HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY
Introduction and Theory: It is called as high
performance liquid chromatography due to its high
performance when compare to classical column
chromatography. It is also high pressure liquid
chromatography as high pressure is used when
compared to classical column chromatography.
The development of HPLC attributed to the
development of smaller particle size. Smaller
particle size is important since they offer more
surface area
INSTRUMENTATION
 Pumps-These are used as the solvent delivery
system
 Mixing unit, gradient controller and solvent
degassing
 Injector- manual or auto injector type are used
 Guard column
 Analytical column
 Detectors
 Recorders and integrators.
Pump- The solvents or the mobile phase used
must pass at a high pressure at about 1000 to
3000psl due to the less particle size of the
stationary phase (5-10µ). The resistant to the
flow of solvent is high. So, such high pressure is
required
There are different types of pumps available like
mechanical and pneumatic pumps. Mechanical
pumps operate with constant flow rate using a
sapphire piston. These are used in analytical
scale.
The pneumatic pump operate with constant
pressure and uses highly compressed gas.
Solvent of high purity are used with HPLC
grade filtered through 0.45µ filter.
Check valves: These are used to control the
back pressure and flow rate of solvents.
Pulse dampners: These are used to dampen the
pulses obtained from the wavy baseline caused
by the pumps.
Mixing unit: These are used for mixing the
solvents at different proportions and pass
through the column
There are two types of mixing units like the low
pressure mixing unit chamber which uses helium
for degassing and a high pressure mixing unit
chamber which do not use helium for degassing
solvents
The process of mixing of solvents may be carried
by
# Static mixer packed with beads
# Dynamic mixer using magnetic stirrer and
operates at high pressure
Gradient Controller: In isocratic separation
solvents of same eluting power or polarity are
used. But, in gradient elution technique the
polarity of the solvent is gradually increased
so, the solvent composition is changed.
Gradient controller is used when two or more
solvent pumps are used for the separation.
Solvent degassing: Many types of gases are
soluble in in organic solvents which should be
removed or else they may interfere with the
Separation, steady base line or shape of the
peak
There are three types of techniques that are
employed for degassing:
• Vacuum filtration: It is used to remove air
bubbles , but it is not reliable and complete
technique.
• Helium purging: It is done by passing helium
through the solvent, helium is expensive
• Ultra sonication: It is employed using
ultrasonicator which converts ultra high
frequency to mechanical vibrations, causing
removal of air bubbles.
Injection systems (manual or auto ) injectors:
There are different types of injection devices
available like
Septum injectors: This is done by injecting the
sample through a rubber septum. The septum
has to with stand high pressure, so this is not
commonly used technique.
Stop flow/on line: This is done by stopping the
flow of the mobile phase for a while and the
sample is injected through valve device.
Rheodyne injector (loop valve type):It is the
most popular type which has fixed volume like
20µl or 50µl or more. This injector has two
modes.
Load position: The sample is loaded in the loop
Inject mode : The sample is injected
Column: These are two types
Guard column: It improves the life of analytical
columns as it has very small amount of
adsorbent. It does not helps in separation but
acts as a prefilter to remove particulate matters.
• Analytical column: This part is important and
decides the efficiency of separation.
Depending on techniques and mode of
separation used there are several stationary
phases available.

• Column material: made of stainless steel

which is widely used as it can withstand high
pressure. Glass, polyethylene and PEEK (poly
ether ketone) which is the latest one in use.
• Column length: Varies from 5cm to 30cm.
• Column diameter: 2mm to 50mm
• Particle size: 1µ to 20µ
• Particle nature: Spherical, uniform and porous.
• Surface area: 1g of stationary phase gives 100-
860sq.m with average of 400sq.m of surface
area.
• Functional group: It reflects the type of
chromatographic separation.
• Silanol group(hydroxy group) in normal phase.
In reverse phase mode:
C18-Octa Decyl silane (ODS) column
C8- Octyl column
C4- Butyl column
CN – Nitrile column
NH2- Amino column
Ion exchange column, gel columns, chiral
columns, affinity columns etc are also available.
Detectors: Depending on the compounds to be
separated different types of detectors are used.
Different types of detectors available are:
UV detector: There are two types of detectors
available in this model they are
Fixed wavelength detector: This is operated at
254nm which is absorbed by the most of the
drugs .
Variable wavelength detector: This is operated at
190nm to 600nm.
Both these detectors are based upon the light
absorption characteristics of sample compound.
Refractive index detector: This is having low
sensitivity and low specificity due to which it
has got no analytical applications. But it is a
non-specific or universal detector.
Flourimetric detector: This detector is based on
the fluorescent radiation emitted by some class
of compounds. This is with more specificity
and sensitivity. This is a type of detector in
which exitation and emission wavelength can
be selected for each compound.
Demerit: Non flourescent compounds cannot be
used.
Conductivity detector: The response is based
upon the electrical conductivity. This is used
only when the sample is having cations and
anions as conducting ions.
Amperometric detector: This is based on the
reduction or oxidation of the compounds on
the application of external potential. The
recorded diffusion current is proportional to
the concentration of the compound eluted.
This is highly sensitive detector.
This is used when compounds have functional
groups that can be oxidised or reduced
Photodiode array detector: This is similar to UV
detector. It is a recent detector operating
from190nm- 600nm. Radiations of all the
wavelengths fall on the detector simultaneously.
This gives a 3- dimensional plot response Vs
time Vs wavelength.
Merits: The wavelength need not be selected, but
the detector detects the response of all the
compounds.
Data systems: There are different data systems to manage
the data collected.
Recorders: These are used to record the responses obtained
from detectors after amplification. They record the base
line and all the peaks if necessary with respect to time.
Merit-Retention time of all peaks can be found out.
Demerits: Area of individual peaks cannot be known.
Integrators: These are improved version of recorders with
advanced data processing capabilities. They can record
the individual peaks with retention time, height and width
of the peaks, peak area, percentage of peak area etc.
 Integrators provide more information on peaks
than the recorders. In the current trend
computers, printers are used for recording and
processing the obtained data.

Stationary phase and mobile phase:

Normal phase mode: In this case the stationary
phase is polar and the mobile phase is no-polar
in nature. In this technique the non-polar
compounds travel faster and elute first due to
less affinity between the solute and the
stationary phase.
The polar compounds are retained for longer
time due to more affinity towards the
stationary phase and elutes slowly. It is not
used in pharmaceutical applications since most
of the drug molecules are polar in nature and
takes longer time to elute and detect.
Reverse phase mode: In this case a non-polar
stationary phase and a polar mobile is used
hence polar components are eluted first and
non-polar components are eluted later. Since
most of the drugs and pharmaceuticals are
polar this mode is used.
Structural factors governing the rate of elution of
the compounds
The rate of elution of a compound depends on the
column efficiency. The factors effecting the
column efficiency are give as:
Dimensions of the column: The ratio of length and
diameter for a column should be monitored for
the best results.
The ratio is given as length: diameter is 20:1 or
30:1 are ideal 100:1 is satisfactory
Nature of solvent: The flow rate of solvent is
affected by its viscosity. Less viscous solvents are
efficient than more viscous solvents. Hence, flow
rate is inversely proportional to viscosity.
Pressure: High pressure above and low pressure
below the column will increase the efficiency of
the column.
The high pressure above is maintained by using a
column of liquid at top or maintaining pressure
devises like pumps. Pressure below the column is
decreased by applying vacuum using vacuum
pumps.
Particle size of the adsorbent: Adsorbent activity is
based on the surface area of the adsorbent. For
increasing the surface area, particle size can be
reduced and hence the adsorbent activity
increases.
Temperature of the column: The elution speed
increases at higher temperature. But, the
adsorption decreases. Normally room temperature
is used for all the samples only complex samples
are separated at higher temperature.
Evaluation of column performance: It is done
by different parameters like
Retention time
Retention volume
Separation factor
Resolution
Theoretical plates
HETP-Height equivalent to theoretical plates.
Asymmetry factor
Retention time: It is the difference of time between the
point of injection and appearance of peak maxim.
Retention volume:retention volume is proportional
to retention time, based on the constant flow rate of a
chromatography system. If the retention time is 5 min
and the volume flow rate (uv) is 5 mL/min, then Vr is 25
mL
Separation factor:
The selectivity (or separation) factor (α) is the ability of
the chromatographic system to 'chemically' distinguish
between sample components. It is usually measured as a
ratio of the retention (capacity) factors (k) of the two
peaks
Resolution:The resolution of a elution is a quantitative measure of
how well two elution peaks can be differentiated in a
chromatographic separation.
Theoretical plates :A theoretical plate in many separation processes
is a hypothetical zone or stage in which two phases, such as the
liquid and vapor phases of a substance, establish an equilibrium
with each other.
HETP: HETP is an acronym for the Height Equivalent to the
Theoretical Plate. The HETP is the theoretical link between the
Plate Theory and the Rate Theory as the HETP is numerically
equal to the variance per unit length of the column as determined
from the Rate Theory.
Asymmetry factor: Asymmetry factor. The asymmetry factor is a
measure of peak tailing. It is defined as the distance from the
center line of the peak to the back slope divided by the distance
from the center line of the peak to the front slope, with all
measurements made at 10% of the maximum peak height.
 Applications of HPLC
 HPLC is routinely used for both qualitative and quantitative
analysis of environmental, pharmaceutical, industrial forensic,
clinical, and consumer product samples.
 Separation and analysis of non-volatile or thermally-unstable
compounds
 Typical non-volatile compounds are:
Pharmaceuticals like aspirin, ibuprofen, or
acetaminophen (Tylenol)
Salts like sodium chloride and potassium phosphate
Proteins like egg white or blood protein
Organic chemicals like polymers (e.g. polystyrene,
polyethylene)
Heavy hydrocarbons like asphalt or motor oil
Thermally unstable compounds such as trinitrotoluene
(TNT), enzymes
Applications of HPLC
 Preparation of Pure Compound(s)
 By collecting the chromatographic peaks at the exit of the
detector, and concentrating the compound (analyte) by
removing/evaporating the solvent,
 A pure substance can be prepared for later use (e.g. organic
synthesis, clinical studies, toxicology studies, etc.).