Prof.Anjali Yadav Department of Pharmaceutical Chemistry Govt Holkar Science College

1. Anjali Yadav
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY
GOVT HOLKAR SCIENCE COLLEGE
INDORE
HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)

2. HPLC(HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY)
 HPLC is a High Performance liquid Chromatography or High Pressure
Liquid Chromatography.
 High performance liquid chromatography (HPLC) is a chromatographic
technique used to separate a mixture of compounds in analytical
chemistry and biochemistry with the purpose of identifying, quantifying
or purifying the individual components of the mixture
 HPLC is a separation technique that involves:
i)The injecting a small volume of liquid sample into a tube packed
with tiny particles (3 to 5 micron (µm) in diameter called the stationary
phase)
ii) Individual components of the sample are moved down the packed
tube (column) with a liquid (mobile phase) forced through the column by
high pressure delivered by a pump.

3. These components are separated from one another by the column packing
that involves various chemical and/or physical interactions between their
molecules and the packing particles.
These separated components are detected at the exit of this tube (column) by
a flow-through device (detector) that measures their amount. An output from
this detector is called a “liquid chromatogram”.

4. PRINCIPLE
 High Performance Liquid Chromatography [HPLC] is principle is based
on adsorption as well as partition chromatography is depending on the
nature of stationary phase, if stationary phase is solid principle is
based on adsorption chromatography and if stationary phase is liquid
principle is based on partition chromatograph.
 It is important for determination of volatile and non volatile compounds.
 It is important for determination of Retention Time (the time is required
after sample injection maximum angle peak reaches to detector)

5. ADVANTAGES
 High sensitivity & High performance.
 Rapid process and hence time saving.
 It is having a high resolution and separation capacity.
 Less requirement of mobile phase in developing chamber.
 Easy visualization of separated components.
 It is having good reproducibility and repeatability.
 It is analytical technique is important for validation of product, quality
control studies of product.
 It is important for qualitative and quantitative analysis. It is used for
both analytical and preparative purpose.

6. Chromatographic separations, including HPLC operate under
the same basic principle-
Every compound interacts with other chemical species in a
characteristic manner.
Chromatography separates a sample into its constituent parts
because of the difference in the relative affinities of different
molecules for the mobile phase and the stationary phase used
in the separation.
AFFINITIES FOR MOBILE AND STATIONARY
PHASES

7. RETENTION FACTOR
• Since Kc is a factor that is wholly dependent on a particular column and
solvent flow rate, a quantitative measure of the affinity of a compound
for a particular set of mobile and stationary phases that does not
depend on the column geometry is useful. The retention factor, k, can
be derived from Kc and is independent of the column size and the
solvent flow rate.
• The retention factor is calculated by multiplying the distribution
constant by the volume of stationary phase in the column and dividing
by the volume of mobile phase in the column

8. 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.
 Adsorption: In Between Normal and Reversed. Separation of
moderately polar analytes using adsorption onto a pure stationary
phase (e.g. alumina or silica)
 Ion Chromatography: Separation of organic and inorganic ions by their
partitioning onto ionic stationary phases bonded to a solid support.
 Size Exclusion Chromatography: Separation of large molecules based
in the paths they take through a “maze” of tunnels in the stationary
phase.

10. HPLC USED FOR -
• SEPARATION AND ANALYSIS OF NON-VOLATILE OR THERMALLY-
UNSTABLE COMPOUNDS.
• HPLC IS OPTIMUM FOR THE SEPARATION OF CHEMICAL AND
BIOLOGICAL COMPOUNDS THAT ARE NON-VOLATILE.
Typical non-volatile compounds are:
o Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)
o Salts like sodium chloride and potassium phosphate
o Organic chemicals like polymers (e.g. polystyrene, polyethylene)
o Heavy hydrocarbons like asphalt or motor oil
o Many natural products such as ginseng, herbal medicines, plant
extracts
o Thermally unstable compounds such as trinitrotoluene (TNT), enzymes
NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline,
etc.), gas chromatography is a better separation technique.

11. INSTRUMENTATION-

12. SOLVENT RESERVOIR-
 Glass or stainless-steel containers capable of holding up to 1 liter
mobile phase (pure organic solvents or aqueous solutions of salts and
buffers)
 Inert to a variety of aqueous and non aqueous mobile phases.
 Stainless steel should be avoided for use with solvents containing
halide ions

14. DEGASSING & FILTRATION OF MOBILE PHASE
 In many cases, aqueous solvents & some organic solvents are
degassed prior to use.
 Degassing is done to prevent formation of gas bubbles in the pump or
detector ( Mobile phases are degassed by stirring of the mobile phase
under vacuum, sonication or sparing with helium gas)
 The mobile phase are filtered to remove particulate matter that may
clog the system
Tubing
 Should be inert.
 Have ability to withstand pressure
 Able to carry sufficient volume

15. PUMPS-
 The solvents or mobile phase must be passed through a column at
high pressures at up to 6000 psi(lb/in²) or 414 bar.
 As the particle size of stationary phase is smaller (5 to 10µ) the
resistance to the flow of solvent will be high.
 That is, smaller the particle size of the stationary phase the greater is
the resistance to the flow of solvents.
 Hence high pressure is recommended.

16. REQUIREMENTS OF PUMPS
 Generation of pressure of about 6000 psi.
 Pulse free output & all materials in the pump should be chemically
resistant to solvents.
 Flow rates ranging from 0.1 to 10 mL/min.
 Pumps should be capable of taking the solvent from a single reservoir or
more than one reservoir containing different solvents simultaneously.
Types of pumps
1. DISPLACEMENT PUMP
2. PNEUMATIC PUMPS
3. RECEPROCATING PUMPS

17. DISPLACEMENT PUMPS
 It consists of large, syringe like chambers equipped with a plunger
activated by a screw driven mechanism powered by a stepping motor.
 So it is also called as Screw Driven Syringe Type Pump.
 Advantages:- It produces a flow that tends to be independent of
viscosity & back pressure.
 Disadvantages:- It has a limited solvent capacity(~250) & considerably
inconvenient when solvents must be changed.

18. RECIPROCATING PUMPS
 This pump transmits alternative pressure to the solvent via a flexible
diaphragm ,which in turn is hydraulically pumped by a reciprocating
pump.
 Disadvantages .
1. Solvent is pumped back and forth by a motor driven piston
2. Two ball check valves which open & close which controls the flow
3. The piston is in direct contact with the solvent
4. Small internal volume 35-400μL
5. High output pressure up to 10,000 psi
6. Ready adaptability to gradient elution and constant flow rate

19.  ADVANTAGES
1. HIGHER OUTPUT PRESSURES UP TO 10,000 PSI.
2. ADAPTABILITY TO GRADIENT ELUTION.
3. LARGE SOLVENT CAPACITIES & CONSTANT FLOW RATES.
4. LARGELY INDEPENDENT OF COLUMN BACK PRESSURE & SOLVENT
VISCOSITY

20. PNEUMATIC PUMPS
 In this pumps, the mobile phase is driven through the column with the
use of pressure produced from a gas cylinder.
 It has limited capacity of solvent .
 Due to solvent viscosity back pressure may develop.

21. PUMP MODULE
• Isocratic pump - delivers constant mobile phase composition. Solvent
must be pre-mixed,lowest cost pump.
• Gradient pump - delivers variable mobile phase composition . It can be
used to mix and deliver an isocratic mobile phase or a gradient mobile
phase
• Binary gradient pump – delivers two solvents
• Quaternary gradient pump – four solvents

22. SAMPLE INJECTOR
Several injector devices are available either for manual or auto injection of the sample.
Manual Injector –
 User manually loads sample into the injector using a syringe
 then turns the handle to inject sample into the flowing mobile phase .
 which transports the sample into the beginning (head) of the column

23. Auto sampler-
 User loads vials filled with sample solution into the auto sampler tray
(100 samples) and the auto sampler automatically measures the
appropriate sample volume, injects the sample, then flushes the
injector to be ready for the next sample, etc., until all sample vials are
process

24. HPLC COLUMNS
 Within the Column is where separation occurs.
 Key Point – Proper choice of column is critical for success in HPLC
 Types of columns in HPLC:
1. Analytical [internal diameter (i.d.) 1.0 - 4.6-mm; lengths 15 – 250 mm] •
2. Preparative (i.d. > 4.6 mm; lengths 50 – 250 mm) •
3. Capillary (i.d. 0.1 - 1.0 mm; various lengths)
4. Nano (i.d. < 0.1 mm, or sometimes stated as < 100 µm)
Materials of construction for the tubing -
 Stainless steel (the most popular; gives high
pressure capabilities)
 Glass (mostly for biomolecules)

25. HPLC COLUMNS PACKING MATERIAL
• Columns are packed with small diameter porous particles –
 The most popular sizes are: 5-μm, 3.5- μm and 1.8-μm
 Columns are packed using high-pressure to ensure that they are stable
during use – most users purchase pre-packed columns to use in their
liquid chromatographs •
 These porous particles in the column usually have a chemically bonded
phase on their surface which interacts with the sample components to
separate them from one another – for example- C18 is a popular bonded
phase
 The process of retention of the sample components (analytes) is
determined by the choice of column packing and the selection of the
mobile phase to push the analytes through the packed column.

26.  Precolumn
1 It contains a packing chemically identical to that in analytical column.
2 Mainly used to remove the impurities from the solvent and thus prevents
contamination of the analytical column, it can protect analytical column.
3 It is also called as guard column or protective column.
4 It is having large particle size.
Analytical column
1 The success or failure of analysis depends upon choice of column and
actual separation is carried out here.
3 Stainless –steel tube ,Size – length -25 to 100 cm ,Internal diameter – 2 to
4.6 mm
4 Column is filled with small particles 5 – 10 micron. The solid support can be
silica gel, alumina.

27. TEMPERATURE CONTROL (WHY IS IT NEEDED?)
 Reproducibility
1. Retention in HPLC is temperature-dependent.
2. If temperature varies, then it is difficult to assign “peaks” to specific
compounds in the chromatogram and the peak areas/heights may
vary.
 Solubility
1. Certain chemical compounds may have low solubility in the HPLC
mobile phase.
2. If they are injected into the flow stream they may precipitate or other
difficulties may arise .
 Stability
1. Certain chemical & biological compounds such as proteins, may not be
stable at room temperature or higher.

28. HOW IS TEMPERATURE CONTROL ACHIEVED?
 Three (3) ways the temperature of a column could be
controlled, use:
1. Oven
2. Heater Block
3. Water bath

29. DETECTORS
THERE ARE MANY DETECTION PRINCIPLES USED TO DETECT THE
COMPOUNDS ELUTING FROM AN HPLC COLUMN.
IDEAL PROPERTIES OF DETECTOR -
 High Sensitivity
 Specificity
 Large linear response range
 Non-Destructive
 Insensitive to temperature & mobile phase
 Continuous operation
 Reliable and easy to use

30. TYPES OF DETECTORS
There are 10 types of detectors-
1. UV, VIS, and PDA Detectors
2. Refractive-Index Detector
3. Evaporative Light Scattering Detector
4. Multi-Angle Light Scattering Detector
5. Mass Spectrometer
6. Conductivity Detector
7. Fluorescence Detector
8. Chemiluminescence Detector
9. Optical Rotation Detector
10. Electro Chemical Detector

31. UV VISIBLE DETECTORS
 UV visible detector is widely used as it detects large number of compounds
because most drugs have appropriate structural characteristics for light
absorption.
 These are useful for aromatic compounds and other type of unsaturated
systems.
These are classified as fixed or variable wavelength detectors-
 Fixed wavelength detectors employ filter as a source to provide appropriate
wavelength.Most common fixed wavelength detectors are based on 254 nm.
 Variable wavelength detectors are employ a spectrophotometer to provide
dispersion of light and selection of any wavelength in UV visible regions.
 Diffraction gratings are frequently used for wavelength dispersion.

32. UV-light with a specific wavelength from a deuterium or quartz lamp in the detector
passes through the flow cell. Followed by detection via a photodiode it is passed
on as an electric signal to the HPLC software. The UV detector constantly measures
the light extinction after transmission through the eluent with the sample
molecules, flowing through two glass plates with defined constant distance, via a
photo meter cell in the flow cell

33. FLUORESCENCE DETECTORS
 It is based on the fluorescent radiation emitted by some compounds.
 The excitation source passes through the flow cell to a photo detector
while a monochromatic measures the emission wavelengths.
 More sensitive and specific.
 The disadvantage is that most compounds are not fluorescent in
nature.
 Fluorescence is a type of luminescence in which the light energy is
released in the form of a photon in nanoseconds to microseconds.

34. REFRACTIVE INDEX DETECTORS
 These bulk property detectors are based on the change of refractive
index of the eluent from the column with respect to pure mobile phase.
 The refractive index detectors suffer from several disadvantages - lack
of high sensitivity, lack of suitability for gradient elution, and the need
for strict temperature control (±0.001 °C) to operate at their highest
sensitivity.
 A pulseless pump or a reciprocating pump equipped with a pulse
dampener, must also be employed. The effect of these limitations may
to some extent be overcome by the use of differential systems in which
the column eluent is compared with a reference flow of pure mobile
phase.

35. DEFLECTION REFRACTOMETER

36. RECORDERS AND INTEGRATORS
 Recorders are used to record responses obtained from the detectors
after amplification, if necessary.
 They record the baseline & all the peaks obtained with respect tot time.
Retention time can be found out from this recordings, but area under
curve cannot be determined.
 The Integrators are improved versions of recorders with some data
processing capabilities.
 They can record the individual peaks with retention time, height, width
of peaks, peak area, percentage area, etc.
 Integrators provides more information on peaks than recorders.
 In recent days computers and printers are used for recording and
processing the obtained data & for controlling several operations.

37. CHROMATOGRAM
This is the chromatogram resulting from the injection of a small volume of liquid
extracted from a Sample was dissolved in an organic solvent. Modern HPLC
separations usually require 10- to 30-minutes each.

38. HPLC IS USED FOR QUANTITATIVE ANALYSIS
 The measurement of the amount of a compound in a sample
(concentration).
 There are two main ways to interpret a chromatogram (i.e. perform
quantification)
1. Determination of the peak height of a chromatographic peak as
measured from the baseline
2. Determination of the peak area.
In order to make a quantitative assessment of the compound, a sample
with a known amount of the compound of interest is injected and its peak
height or peak area is measured. In many cases, there is a linear
relationship between the height or area and the amount of sample

39. APPLICATIONS OF HPLC
 Drug Discovery
 Clinical Analysis
 Forensic Chemistry
 Drug Metabolism study
 Environmental chemistry
 Diagnostic studies
 Cosmetic analysis
 Structural Determination
 Pharmaceutical Applications
 Qualitative and quantitative analysis