HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

05-MARCH-2019 CHROMATOGRAPHY TECHNIQUE:

 Basics of Chromatography: Separation, Isolation, Identification
and purification
 Examples : Column chromatography, Gas chromatography,
Paper chromatography, Liquid chromatography, TLC etc
 Column chromatography classify on the basis of packaging
material/stationary phase
 Major types: Open column and Closed column
 Two phases are required for chromatography: stationary phase
& mobile phase
 Major differences between open and closed column
 General intro about HPLC
12-MARCH-2019 HPLC:
 Developed in late 1960’s & early 1970’s
 Major Principle: Adsorption, Partition, Ion exchange, Size
exclusion
 Difference between Open and Closed column: Highly accurate,
precise, and sensitive, fast separation, High resolution
 Other differences about Open and Closed column about column
and working
INSTRUMENTATION:
 Mode of operation:
o Isocratic elusion: use same (ratio; fix) solvent throughout
the working (fixed solvent)
o Gradient elusion: use different (ratio; different) solvent
throughout the working (gradient solvent)
 Requirement for running HPLC:
o HPLC grade (highest grade) or Analytical grade solvent
o Filtration must be required under reduce pressure
o Sp. pH maintained
 If you require basic solvent system, add few drops
of 10% liq. NH3
 If you require acidic solvent system, add few
drops of dil. orthophosphoric acid
o Pass through sonicator to remove bubbles
 Ready HPLC for study:
o Firstly wash (by run the HPLC under pressure) with
compatible solvent with the previously studied sample
(either water or organic solvent)
o Then wash (under pressure) with methanol/ethanol to
prevent mould growth in it (before and after study)
o Prepare solvent system for the study and saturate the
column with solvent
13-MARCH-2019  Elusion : means to comes out
  Flow rate: to travel solvent in the column per min
o For Analytical column: 0.5-2 ml/min
o For Microborne column: 10-100ul/min
o For Semi-preparative and Preparative column:
2-10ml/min
 Pressure: it is the main indicator of your working condition
o If pressure drops suddenly it means working is disturb:
flow leakage
 We use tissue for detect leakage and then wrap
HPLC tape around it
o If pressure increases suddenly it means we have to stop
working: blockage/choking occur
 We have to run solvent ( either the same used in
working or different from that) under high
pressure
 Pump: provide a constant flow rate and pressure
o Single piston pump: one piston is used for both firstly
sucking and then pump the solvent to the column
 Issue: This will create problem in the signalling in
the form of noises/pulsation.
 Solution: This can be avoided by connecting
dumping device which will reduce the noises
(pulsation) and create proper signals
o Double piston pump: In this one piston is responsible for
sucking the solvent while other is for pumping this will
occur simultaneously avoid gaps and noises in the signals.
 It will highly useful for the flow sensitive detector
like photometric or refractive index detector.
14-MARCH-2019  Injections: These are for inject the sample in the HPLC
apparatus
o There are two major modes of injection
 On the column
 On preload system or pre-column
 For the above two system two syringes are used:
o With high pressure syringes: it can be inserted in the
column while running of the solvent
o With low pressure syringes: it can be inserted after
stopping the running of the solvent
 Now a day, modern injection systems are used in which there is
the injection port, a loop, a lever.
o Working: Firstly you have to insert the syringe in the
injection port, then move the lever to 60o toward left and
then inject the sample in the loop (loop capacity is 20ul if
excess solution is inserted it will flushed out of the loop
not get into the column. Then move the lever back to the
60o right and eject out the syringe from the port.
 There are two further techniques for the use of modern
 injection.
o Full filling of syringes: if the syringes are completely filled
this will increase the highest reproducibility of the results
and capable of giving relative standard deviation of less
than 0.2%
o Partial filling of syringes: if the syringes are being partial
filled. With the proper handling and procedure it give
precise results of about relative std deviation of about 1%
19-MARCH-2019  Detectors: There are two major types of detector based on
property it determined.
o Solute property: it is the property of the individual
components of the sample. e.g. ƛmax, flourescence
o Bulk property: it is the property of the solution rather
than of individual components e.g. refractive index
o Characteristics of HPLC Detector:
 Should respond to specific property like
fluorescence of the compound.
 Should be sensitive to at least 10-8g/ml.
 Give linear response to a wide range of
concentration.
 Should have low dead volume.
o Solute property detector:
 Photometric detector: UV Visible
spectrophotometer. It detects the ƛmax of the
component isolated from the sample. It of five
types:
 Single wavelength detector: equipped
with low pressure mercury discharge lamp
provide wavelength of 254nm
 Multi wavelength detector: employ with
mercury discharge lamp with combination
of interference filter which allow the
wavelength of 206, 226, 280, 313, 340 or
365nm
 Both above detectors are stable
 Variable wavelength detector: equipped
by deuterium lamp provide variable no. Of
wavelength within the range of 200-
360nm. Used for the unknown compound
whose absorption wavelength is unknown
 Programmable detector: automatic
changes of wavelength between and
during the analysis can be done.
 Diode array detector: are microprocessor
controlled photodiode in which the
provided light from UV source is passed
through the flow cell then dispersed by
 polychromator, then falls on array of diode
 Precautions:
 Analytical grade solvent must be used
 Solvent should be used on the basis of
their cutoff point (solvent should be inert
in the region of interest)
26-MARCH-2019  Fluorescence detector: They are equipped either
with filters (fluorimeters) or grating monochromators
(spectrofluorimeter), more sensitive than
photometric detector detect fluorescence of the the
fluoriscents compound.
 Electrochemical detector: They are based on the std
electrochemical principle involve amperometry,
voltammetry, and polarigraphy. Majorily they are
sensitive to those substances which undergo
Reduction or Oxidation reaction at suitable potential
like biological molecules.
o Bulk property detector:
 Refractive index detector: It detects the difference of
refractive index of solvent and solution. It is the
universal detector. Although they are highly flow
sensitive but the sensitivity for the conc. is either less
than solute property detector.
 Columns: Classification of column is based on flow rates as
already discussed above, and size and diameter and packaging
material discussed below:
 Size and diameter basis:
Column Length Diameter Particle Function
size
Analytical 10-30cm 4-5mm 3-10um Simple
column analysis
Short column 3-6cm 4-5mm 3 or 5um For better
efficiency
by
theoretical
plates(N)
simple
analysis
Microborne 10-25cm 1-2mm LOD for less
column quantity
separation
used less
solvent
Semipreparative -- 7-10mm For
column isolation of
multiple
component
 on large
scale
Preparative -- 20-40mm For only
column one drug
separation
on large
scale
27-MARCH-2019  Packaging Material basis:
o Adsorption HPLC:
 Stationary phase: Unmodified silica is used for
packaging material to avoid intramolecular spaces
in between particles. Silica has capacity of 1-
5mg/g to adsorb.
o Particle size must be less than 10um although large
particle size about 70um was used anciently in 1970 or
before while still used in guard column now days.
o Advantages of advancement by using small particle size:
1. Separation efficiency is high
2. Separation performance is highly improved by maintain
retention time, AUC, and theoretical plates
3. Retention time is improved for the proper separation
 Mobile phase: Mobile phase may be used as a
less polar solvent either entirely organic, a single
binary solvent as ethanol or mixture of solvent of
correct polarity according to the requirement
 Working principle: As adsorption principle the
sample component are bind with silica by silanol
(OH) grp according to their polarity
 Elution pattern: Less polar may be elute out first
or more polar elute out last

28-MARCH-2019 o Partition HPLC: Normal phase partition HPLC

 Stationary phase: Before the advancement the
stationary phase used is ethan1,2diol coated on the
silica which is used as a support but it is seen that the
solvent coating is washout during the procedure
under pressure.
So now silica is chemically treated with the less polar
compound like lichrosorb diol, Bondpak CN, polygosil
NH3 the order of polarity increases as diol<CN<NH3 to
make the phase more polar
 Mobile phase: Mobile phase should be less polar
than stationary phase
 Working principle: as partition as a main principle the
sample component attached to the stationary phase
by siloxane linkage
 Elution order: least polar elute out first or most polar
 in last

2-APRIL-2019 o Partition HPLC: Reversed phase partition HPLC

 Stationary phase: The stationary phase used in
reversed phase is silica as a support is treated with
organochlorosilicane (organo grp may be hexyl or
octyl or octadecyl /C6 or C8 or C18) and for untreated
silanol groups it is further capped with trimethyl
chlorosilicane. This treatment is done to make the
silica non polar.
 Mobile phase: The mobile phase is used as polar
solvent like water with less polar organic solvent
modifier as ethanol or acetonitrile. The mobile phase
is more polar as compared to stationary phase. The
organic solvent modifier is added to improve the rate
of elusion.
 Working principle: The elution pattern is based on
hydrophobicity of sample components.
 Elusion pattern: The most polar component elute out
first or least polar at last.
o Modification in Partition HPLC:
 Ionization suppression: To avoid ionization of the
sample the pH of solvent is adjusted according to the
nature of sample as for acidic drug the pH of the
solvent should be acidic to avoid ionizing sample and
vice versa. By this modification, the polarity is
reduced and the retention time is increases of the
non-ionized form.
 Counter ion effect: It is used in ion pair reversed
phase HPLC. Through this the counter ion is added to
the mobile phase which form a ion pair with the
solute ions this will render to be more hydrophobic to
be retained on the stationary phase for a short time
period.
3-APRIL-2019 o Ion exchange HPLC:
 Mobile phase: Mostly aqueous medium is used
although bonded mixture of aq. Buffers and organic
solvent is used if sample is less water soluble
 Stationary phase: Based on cross linked polystyrene-
divinylbenzene resin or ion exchange residue
chemically bonded to silica. The strong cation
exchanger containing sulphonate (e.g sulphopropyl)
group or strong anion exchanger containing
tetraalkylammonium (e.g. N-propyl-N-N-N-
trimethylammonium) groups
 Elution: Retention can be controlled by ion strength
or pH of the mobile phase
 o Size exclusion HPLC:
 Stationary phase: Based on cross linked polystyrene-
divinylbenzene resin or silica microspheres (5-15um
in diameter) are used to fractionate material of high
molecular wt. The high resolution given by the small
particles allows the separation of closer molecular
mass than classical gel chromatography where
Sephradex is use as stationary phase.
 Mobile phase: Single aq. or organic solvent is used
o Special techniques:
 Chemical derivatisation: To improve the selectivity
and/or sensitivity of the analysis we should convert
the substance into its derivative
It may be pre column or post column derivatisation
Pre column derivatisation is used to improve the
chromatographic performance (resolution or peak
symmetry) to increase the stability of labile solutes or
alter the retention time of solutes
Post column derivatisation is used to improve the
sensitivity of the detection of the solutes.
Example are to convert carboxlic acids, phenols,
amines or carbonyl compound to their dansyl (5-N,N-
dimethylamino-napthalene-1-sulphonyl) derivatives
to their detection through photometric of
fluorescence detector
 Peak identification and purity: It can be achieved by
connecting photometric or mass spectrophotometer.
The separation in peaks is detected to be pure,
impure or degradation in the sample components like

APPLICATIONS OF HPLC:
o General Applications:
1. The wide variety of packaging material allows the
separation most chemical compound. Majorily
used stationary phase is silica for adsorption and
octyl silyl or octadecyl silyl bonded with silica for
reversed partition while ion exchange and size
 exclusion have limited number of application
2. Different types of detectors available permit the
sensitive detection of most chemical types,
quantifies with highest accuracy and precision.
3. Microparticulate packaging material give highly
resolution peaks of similar substances this is
achieved by number of theoretical plates(N) in
the column (5000-10000)
4. The short column (3-10cm) in routine use allow
fast separation achieved within few minutes.
5. A combination of HPLC and spectrometric
technique (e.g UV, IR, mass spectrometer) allows
the quantitation and identification
simultaneously.
o Qualitative and Quantitative analysis:
1. HPLC is used in routinely quality control testing of
drugs and medication for compliance with
specification, stability studies, therapeutic
monitoring, drug metabolism studies, and
pharmacokinetic studies.
2. It is specifically useful in providing compound
specific assays e.g Oestradiol Benzoic Injection
and in the separation and control of impurities
e.g. related substances of Flurbiprofen
3. HPLC assays are used increasingly for assay of
natural medicinal product such as antibiotics and
hormones.
4. HPLC methods also effective in the separation of
geometrical isomers and racemates.