A Brief Introduction

1. HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
BINOD BASHYAL
1ST YEAR
MSc. BIOCHEMISTRY
bbashyal75@mail.com

2. CONTENTS
1. INTRODUCTION
2. PRINCIPLE
3. HISTORY
4. TYPES
5. INSTRUMENTATION
6. PROCEDURE
7. ADVANTAGES
8. DISADVANTAGES
9. APPLICATIONS
10. RECENT EXPERIMENTS
11. REFERENCES

3. INTRODUCTION
 Most advanced form of chromatography, in terms of performance and speed.
 Different from others in the sense that, rather than gravity, a high pressure of
about 300-400 atm. is applied to the mobile phase which makes the separation,
faster and efficient.
Mobile Phase:Water based solvent, organic solvent or the mixture of both
Stationary phase: Silica, cellulose, organic resin
(Silica is mostly preferred)

4. WHY IS SILICA PREFERREDAS STATIONARY PHASE PARTICLE?
 Chromatography performs better with smaller size particles as:
- it will have greater area, to get exposed to sample molecule
- inter-particle space decreases
 1ST Problem:
When small sized particles was used,
- back pressure of column increases, as space between particle decreases
 Hence, a high pressure is to be applied for effective performance and separation.
 2nd Problem:
At high pressure, solid phase particles of cellulose and organic resin would
collapse.
 To withstand such high pressure with small size, silica was preferred, which could easily
accommodate high pressure of 300-400atm beside having size of about 5µm-10µm.

5. HISTORY
 In 1960, Cal-Gidding, Josef Huber and other colleagues improvised the technique of liquid
chromatography by the addition of external pressure, in order to increase the mobile phase
velocity.
 This newly introduced technique underwent several experimentation in 60s and 70s, not
only in terms of procedure but also in terms of instrumentations(such as pumps and injectors)

6. PRINCIPLE
 The basic principle of HPLC, actually involves the types of HPLC we are going to perform.
 In most of the process, adsorption, polarity and size are the basis of separation.
 Separation of molecules, also depends upon:
- difference of solubility in water
- difference of solubility in organic solvent
- net positive or negative charge of the components
- size of the components

7. TYPES
1. Ion-Exchange Chromatography
2. Size ExclusionChromatography
3. Normal Phase Chromatography
4. Reverse Phase Chromatography
5. Bio Affinity Chromatography

8. INSTRUMENTATION
It has classified as:
a. Delivery of solvent and sample to column
i. Solvent Reservoir
ii. Pump
iii. Injector
iv. Column
b. Detection of molecules emerging from column
i. Detector
ii. Chromatogram
Solvent Reservoirs
Quaternary Pump
Autosampler
Column Compartment
DAD

10. SOLVENT RESERVOIR
 Hold HPLC solvent i.e. mobile phase, which is further connected to pump
Requirements:
- materials must not contaminate mobile phase
e.g.Teflon, glass or stainless steel
- mouth must be covered.
- mouth must not be too tight, as vacuum will form and prevent the flow
of mobile phase

11. PUMP
 mobile phase solvent is pressurized by pump, which passes to column .
 here, high pressure of about 300-400 atm. is applied.
Requirements:
- generate sufficient and enough pressure without fluctuations
Types:
On the basis of requirements of the types of HPLC:
1. Screw driven syringe pump
2. Direct gas-pressure system pump
3. Pneumatic pump
4. Constant flow pump

12. INJECTOR
 port from where the sample mixture is injected, lies prior to column
 mix with solvent and enters the column, where separation takes place
 can be manual or automated
Requirements:
- Injection sample should not contain any bubbles
- Injector should be able to inject small volume of sample per unit time at the
rate of µl

13. COLUMN
 most important part of HPLC, as the major task takes place here i.e. the separations of the components
from a given mixture
 usually made up of thick glass or steel
 Strong enough to withstand the high pressure of the solvent mixture
Types:
On the basis of method of separation and the composition of the mixture taken:
1. Normal phase column
2. Reverse phase column
3. Ion-exchange column
4. Size exclusion column

14. HPLC ELUTION
 Based on the composition of mobile phase throughout HPLC experiments, HPLC elution can be:
a) Isocratic elution: - mobile phase composition is constant throughout the HPLC separation
- used for simple mixtures
b) Gradient elution: - mobile phase composition gradually changes from polar to non polar
- separation of complex mixtures
(analysis of complex herbal extracts where analytes are having mix polarities)

15. DETECTOR
 located at the end of the column
 detects the components of sample mixture eluted at different time interval, from the column
Types:
On the basis of, components of the mixture, different detectors can be used as:
1. UV,Vis detector
2. Refractive index detector
3. Evaporative light scattering detector
4. Multi-angle light scattering detector
5. Mass spectrometer
6. Conductivity detector
7. Fluorescence detector
8. Chemiluminescence detector
9. Optical rotation detector
10. Electrochemical detector

16. DATA COLLECTOR(CHROMATOGRAM)
 signal from detector is received by recorders
 used to progress, store and reproduce chromatographic data
 data is interpreted by the computer and represent in the forms of graph, known as chromatograph

17. PROCEDURE
 Mobile phase solvent taken from reservoir is pumped with a high pressure towards the column.
 Sample is mixed with solvent with the help of injector, before reaching the column.
 Column contains small sized particles(stationary phase) and the sample components are separated on the
interactions between the mobile phase and the stationary phase
 Separated different eluates are eluted out of the column at different time intervals
 Detectors detect the components, based with their polarity, size and comparing with the retention time.
 Data from detector is processed and chromatograph is produced by the chromatogram.

18. ADVANTAGES AND DISADVANTEGES
Advantages:
 Identify and simultaneously quantify large number of components of mixture such as, amino acids, steroids,
drug metabolites (large advantage over RIA, in which separate assay is required)
 For a limited samples, even automated systems of sample injections can be used
 Running cost is low (although it seems expensive initially)
Disadvantages:
 Initial cost is high
 Being quite sensitive to a number of components, sometime costs much, as it may show its sensitivity to some
of the components in the mixture, thus contaminating the separation.
 HPLC is not so useful for the separation and purification of complex biopolymers such as protein in active form.

19. APPLICATIONS
 Molecular Weight Determination in:
- Analytical Chemistry
- Pharmaceutical and Drug Science
- Clinical Science
- FoodTechnology and Consumer Products
- Polymer Chemistry
- Environmental Chemistry
 ForensicApplications:
- toxicology quantification
- environmental components analysis
 Pharmaceuticals Studies:
- detail drug specification, along with entire drug development
- latest advancement in HPLC techniques in the form of LC-NMR,
preparative HPLC, high temperature HPLC for pragmatic and practical
approaches in Pharma industry

20.  Immobilization of nucleic acids bases on silica gel, for the selective separation
and study of nucleosides.
 It can also be used for analysing air and water pollutants and also monitoring the pesticide
levels in the environment.

21. RECENT EXPERIMENTATION
1. HPLC- UV was found comparatively more better tools than ELISA in determining the variations among
Vitamin D2, D3 and their 25-hydroxyl metabolites status in plasma.
2. HPLC was used to identify and quantify the Cholesterol Oxidation Products(COP), in salted and unsalted meat.
3. Comprehensive 2-D HPLC has been used successfully in proteomics biomolecules applications including the
analysis of pharmaceutical materials for ingredient purity and degradation profiling.
4. A novel fingerprinting method for glycosides in Nakshatra plants by HPLC.
5. Determination of acidic and basic-neutral pesticides in water at precipitated concentration
level by ion-interaction micro-HPLC.

22. REFERENCES
 www.ncbi.nlm.nih.gov
 www.news-medical.net
 www.waters.com
 www.hplc.chem.shu.edu
 www.shodex.com
 link.springer.com

23. THANKYOU!