Modern analytical System and Bioanalysis

3.  Bioanalysis is crucial in the pharmaceutical industry to measure
the quantitative measure of the drugs or its metabolites for the
study of pharmokinetics,toxicokinetics,bioequivalence and
exposure-response like pharmacodynamic studies.
 Bioanalysis is a progressive discipline for which the future
holds many exciting oppurtunities to further improve
sensitivity,specificity,accuracy,assay throughout,data
quality,data handling and processing, analysis cost, and
environmental impact.
BMV-Bioanalytical Method Validation

4. BMV Guidance to Industry

6. High-performance liquid chromatography (HPLC) has
become a very versatile and powerful separation and
analytical method over the years.
It is an advanced form of liquid chromatography(LC).
Instead of introducing the solvent into the column and
allowing it to drip down under the influence of gravity, in
HPLC, the sample is forced through the column under high
pressures of nearly 400 atm, resulting in faster and more
efficient separation.
This technique is also called High-Pressure Liquid
Chromatography.

7. HPLC-Instrumentation

8. THE COMPONENTS OF HPLC:
 COLUMNS: HPLC columns are normally made of stainless steel and are
50 - 300mm long with an internal diameter of 2 - 5mm. They are filled
with the adsorbents (stationary phase) of particle size 3 – 10µm.
 SAMPLE INJECTOR: The sample is injected into the column by an
injector which is capable of handling sample volumes in the range of 0.1
- 100mL under high pressures of up to 4000psi.

9. RESERVOIR: The solvent or the mobile phase is placed
in a glass reservoir. It is usually a blend of polar and
non-polar liquids whose concentrations depend on the
sample composition.
PUMP: The solvent in the mobile phase is aspirated by
a pump from the reservoir and forced through the
HPLC column and then the detector.
DETECTOR: The detector in an HPLC system is located
at the end of the column and it detects the components
of the sample that elute from the column. Different
types of detectors such as fluorescence, mass-
spectrometric, UV-spectroscopic, and electrochemical
detectors are used.

11. DATA COLLECTION SYSTEMS: The signal from the detector is received
by recorders which are used to process, store, and reproduce
chromatographic data. The data is interpreted and integrated by a
computer that produces a user-friendly chromatograph.

13. The key steps in the HPLC separation technique are as follows:
 Injection of the liquid sample into the column containing the
stationary phase.
 Individual sample components are forced down the tube by high
pressure from the pump.
 Components are separated under the influence of various
chemical/physical interactions with the particles in the stationary
phase.
 The separated analytes are identified by the detector present at the
end of the column.
 The detector measures the concentration of the components.
 Data from the detector is processed and a chromatogram is
produced.
 Investigating a wines character using chromatography.

15. HPLC follows the same basic principle as
chromatography. Different components in the sample
have varying affinities to the adsorbent material. This
causes a difference in the flow rate for each component
which leads to their separation as they come out of the
column. The only difference is that the speed and
sensitivity of HPLC are much higher than that of LC due
to the application of high pressure.
The magnitude of pressure applied depends on several
factors such as the length and diameter of the column,
flow rate, size of particles in the stationary phase, and
mobile phase composition.

17. HPLC is widely used in the following applications:
 QUALITATIVE ANALYSIS - Separation of thermally unstable
chemical and biological compounds, e.g., drugs (aspirin and
ibuprofen), salts (sodium chloride), proteins (egg white or
blood), organic chemicals (polystyrene and polyethylene),
herbal medicines, and plant extracts.
 QUANTITATIVE ANALYSIS - To determine the concentration of
a compound in a sample by measuring the height and area of
the chromatographic peak.
 Preparation of pure substances for clinical and toxicology
studies and in organic synthesis. This is also called
preparative chromatography.
 TRACE ANALYSIS – this is the analysis of compounds
present in very low concentrations in a sample. This is very
useful in pharmaceutical, toxicology, environmental, and
biological studies.