Liquid chromatography is a technique used to separate ions or molecules dissolved in a solvent. Early LC used glass columns that were long and had low efficiency, requiring separation times of hours. This led to the development of high-pressure liquid chromatography (HPLC), which uses smaller particle sizes and higher pressures to improve efficiency and reduce separation times. HPLC is widely used for analytical separations in industries like pharmaceuticals due to its high sensitivity, adaptability, and ability to separate non-volatile compounds. The basic components of an HPLC system are solvent reservoirs, pumping systems, sample injection systems, chromatographic columns, detectors, and data recorders or processors.

2.  Liquid chromatography (LC) is an analytical
chromatographic technique that is useful for
separating ions or molecules that are dissolved in
a solvent
 Oldest method
 Not quietly used due to non availability of highly
sensitive detectors at that times

3.  Early LC was carried out in glass columns with
dia-1 to 5 cm and length 50 to 500 cm
 Stationary phase particle dia-150 to 200µm
 Flow rates are low
 Separation time taken were very long (hours)
 Lower efficiency
 Lead to the development of HPLC-High Pressure
Liquid Chromatography

4.  Needs high pressure for the separation of species
 Can be performed in Column and not on open
bed
 Widely used in all analytical separation process
 High sensitivity, ready adaptability, sustainability
for separating non volatile species, applicability
to substances that are of prime interest to
industries.
 Examples include amino acids, drugs,
hydrocarbon, proteins, antibiotics, steroids and a
variety of inorganic substances

5.  Accurate maintenance of a solvent flow is
difficult
Basic Components of HPLC
Solvent Reservoir
Pumping System
Sample Injection System
Chromatographic Column
Detecting System
Microprocessor or Recorder

7.  Solvent-Mobile phase
 Glass or stainless steel reservoirs with 200 to 1000ml
capacity
 Equipped with pulse damper, filter and degasser
 Flow of solvent is in the form of pulses
 To smooth out ripples in pulses pulse damper is required
 Oxygen and nitrogen are dissolved in the solvent which forms
bubbles causing band spreading
 To improve the performance these gases are removed from
the solvent using a degasser
 Filter to remove dust and particulate matter to prevent
damage to the pumping or clogging of the column

9. The following are the characteristics of good
pumping system
 Generation of pressure up to 6000 psi
 Moderate flow rates of 0.5 to 2ml/min
 Pulse free output
 Corrosion resistant components
 Should have small hold up volume

10.  Reciprocating Piston Pumps
 Syringe or Displacement type pumps
 Pneumatic or constant pressure
pumps

12.  Consists of small motor driven piston to move
back and forth in an hydraulic chamber (35-
400µl in volume)
 On back stroke ball check valve is closed
 Piston pulls in solvent on the forward stroke
 Pump pushes solvent out to the column
 Wide range of flow rate is obtained by altering
piston stroke volume
 Drawback-Pulsed flow must be damped before
affecting the column

14.  More suitable for bore columns as it delivers a
finite
 Volumes between 250 to 500ml
 Consists of large syringe like chambers equipped
with plunger activated by screw mechanism
powered by stepper motor
 Advantages: Flow is independent on viscosity
and back pressure, pulse less flow
 Disadvantages: Limited solvent capacity and
inconvenience when solvents are changed

16.  Solvent is driven through the column with the use
of pressure from gas cylinder
 Low pressure gas source (1-10atm) is needed to
generate high liquid pressure(1-400atm)
 Large piston drives the mobile phase
 Advantage: Inexpensive and pulse free
 Disadvantage: Flow rate dependant on solvent
viscosity and column back pressure

17.  Detectors are sensitive to variations in flow
 To attain highly stable signal in the detector flow
must be smoothened
 Pulse damper consists of a rigid vessel filled
with fluid of known compressibility
 Compressible fluid is separated by semi
permeable membrane
 Solvent mixture passes through the separated
space with constant flow

18.  Injection should be a narrow plug to avoid band
broadening
Two methods to introduce samples
 Sampling valves and loops method
 Stopped flow injection method

20.  Flow of solvent is stopped momentarily by
turning off the pump
 Syringe containing the sample is injected and
then the pump is turned on

21.  Columns are constructed by heavy wall glass lined
metal tubing or stainless steel tubing to withstand
high pressure
 Uniform bore diameter and smooth inner wall
 Straight columns operated in vertical positions
 End fitting designed to avoid stagnant of mobile
phase

22.  Analytical columns
Internal diameter – 1cm or more
High separation efficiency of minute samples
Practical problems due to smaller diameters
 Guard columns
Designed to remove particles that
 Clog the separation column
 Compounds and ions that cause precipitation
 Compounds that cause decrease resolution and create false peaks

23.  Maintaining column temperature may improve
column efficiency
 Most instruments are equipped with column
heaters and water jackets to give precise
temperature control

24. Ideal Chromatographic detector should have the
following characteristics
 Good sensitivity, stability, reproducibility
 A linear response to solute
 Short response time
 Nondestructive sample
 High reliability
 Insensitive to changes in flow rate

25.  Bulk Property Detectors
 Solute Property Detectors
Mainly used Detectors
 UV Spectro photometric detector
 Fluorescence detector
 Refractive index detectors
 Electro chemical detectors

26.  Used to monitor chemical process online
 For automatic operation –already programmed in
microprocessor or micro computer
 Electrical output from the detector is applied to
recorder