HPLC, or high performance liquid chromatography, is an analytical technique used to separate compounds in a mixture. It works by injecting a sample onto a column containing a stationary phase, which causes the different compounds in the mixture to pass through the column at different rates based on their interactions with the stationary and mobile phases. This separation allows for the individual quantification and identification of compounds in the sample. Key aspects of HPLC include the use of high pressure to allow for small particle sizes in the stationary phase, which enables better separation. Common applications of HPLC include the simultaneous analysis of multiple compounds, analysis of compounds at low concentrations, and fractionation of samples for further analysis or purification.

1. HPLC
B.V.Rao

2. 2
Concept of Chromatography
Chromato
graphy
Supercritical-fluid
Chromatography
• Chromatography is an analytical method that the compounds are
physically separated prior to measurement
• The main purpose of chromatography is to separate and quantify
the target sample in the matrix
Gas
Chromatography
Liquid
Chromatography

3. HPLC
High Pressure Liquid Chromatography
 High pressure to be able to use small particle size to
allow proper separation at reasonable flow rates
High Performance Liquid Chromatography
 High performance due to its reproducibility
currently refers to:
High Precision Liquid Chromatography

4. 4
Why use HPLC?
• Simultaneous Analysis
• High Resolution
• High Sensitivity (ppm-ppb)
• Good repeatability
• Small sample size
• Moderate analysis condition
• no need to vaporize the sample like GC
• Easy to fractionate the sample and purify
• No destructive for many detectors

5. Introduction
 Compounds are separated by injecting a sample
mixture onto the column. The different component
in the mixture pass through the column at different
rates due to differences in their partition behavior
between the mobile phase and the stationary phase.

6. 6
2
1
Separation Mechanism
Compounds are separated because the molecules move
at different rates in the column.
column

7. 7
Separation Mechanism
Due to different interaction between stationary phase and
different sample, the molecules move at different rate,
therefore separation can be done.
Stationary Phase
Stronger
interaction
Weaker
interaction
Mobile Phase
2
1

8. 8
What Is the Interaction?
Hydrophobic Interaction
Less polar (more hydrophobic) analytes are more attracted and
spend more time associated with the hydrophobic bonded phase,
therefore, they are eluted last.
A
B
B
B
B
B
A
A
A
A
A
B
Support
particle
Nonpolar
bonded phase
Interstitial area
(mobile phase)
Less polar analyte
More polar analyte
B
A

9. 9
Chromatogram
tR : Retention time
A : Area
h : Height
tR
Signal
Time
Peak
h
A

10. 10
Some Important Terms
• Chromatogram: A plot of detector signal output versus time or
elution volume.
• Mobile phase: The liquid that moves the solute through the column.
• Stationary phase: The packing material of the column, which is the
immobile phase involved in the chromatographic process.
• Peak: The visual representation on the chromatogram based on the
detector's electrical response due to the presence of a sample
component inside the flow cell.
• Retention time: The time taken by the analyte peak to reach the
detector after sample injection.
• Qualitation: An analysis process which is designed to identify the
components of a substance or mixture.
• Quantitation: An analysis process which is designed to determine
the amounts or proportion of the components of a substance.

11. Types of HPLC Techniques
Based on modes of Chromatography
 Normal Phase mode
 Reverse Phase mode
Based on principle of separation
 Adsorption – solid-liquid, Silica, Alumina
 Partition – Liquid-liquid, PEG coated on silica
Based on elution technique
 Isocratic separation –same composition
 Gradient Separation – different composition
Dis-adv :Time taking for reconditioning for next run
Base line disturbance because of different solvents
Based on Scale of operatioon
 Analytical HPLC
 Preparative HPLC

12. 12
Separation Modes
• Normal phase chromatography
• Reversed phase chromatography
• Ion exchange chromatography
• Size exclusion chromatography
• Affinity chromatography

13. 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.
Ex :Diamond, hydrocarbon polymers.
 Ion Exchange Chromatography: Separation of organic and
inorganic ions by their partitioning onto ionic stationary phases
bonded to a solid support.
Quaternary ammonium group – seperation of anions
Sulfonic acid - seperation of cations
Ex : Nucleic acids, amino acids, sugars
 Size Exclusion Chromatography: Separation of large molecules
 Affinity Chromatography : Compound binds to a ligand
Ex : Biological mixtures , enzymes

14. 14
Flow Diagram of HPLC
Pump
Injector
Column
Oven
Detector
Mobile Phase
Data
processor

15. 15
Isocratic System
Simple system with one pump and one solvent reservoir.
If more than one solvent is used, solvents should be premixed.
Data
processor
Pump
Injector
Column
Oven
Detector
Mobile Phase

16. 16
Low-pressure Gradient System
•One pump used to control 4 reservoirs;
•Mixing is done before pump.
•On-line degasser is necessary.
low pressure
gradient valve Data
processor
A B D
C
Pump
Injector
Column
Oven
Detector

17. 17
High-pressure Gradient System
Data
processor
pump
pump
pump
A
B
C
Injector
Column
Oven
Detector
Mixer
• Excellent gradient accuracy.
• 2-3 pumps required - one pump per solvent used.
• On-line degassing may not be critical.
• The sum of the flow rates delivered by each pump is maintained
constant

18. HPLC Basic Instrumentation
Mobile
phase
Pump
Solvent Delivery
Injector
Sample Injection
Column
Separation
Detector
Data Processor

19. Components Of A Liquid Chromatography
System
 Mobile Phase / Solvent Reservoir
 Degasser
 Solvent Delivery System (Pump)
 Injector
 Precolumn
 Column
 Temperature Control
 Detectors
 Recorder (Data Collection)

20. The Mobile Phase in HPLC
 Must do the following:
 solvate the analyte molecules and the solvent they are in
 be suitable for the analyte to transfer “back and forth”
between during the separation process
 Must be:
 Compatible with the instrument (pumps, seals, fittings,
detector, etc)
 Compatible with the stationary phase
 Readily available (often use liters/day)
 Adequate purity
 Free of gases (which cause compressability problems)
 Low viscous – methanol than ethanol

21. Mobile Phase for Reversed Phase HPLC
• Water / buffer + Organic solvent
– Organic solvents:
– Methanol
– Acetonitrile
– THF
– Buffer:
– Phosphate buffer
– Acetate buffer
– Ammonia buffer
• Ratio of aqueous and organic solvents is important

22. Degasser
 Problems caused by dissolved air(O2, N2)in mobile phase
(Deaerated mobile phase)
 Unstable delivery in pump
 Bigger noise and large baseline-drift in detector cell
 In order to avoid causing the problems,
mobile phase should be degassed.
 vacuum pumping systems
 distillation system
 a system for heating and stirring the solvents
 sparging system – Passing an inert gas of low
solubility through the solvent

23. PUMPS
Four basic types of LC Pumps are:
 Pneumatic pumps – Preparative purpose only
 Motor driven syringe type pumps
 Reciprocating pumps
 Hydraulic amplifier pumps – not in use

24. Motor driven syringe type pumps
Works on the principle of positive solvent displacement
Double syringe pumps are available:
one for column
one from reservoir
 Advantages:
 Simple
 Inexpensive
 Pulse free
 Stable flow rate
 Lowest dead volume
 Disadvantages
 Limited capacity
 Not suitable for gradient elution

25. 25
Plunger Reciprocating
Pump
motor and cam
plunger
plunger seal
check valve
pump head
5 - 50µL
out
in
Mobile phase
check valve

26. 26
• Consists of a small chamber in which the solvent
is pumped by the back and forth motion of a
motor-driven piston
• Advantage
– Low pressure fluctuation
– Very easy to replace other solvent
• Disadvantage
– Change the plunger seal
– Flow variation
– Small volume of solvent delivery is possible
Plunger Reciprocating Pump

27. Sample Injection Systems
 Convenient to use
 For injecting the solvent through the column
 Minimize possible flow disturbances
 Volumes must be small
 .1-500 L
 Sampling loops
 interchangeable loops (5-500 L at pressures up to
7000 psi)
• Chemically inert
• Reproducible

28. Different ways of sample injection
 Fixed volume valve injection
 First HPLC sample injection system
 Valve loop is filled with sample
 Reproducible sample amounts can be injected
 Variable injection valve injection
 Flow restrictors are used b/w pump-column
 On column injection
 Injected by means of syringe through septum
 Simple method of injection
 Dis-adv :Leaching effect of the mobile phase in contact with
septum , leads to a ghost peak

29. Peak broadening depends on
 Type of injection system used
 Connection between injector and column
 Injection volume and time taken for injection
• Commonly used Injectors
Syringe injection - Trouble is more
- Peak broadening
Valve injection – Automation is possible
-- Highly précised one
-- Constant pressure is maintained

30. Manual injector
• Valve injectors
(a) Isolated from the pump eluent
Stream (LOAD position)
(b) Positioned in it (INJECT position)

31. Direct injection auto sampler
from Pump from Pump to Column
Vial
Needle
Measuring Pump
to Column
LOAD INJECT

32. Guard column
 Protection device, often included just prior to the analytical column
to chemically remove components of the sample that would foul the
main column
 Guard column filled with removable protective cartridge
Troubleshooting: if pressure in system high check the cartridge in the
guard column
Guard - Protects the analytical column.
 Particles
 Interferences
 Prolongs the life of the analytical column

33. Columns and Stationary Phases.
 HPLC is largely the domain of packed columns
some research into microbore/capillary columns is
going on.
 Stationary phases are particles which are usually
about 1 to 20 m in average diameter (often irregularly
shaped)
 In Adsorption chromatography, there is no additional
phase on the stationary phase particles (silica,
alumina, Fluorosil).
 In Partition chromatography, the stationary phase is
coated on to (often bonded) a solid support (silica,
alumina, divinyl benzene resin)

34. 34
Stationary Phase in
Reversed Phase Column
• C18 (ODS) type
• C8 (octyl) type
• C4 (butyl) type
• Phenyl type
• TMS type
• Cyano type
C18H37
Si O Si
CH3
CH3
Non-polar

35. Stationary Phases
 Polar (“Normal” Phase):
 Silica, alumina
 Cyano, amino or diol terminations on the bonded phase
 Non-Polar (“Reversed Phase”)
 C18 to about C8 terminations on the bonded phase
 Phenyl and cyano terminations on the bonded phase
 Mixtures of functional groups can be used!!
 Packed particles in a column require:
 Frits at the ends of the column to keep the particles in
 Filtering of samples to prevent clogging with debris
 High pressure pumps and check-valves
 Often a “Guard Column” to protect the analytical column

37. Properties of Detector
 Adequate sensitivity
 Stability and reproducibility
 Short response time
 Minimum volume for reducing zone
broadening
 High reliability and ease of use
 Similarity in response toward all analytes
 Non-destructive

38. 38
Detectors for HPLC
• UV-VIS Ultraviolet / Visible detector
• PDA Photodiode Array detector
• RF Fluorescence detector
• CDD Conductivity detector
• RID Refractive Index detector
• ECD Electrochemical detector
• ELSD Evaporative light scattering detector
• MS Mass spectrometer detector

39. 39
Ultraviolet / Visible Detector (4)
Advantage:
• Sensitivity is high
• Relative robust to temperature and flow rate change
• Compatible with gradient elution
Disadvantage:
• Only compounds with UV or visible absorption could
be detected.
Additional Functions
• Dual Wavelength mode
• Wavelength Time Program mode
• Wavelength Scan mode

40. 40
Photodiode Array Detector (1)
Sample Cell
512 Elements Photodiode Array
Grating
D2 / W lamp
One element detects
one absorbance at
one wavelength.

41. 41
PDA Detector
Advantages:
• PDA Detector could analyze a sample simultaneously at many
different wavelengths.
• UV Visible spectra are useful for compound identification,
checking peak purity, as well as finding the optimum
absorbance for the compounds.
• UV Visible spectra of many compounds could be stored in the
spectrum libraries, which are useful for compound
identification.
• Relatively robust to temperature and flow rate fluctuations
• Compatible with gradient elution.
Disadvantages:
• Slightly less sensitive than UV-Visible detector.

42. 42
Refractive Index Detector
(1)
Sample
Reference
Photodiode
W Lamp
Refraction

43. 43
Refractive Index Detector (3)
Advantage
Responds to nearly all solutes
Unaffected by flow rate
Disadvantage
Not as sensitive as most other types of detectors
Could not be used with gradient elution

44. 44
Selection of Detectors
Detectors Type of compounds can be detected
UV-Vis &
PDA
Compounds with chromophores, such as aromatic rings
or multiple alternating double bonds.
RF Fluorescent compounds, usually with fused rings or
highly conjugated planar system.
CDD Charged compounds, such as inorganic ions and
organic acid.
ECD For easily oxidized compounds like quinones or
amines.
RID & ELSD For compounds that do not show characteristics usable
by the other detectors, eg. polymers, sccharides.

45. Parameters used in HPLC
CAPACITY FACTOR
RESOLUTION
ASYMMETRY FACTOR ( TAILING FACTOR )
EFFICIENCY
Retention : When a component in a sample
interacts with the stationary phase in the column
and a delay in elution occurs.
Column efficiency : Goodness of a column

46. Parameters used in HPLC
Retention parameters
tR : retention time (the time between the injection point and the maximum
detector response for correspondent compound)
vR : retention volume (tR x eluent flow rate)
k’ : capacity factor
t0 : the time required for the component not retained by the column to pass
through the column
tR
tR - t0
t0 k’ =
tR - t0
t0

47. Resolution
tR1
tR2
k’1
k’2
W1 W2
Resolution :
Separation factor :
Rs = 2 x
W1 + W2
tR2 - tR1
=
k’1
k’2
The resolution of two bands is a function of
both their relative Retentions and peak width.

48. Peak symmetry
S : Symmetry factor ( T : Tailing factor )
S =
2f
W0.05
f
W0.05
h x 0.05
h
S = 1 : The peak is completely symmetric.
S > 1 : Tailing
S < 1 : Leading

49. 3.) Efficiency:
Efficiency is related experimentally to a solute’s peak width.
- an efficient system will produce narrow peaks
- narrow peaks  smaller difference in interactions in order to separate two
solutes
Efficiency is related theoretically to the various kinetic processes that are
involved in solute retention and transport in the column
- determine the width or standard deviation (s) of peaks
Wh
Estimate s from peak widths,
assuming Gaussian shaped
peak:
Wb = 4s
Wh = 2.354s
Dependent on the amount of time that a solute spends in the column (k’ or tR)

50. Number of theoretical plates (N): compare efficiencies of a system for
solutes that have different retention times
N = (tR/s)2
or for a Gaussian shaped peak
N = 16 (tR/Wb)2
N = 5.54 (tR/Wh)2
The larger the value of N is for a column, the better the column will be
able to separate two compounds.
- the better the ability to resolve solutes that have small
differences in retention
- N is independent of solute retention
- N is dependent on the length of the column

51. Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of
columns with different lengths:
H = L/N
where: L = column length
N = number of theoretical plates for the column
Note: H simply gives the length of the column that corresponds to one theoretical plate
H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size,
etc.) to the kinetic processes that give rise to peak broadening:
Why Do Bands Spread?
a. Eddy diffusion
b. Mobile phase mass transfer
c. Stagnant mobile phase mass transfer
d. Stationary phase mass transfer
e. Longitudinal diffusion

52. a.) Eddy diffusion – a process that leads to peak (band)
broadening due to the presence of multiple flow paths
through a packed column.
As solute molecules travel through
the column, some arrive at the end
sooner then others simply due to the
different path traveled around the
support particles in the column that
result in different travel distances.
Longer path arrives at end of column after (1).

53. A solute in the center of the
channel moves more quickly
than solute at the edges, it
will tend to reach the end of
the channel first leading to
band-broadening
The degree of band-broadening due to eddy diffusion and
mobile phase mass transfer depends mainly on:
1) the size of the packing material
2) the diffusion rate of the solute
b.) Mobile phase mass transfer – a process of peak
broadening caused by the presence of different flow profile
within channels or between particles of the support in the
column.

54. 54
Applications of HPLC
Field Typical mixtures
Pharmaceuticals Antibiotics, sedatives, steroids, Amino analgesics,
crude drugs, cosmetics
Biochemical acids, proteins, peptides, carbohydrates, lipids,
enzymes, medicines, hormone
Food products Mycotoxins, additives, saccharides, amino acids,
vitamins, fatty acid, coloring agents, antibacterials
Industrial
chemicals
Condensed aromatics, surfactants, propellants, dyes,
polymers, plasticizers
Forensic chemistry Drugs, poisons, blood alcohol, narcotics
Environmental field Inorganic ions, organic acids, agricultural chemicals,
pesticides, herbicides, phenols,
Clinical medicine Bile acids, drug metabolites, urine extracts,
estrogens