All about HPLC

1. High-Performance Liquid
Chromatography (HPLC)
Ajah Anayochukwu Kc.
B. Pharm(Hon’s), M. Pharm

2. • HPLC is an advanced form of column
chromatography:
• Packing materials 5-10 μm diameter
• Column has an internal diameter of 5mm or less
• Operation under high pressure liquid flow
• Sophisticated instrumentation
HPLC was introduced in late 1960s.
It is now regarded as the most versatile and widely
used of all chromatographic methods.

4. Basic components of HPLC
• Solvent reservoir and Degasser
• Pump and Pulse damper
• Sample loop and Injector port
• Column and Guard column (protects the HPLC
column)
• Detector (UV / fluorescence)
• Recorder (connected to the detector and
generates the chromatogram plot)

7. Flow diagram showing the components of an HPLC instrument

8. How HPLC works
• The solvent present in the reservoir is the mobile phase.
• The high-pressure pump is used to generate and maintain a
specified flow rate of the mobile phase (mm/s).
• The injector is used to introduce the sample into the
continuously flowing mobile phase stream that carries the
sample into the HPLC column.
• The HPLC column contains the chromatographic packing
material which is the stationary phase.
• The stationary phase is needed to effect the separation.
• A detector is needed to observe the separated compound
bands as they elute from the HPLC column.
• The mobile phase exits the detector and can be sent to waste
or collected as desired.

10. • HPLC Operation:
• Mobile phase enters the column from the left, passes through the particle bed, and exits at
the right.
• The top image represents the column at time zero [the moment of injection], when the sample
enters the column and begins to form a single black band.
• After a few minutes, as the mobile phase flows continuously and steadily past the packing
material particles, bands are observed as individual analytes travel at different speeds through
the column.
• This is because there is a competition between the mobile phase and the stationary phase for
attracting each of the analytes.
• Since the yellow dye moves the fastest, it has a higher affinity for the mobile phase than the
stationary phase.

11. • On the other hand, the blue dye having a higher affinity for the stationary
phase, moves significantly slow.
• The red dye has an intermediate attraction for the mobile phase. Hence, it
moves at an intermediate speed through the column.
• Since each dye band moves at different speed, they are separated
chromatographically.
• However, if the sample is colourless, a detector is needed to observe the
separated compounds as they elute.
• The detector has the ability to sense the presence of the compound and its
corresponding electrical signal to a computer data station.
• Several different types of detectors can be used. The detector selected
depends on the compound being tested.
• For example, if a compound can absorb UV light, a UV-absorbance detector is
used. If the compound fluoresces, a fluorescence detector is used.

14. • The recorder records the electrical signal needed to generate the
chromatogram on its display.
• The chromatogram is a representation of the separation that has
chemically [chromatographically] occurred in the HPLC system.
• It helps to identify and evaluate concentration of the sample
constituents.
• A series of peaks rising from a baseline is drawn on a time axis.
Each peak represents the detector response for a different
compound.
• The chromatogram is plotted by the computer data station.

15. A typical HPLC system

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SeparationsSeparation in based upon differential
migration between the stationary and
mobile phases.
Stationary Phase - the phase which
remains fixed in the column, e.g. C18,
Silica
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid Chromatograph
Waste

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SeparationsInjector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph

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SeparationsInjector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
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time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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SeparationsInjector
Detector
Column
Solvents
Pumps
Mixer
Chromatogram
Start Injection
mAU
time

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HPLC Analysis Parameters
Mobile Phases
Flow Rate
Composition
Injection Volume
Column
Oven
Temperature
Wavelength
Time Constant

34. Adsorption chromatography
• Adsorption chromatography is also known as normal phase chromatography or liquid-solid
adsorption chromatography.
•Difference between Adsorption versus Absorption:
•In absorption, one substance penetrate into the bulk of another substance.
• Adsorption is a surface phenomenon where interaction takes place only on the surface of
one substance.
• Adsorption chromatography is the oldest type of chromatography, which was used by
Tswett.
• The stationary phase is a solid on which the solutes are adsorbed. The stationary phase is
called the adsorbent.
• The solutes distribute between the two phases through a combination of adsorption and
desorption process.
• Desorption is a phenomenon whereby a substance is released from or through a surface.
• Separation is based on differences between the adsorption affinities of the solutes for the

35. Silica and alumina are the only stationary
phases that find extensive usage
in adsorption chromatography.
Silanol group

36. Characteristics of Normal Phase Chromatography:
• Stationary phase is polar. E.g., silica, alumina.
• Mobile phase is usually non-polar. E.g., n-hexane, chloroform, ethyl acetate, etc.
• Suitable for non-polar/water insoluble analytes of molecular weight < 1000.
• Hence, least polar component is eluted first in Normal Phase chromatography.
• When the packing material is silica (SiO2):
•Silica surface has strong adsorption
properties.
• Highly active silanol sites (Si-OH) will
interact with polar functions on analytes.
• Hence, polar analytes are better retained
due to strong interaction with the silanol
group.

37. • The usual order of elution from longer to shorter retention times:
Carboxylic acids
Amides
Alcohols, amines
Esters, aldehydes, ketones
Ethers
Aromatic hydrocarbons
• pH working range of silica is 2-8.
Longer
Shorter

38. Partition Chromatography
• Partition chromatography is also known as Reversed-Phase Chromatography or
Liquid-Liquid Chromatography.
• The stationary phase is a thin liquid film coated on an inert solid support (made of
silica).
• Separation is based on a difference in the equilibrium partitioning of solutes
between the liquid stationary phase and the mobile phase.

39. Characteristics of Reversed-Phase Chromatography:
• Stationary phase is non-polar. E.g., C18, C8, phenyl bonded silica, etc.
• Mobile phase is usually polar. E.g., methanol, acetonitrile, water, etc.
• Suitable for polar, non-ionic, ionisable and water soluble analytes.
• However, too polar compounds may not be
retained in Reverse Phase Chromatography.
• Reverse-Phase Chromatography is generally
superior to Normal-Phase Chromatography, and
most popular form of HPLC.
• Polar analytes elute first; non-polar analytes
interact more strongly with the ‘liquid-like’
hydrophobic C18 .
Si
Si

40. • pH working range is 2-8.
•The eluting power is determined by polarity of the mobile phase.
• Eluting power decreases with increasing solvent polarity.

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HPLC Applications
Chemical
Environmental
Pharmaceuticals
Consumer Products
Clinical
polystyrenes
dyes
phthalates
tetracyclines
corticosteroids
antidepressants
barbiturates
amino acids
vitamins
homocysteine
Bioscience
proteins
peptides
nucleotides
lipids
antioxidants
sugars
polyaromatic hydrocarbons
Inorganic ions
herbicides