High performance liquid chromatography

1. High Performance Liquid Chromatography
(HPLC)
Amandeep Singh
Assistant Professor
Department of Biotechnology
GSSDGS Khalsa College
Patiala

2. Introduction
• It is a type of Column chromatography. Traditional
chromatographic techniques have one disadvantage
of long separation time of the components. This
disadvantage can be overcome by HPLC. It have
following advantages over the other chromatographic
techniques:
1. Great speed in separation of compounds
2. High sensitivity
Main Criteria for obtaining Good resolution &
separation of components is Bead Size of material
packed in the column. Bead is the thing which is used
for making stationary phase of the column of HPLC.

3. Introduction
• If we keep the bead size smaller, separation will
become sharper of components. But one main
disadvantages of smaller bead size is the column
become tightly packed which result in reduced
flow rate or sometimes no flow at all.
• This disadvantage in HPLC can be overcome by
applying high pressure using pumps.
• Therefore, very high resolution and faster
separation in HPLC is achieved by using smaller
bead size of column matrix and with high
pressure pumps.

4. Bead Size
Smaller
Column get tightly packed
DisadvantagesAdvantages
Sharper the separation
Reduced flow rate
Disadvantages is overcome by applying high pressure using pumps.
Therefore, very high resolution and faster separation in HPLC is achieved by using;
smaller bead size of matrixes and with high pressure pump.
Smaller particle size is used in HPLC of 3-15 micron, which can withstand high pressure
of approximately 300 Kg/cm2
As compared to 40-300 micron of normal chromatographic column, which can withstand
only pressure of 3 Kg/cm2

5. Advantages of HPLC
• The column size is reduced because it uses smaller
bead size (usually 10-30 cm stainless steel columns
with 1-5 mm diameter are used).
• The running time is reduced significantly because of
smaller columns and high pressure pumps.
• Better resolution is achieved because of very small
particle sizes of column materials.

6. Principle
The principle of HPLC is based on material of Stationary phase used. If silica is
used to make stationary phase of HPLC, then principle of partition is used for
separation. The same principle is used in thin layer chromatography. Following is
the list of materials used for stationary phase of HPLC:
Principle Stationary Phase material
Partition Silica
Ion Exchange DEAE/CM-Cellulose
Gel Filtration Sepharose, Sephacryl, Agarose based matrix
Affinity Protein-A-Sepharose
Reverse Phase Octyl-C8, Octadecyl C-18
Normally Column for HPLC is made by stainless steel and packing of that
column can be done by any of the stationary phase material listed above.
Note: For separation of biological samples using HPLC, corrosive liquids like
NaCl, NH4Cl, HCl used as mobile phase. For that Titanium columns are used
instead of stainless steel. Because titanium is more resistant to these corrosive
materials.

7. Instrumentation

8. Instrumentation
1. Solvent Reservoir (to store the mobile phase)
2. Pumping system (to push the mobile phase
through the column)
3. Sample injection (A device to inject the sample
into the mobile phase)
4. Column (in which separation will take place)
5. Detector (for detecting the concentration of
sample components coming out of the column)
6. Recorder (to produce a chromatogram)

9. 1. Solvent Reservoir
It’s a glass or stainless steel container and it
must possess following properties:
A. It must be Inert to the solvent being used.
B. It must possess a degassing system (by
heating, vacuum, ultrasonic sound, helium
gas spraying) to remove air particles.
C. It must contain volume enough for repetitive
analysis.

10. Solvent
• Solvent act as mobile phase for HPLC.
• Solvent used must be free from dust particles,
fine precipitates etc. As they increase the
pressure of the pump beyond threshold
pressure (set limit).
• Pump stop automatically if pressure crosses
threshold limit.
• Therefore, sample must be filtered first by 1-5
mm nitrocellulose membrane filter.

11. Isocratic and Gradient Separation
• Type of separation desired, dictate the choice of mobile phase
(solvent) used. Separation is either isocratic or gradient.
• This means for the elution of sample components from the
column, either one solvent is enough or mixture of two or more
solvents are required.
Isocratic Separation Gradient Separation
Single solvent is used.
e.g. 100% water
OR
Fixed proportion of 2 solvents is
used.
e.g. 50% ethanol: 50% water
More than one solvent is used.
e.g. ethanol and water
OR
Varied proportion of 2 solvents is
used.
e.g. 90% ethanol:10% water

12. 2. Pumping Systems
Pumping system can be said to be the heart of HPLC. By producing
high pressure, the pump is a major factor in obtaining high
resolution and better separation. A good pump should have the
following qualities:
1. A pulseless stable flow
2. Should provide a constant flow rate of 0.5-1o ml/min
3. A constant flow delivery
4. Should withstand high pressure of upto 6000 psi
5. Should be adaptable to gradient operation.
Types of pumping systems:
• Holding Coil
• Pneumatic amplifier
• Moving Fluid Type
• Reciprocating piston
• Syringe pump

13. 3. Sample Injection
Sample in HPLC column can be injected by
two methods:
1. With Microsyringe 2. With small volume metal loop

14. 4. The Column
• Column for HPLC can be made by stainless
steel, glass, aluminium, copper or PTFE
(Polytetrafluoroethylene).
• Stainless steel is mostly preferred as it can
withstand pressure upto 8000 psi.
• Straight column of 20-50 cm length are
commonly used having 1-4 mm in diameter.

15. Column Packing
Three forms of column packing
materials are available:
(A)Microporous (5-10 mm in
diameter)
(B)Pellicular (Solid inner core,
40 mm diameter)
(C)Bonded phase (Stationary
phase is bonded to inert solid
support)

16. Column Packing Procedure
Column packing has to:
1. Uniform
2. Should be packed as densely as possible
Technique used for column packing normally is: High pressure slurry technique.
Solvent
+
Packing material
(silica e.g.)
Thick suspension
(Slurry)

17. Thick suspension is poured in
the column
Porous plug at the bottom
Column Packing Procedure

18. The Guard Column
• Serum or other biological materials can clog
(block) the column.
• To prevent this, a short column called guard
column (2-10 cm) is attached before the main
HPLC column.
• This guard column, filter out the impurities
before they can reach to the main column.

19. 5. Detectors
• UV-Visible absorption detector (UVD)
• Refractive Index detector (RID)
• Electrochemical Detector (ECD)
• Fluorescence Detector (FLD)
• Mass Spectrometer

20. Detector
• There are several ways of detecting when a substance has passed through the
column. A common method which is easy to explain uses ultra-violet absorption.
• Many organic compounds absorb UV light of various wavelengths. If we have a
beam of UV light shining through the stream of liquid coming out of the column,
and a UV detector on the opposite side of the stream, we can get a direct reading of
how much of the light is absorbed.
• The amount of light absorbed will depend on the amount of a particular compound
that is passing through the beam at the time.
• We might wonder why the solvents used don't absorb UV light. They do! But
different compounds absorb most strongly in different parts of the UV spectrum.
• Methanol, for example, absorbs at wavelengths below 205 nm, and water below
190 nm. If we were using a methanol-water mixture as the solvent, we would
therefore have to use a wavelength greater than 205 nm to avoid false readings from
the solvent.

21. Variants of HPLC
Two variants of HPLC are used.
1. Normal Phase HPLC
Stationary phase = Polar
Mobile phase = Non-polar
2. Reverse Phase HPLC
Stationary phase = Non-polar
Mobile phase = Polar