants, for monitoring materials that may jeopardize

occupational safety or health, and for monitoring pesticide
levels in the environment. Federal and state regulatory
agencies use HPLC to survey food and drug products, for
identifying confiscated narcotics or to check for adherence
to label claims.

Picture of an HPLC column

WHAT AFFECTS SYSTEM

Column Parameters

Instrument Parameters

Column Material
Deactivation
Stationary Phase

Coating Material

Temperature
Flow
Signal
Sample Sensitivity
Detector

WHAT AFFECTS SYSTEM:

Sample Parameters

Concentration
Matrix
Solvent Effect
Sample Effect

Several column types

(can be classified as )
Normal phase
Reverse phase
Size exclusion
Ion exchange

Types of Liquid Column Chromatography

(LCC)

LLC (Liquid Liquid)

LSC (Liquid Solid adsorption)

SEC (Size Exclusion)

GLC

GSC

SFC (Supercritical
Fluid)

Types of Detectors:

Absorbance (UV with

Filters, UV with
Monochromators)

Evaporative Light
Scattering Detector
(ELSD)

IR Absorbance

Electrochemical

Fluorescence

Mass-Spectrometric

Refractive-Index

Photo-Diode Array

EVALUATION PARAMETERS:

EFFICIENCY
RESOLUTION
INERTNESS
RETENTION INDEX
COLUMN BLEED
CAPACITY FACTOR

Advantages:
1.

Separations fast and efficient (high resolution power)

2.

Continuous monitoring of the column effluent

3.

It can be applied to the separation and analysis of very complex

mixtures

4.

Accurate quantitative measurements.

5.

Repetitive and reproducible analysis using the same column.

6.

Adsorption, partition, ion exchange and exclusion column

separations are excellently made.

7. HPLC is more versatile than GLC in some respects, because it

has the advantage of not being restricted to volatile and thermally
stable solute and the choice of mobile and stationary phases is
much wider in HPLC

 Both aqueous and non aqueous samples can be analyzed with

little or no sample pre treatment

9. A variety of solvents and column packing are available,

providing a high degree of selectivity for specific analyses.

10. It provides a means for determination of multiple

components in a single analysis.

Applications:
HPLC is one of the most widely applied analytical
separation techniques.
Pharmaceutical:
Tablet dissolution of pharmaceutical dosages.
Shelf life determinations of pharmaceutical products.
Identification of counterfeit drug products.
Pharmaceutical quality control.

Conclusion:
The selectivity needed for the determination of targeted
analytes in a complex matrix can be achieved anywhere in the
analyticalcycle. With a focus on the sample preparation
portion, the concept of Just Enough sample preparation was
presented. This concept relies heavily on the increased
sensitivity and selectivity that can be achieved with tandem
mass spectrometry coupled with Chromatographic separation.
Provided that ion suppression/enhancement contributions are
held to a minimum, Just Enough sample preparation can
provide the recoveries, minimum detectable limits (MDLs),
and minimum detectable quantities (MDQs) consistent
with the needs of the assay.

References:
D. P. Glavin, J. P. Dworkin, A. Aubrey, O. Botta, J. H. Doty, Z.

Martins, and J. L. Bada, "Amino acid analyses of Antarctic

CM2 meteorites using liquid chromatography-time of flightmass spectrometry," Meteorit. Planet. Sci., vol. 41, no. 6, pp.
889-902, 2014.
S. A. Getty, J. P. Dworkin, D. P. Glavin, M. Martin, Y. Zheng,
M. Balvin, A. E. Southard, S. Feng, J. Ferrance, C. Kotecki, C.
Malespin, and P. R. Mahaffy, "Organics Analyzer for Sampling
Icy Surfaces: A liquid chromatograph-mass spectrometer for
future in situ small body missions," in 2013 IEEE Aerospace
Conference, 2013
C. R. Blakley and M. L. Vestal, "Thermospray interface for
liquid chromatography/mass spectrometry," Anal. Chem., vol.
55, no. 4, pp. 750-754, Apr. 2013.

References (cont.)
Bochra Bejaoui Kefi, Latifa Lustrous EI Atrache, Hafedh

Kochkar, et al. TiO2 nanotubes as solid-phase extraction

adsorbent for the determination of polycyclic aromatic
hydrocarbons in environmental water samples. Journal of
Environmental Sciences. 2011.
Dugo P, Cacciola F, Kumm T, et al. Comprehensive
multidimensional liquid chromatography: Theory and
applications.
Journal
of
Chromatography
2010.

THANK YOU