High- performance Liquid Chromatography”/ (High- pressure Liquid Chromatography) is a powerful tool in analysis, it yields High Performance and high speed compared to traditional columns chromatography

1. Chromatography
Dr. Suji V. S.

2. Chromatography
Term derived from Greek word
• “Chroma” meaning ‘Colour’
• “Graphein” meaning ‘to write’
• collective term for a set of laboratory techniques
for the separation of mixtures.
• The mixture is dissolved in a fluid called Mobile
phase, which carries it through a structure
holding another material called Stationary phase.

3. History
• 1st employed by M. S. Tswett, a Botanist in 1903 for
separation of plant pigments using a column of
alumina

4. Types of Chromatography
Chromatography
Planar
Thin layer
(TLC)
Paper
Column
Liquid (LC) Gas (GC)

5. Types of Chromatography
1. Adsorption
2. Partition
1. Paper
2. TLC
3. Ion exchange
4. Gel filtration
5. Affinity
6. HPLC

6. Adsorption chromatography

7. Adsorption chromatography
• Earliest form of chromatography
• Based on differences in adsorption at the
surface of Solid Stationary Medium (Silica gel-
Common adsorbing substance)
• Relative Lipophilic drugs readily analysed by
Silica gel HPLC with UV detection

8. Partition chromatography
• Developed by Martin & Synge (1941)
(Nobel Prize 1952)
• Use- Separation of mixtures of Amino Acids &
Peptides

9. Partition chromatography
1. Stationary phase- Solid/ Liquid over which
Mobile phase (Liquid/Gas) moves.
2. depends on Partition Co-efficient (solubility)
of the particular substances in the mixture.

10. Partition chromatography
• Types
1. Depending on phases of the components
partitioned
a. Solid-liquid,
b. Liquid-liquid,
c. Gas -liquid, etc.

11. Partition chromatography
2. Based On Mode Of Separation
RPC- More Commonly used in TDM as Drugs are
usually Hydrophilic
Stationary phase Mobile phase
Normal phase
chromatography
(NPC)
Polar (hydrophilic) Non -polar
(hydrophobic)
Reverse phase
chromatography
(RPC)
Non -polar
(hydrophobic)
Polar (hydrophilic)

12. 2A. Paper chromatography
a. Stationary phase - water held on solid
support of filter paper (cellulose)
b. Mobile phase - mixture of immiscible
solvents (water+a non polar solvent+
acid/base) eg. Butanol- acetic acid- water

13. 2B. Thin Layer Chromatography (TLC)
a. Stationary water phase- held on a silica gel
(Kieselguhr) spread on a glass plate
b. Mobile phase- Non Polar Solvent moves up
Advantage of TLC over Paper Chromatography- Separation takes 2-4hrs

14. Visualisation of chromatography
• After chromatographic run is over, paper/ plate has
dried  sprayed with a Location Reagent
Location Reagent
Ninhydrin Proteins
Sulfuric acid Phospholipids
Diphenylamine Sugars

15. Rf value
Ratio of fronts (Rf)
= Distance travelled by solute
Distance travelled by solvent
• Is a constant for a particular solvent system at
a given temperature.

16. Gas Chromatography
• Type of partition chromatography where
Mobile phase is Gas.
• Stationary phase- liquid/solid
– supported by a column of inert material (silica)
in a long narrow column
Separation is based on:
-Solute differences in vapor pressure
-Interaction with stationary phase

17. GLC components
1. Column
2. Supply of carrier gas
3. Flow control apparatus
4. Injector
5. Column oven
6. Detectors
7. Computer

18. GLC (Gas Liquid Chromatography)
• Mixture of substances to be separated is made
volatile at one end of the column & the vapors
are swept over the column by an inert carrier
gas like argon/ nitrogen
• Fractions emerging from the column are
detected & quantified by detecting devices
• Suitable for compounds (eg Lipids) which
resists degradation & temperature.

20. GLC (Gas Liquid Chromatography)
• Advantage –
• Requires only a small sample; Specific.
• Disadv-
1. Time consuming,
2. High potential for Errors
3. Volatile components
• Not suitable for TDM

21. Ion exchange chromatography
• Separation is based on Electrostatic Attraction
between charged Biological molecules to
oppositely charged groups on the ion
exchange resins.
• Use- Separation of proteins & Amino Acids

22. Ion exchange chromatography

23. Gel Filtration Chromatography

24. Gel Filtration Chromatography
• ~ Gel Permeation /Molecular sieving/ Size
Exclusion Chromatography
• Based on Molecular Size of solute
• Use-
• Separation not only of Macromolecules but
also of Small Molecular Weight substances.
– Separation of proteins;
– Purification of Proteins
– Molecular weight determination

25. Affinity chromatography

26. Affinity chromatography
• Based on High Affinity of specific proteins for
specific chemical groups
• Uses
1. Co -enzymes can be used to purify enzymes
– Eg. NAD+ used to purify Dehydrogenases
2. Separating antibodies & antigens

27. HPLC

28. HPLC
“High- performance Liquid Chromatography”/
(High- pressure Liquid Chromatography).
• is a powerful tool in analysis, it yields High
Performance and high speed compared to
traditional columns chromatography
• The liquid phase is passed through the column under
High Pressure.

29. Principle of HPLC
1. Chromatography principle
2. Resolving power (Resolution) of HPLC increases
with
a. Column length and
b. Smaller particle size of stationary phase.
3. High Pressure (500-6000 psi)
– Because of small particle size of stationary phase <10µ
 high resistance to the flow of solvent

30. Factors determining HPLC
1. Mechanical separation power (Efficiency)
– Column length
– Particle size
– Packed bed uniformity
2. Chemical separation power (Selectivity)
– Interaction between Packing materials & Mobile
phase

31. HPLC setup sketch

32. HPLC system

33. UPLC (Ultra Performance HPLC)
• If resolution increased by
1. Decreasing particle size (1-1.7μ);
2. Very high pressure, 15,000-1,00,000 psi is used
to deliver mobile phase.

34. A . Solvent delivery system
(Mobile Phase)
• acts as a carrier to the sample solution

35. B. Pumps
• Force the solvent (mobile phase) through
the liquid chromatograph at a specific flow
rate (1-2mL/min).
• Typical pumps can reach pressures of 6000-
9000 psi.

36. C. Injector
• introduces liquid sample into the flow
stream of the mobile phase for analysis
• Typical sample volumes : 5-20 μL

37. D. Column (Stationary phase)
• “Heart of the Chromatograph”
• Column Inner Diameter & Packing play imp. role in
Efficiency of HPLC
• separates the sample components using various
physical and chemical parameters.
• “Packing”
– Small particles (<10µ) inside the column (Silica gel)
– Resp. for the High Back Pressure at normal flow rates.

38. Column
• Made of Stainless Steel to withstand high
pressure
HPLC Column Dimensions

39. E . Detector
1. Detect & measure the amount of individual
molecules that elute from the column
2. convert the data into an electrical signal 
provides an output to a Recorder (Computer)
 Liquid Chromatogram

40. Types of Detector
• Detector is selected based on the analyte or
the sample under detection
1. Ultraviolet detector
2. Fluorescence detector
3. Refractory index detector
4. Mass spectrometry detector
– coupled with HPLC called LC-MS

41. F. Recorder (Computer)
1. controls all the modules of the HPLC
instrument
2. takes the signal from the detector and uses it
for analysis of sample components
(qualitative analysis) and the amount of
sample (quantitative analysis).

42. HPLC chromatogram
Conc. of each detected component is
calculated from the area or height of the
corresponding peak

43. TYPES OF HPLC
I. Based On Mode Of Separation
RPC- More Commonly used in TDM as Drugs are
usually Hydrophilic
Stationary phase Mobile phase
Normal phase
chromatography
(NPC)
Polar (hydrophilic) Non -polar
(hydrophobic)
Reverse phase
chromatography
(RPC)
Non -polar
(hydrophobic)
Polar (hydrophilic)

44. Types of HPLC
II. Based On Principle of Separation
1. Adsorption (Liquid- solid)
2. Partition
3. Ion exchange
4. Gel permeation
• Selection of proper HPLC mode is necessary
for each drug

45. Types of HPLC
Stattionary
phase
Mobile phase Type of
interaction
Feature
NPC Silica gel (polar) Organic solvent (n-
Hexane/ IPE)
100% organic
(Non polar)
Adsorption Fat soluble
RPC Silica – ODS
(Silica-C18)
Non polar
MeOH/ water (Polar) Partition/
Hydrophobic
MC used
SEC Porous polymer
Aqueous porous
polymer
Organic solvent (THF)
Buffer solution
Gel
permeation
Molecular Wt
distribution,
protein separation
IEC Ion exchange gel Buffer solution Ion exchange Separation of ionic
substance

46. Advantages of HPLC
1. Separations fast and efficient (High Resolution Power)
2. Continuous monitoring of the column effluent
3. 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.

47. Advantages of HPLC
7. HPLC is more versatile than GLC because
– Not being restricted to volatile & thermally stable solute
– Choice of mobile and stationary phases is much wider.
8. Both aqueous and non aqueous samples can be
analyzed with little or no sample pre treatment
9. High degree of Selectivity for specific analyses.
– As variety of solvents and column packing are available
10. Determins multiple components in a single analysis.

48. Applications
• Widely used in Biotechnology, Biomedical,
Biochemical research
• MC used analytical technique for TDM
• In other fields & industries
1. Cosmetics
2. Food & Flavour
3. Forensic
4. Environmental studies

49. Applications of HPLC
• Clinical
• used for assaying or monitoring many
substance like
– Amino acids, peptides, proteins, carbohydrates,
lipids, vitamins, nucleic acids, hormones,
metabolites,
– Drugs- Antiarrhythmics, Antibiotics, Antiepileptics,
Analgesics, Tricyclic Antidepressents, etc.

50. Advantages of HPLC
1. Versatality
2. Efficacy
3. Precision
4. Sensitivity
5. Speed
• Most popular, widely accepted, powerful
form of chromatography for analysis.

51. Thank you