High Performance Liquid Chromatography (HPLC): Working Principle and Applications --- 1. Introduction High Performance Liquid Chromatography (HPLC) is one of the most powerful and widely used analytical techniques in modern chemistry, biochemistry, pharmacy, and environmental science. It serves as an essential method for separating, identifying, and quantifying components in complex mixtures. Compared to traditional column chromatography, HPLC offers high resolution, faster analysis, improved sensitivity, and reproducible results. HPLC became popular in the 1970s with advancements in pump technology, particle size reduction, and detector design. Its core strength lies in the ability to separate analytes based on their interactions with a stationary phase and a liquid mobile phase under high pressure. The method is applicable to both qualitative and quantitative analysis of compounds ranging from small organic molecules to large biomolecules like proteins and nucleic acids. This essay explores in detail the working principle of HPLC, its major components, types, modes of separation, and its wide-ranging applications in scientific and industrial fields. --- 2. Basic Principle of HPLC The fundamental principle of HPLC is the differential distribution of solutes between two phases — a stationary phase and a mobile phase. The stationary phase is a solid or liquid supported on a solid matrix packed inside a column, while the mobile phase is a liquid solvent (or mixture of solvents) that moves through the column under high pressure. When a mixture of compounds is injected into the mobile phase and passed through the stationary phase, each component interacts differently with the stationary and mobile phases. Substances that interact strongly with the stationary phase move more slowly through the column, while those with weaker interactions elute faster. This difference in migration times leads to separation of the mixture into distinct peaks, which are detected and quantified. The process is governed by parameters such as: Partition coefficient (K): The ratio of concentrations of a compound in stationary and mobile phases. Retention time (tₑ): The time taken for an analyte to elute from the column. Selectivity and efficiency: Determine how well two compounds can be resolved. Thus, HPLC separation is based on the principles of adsorption, partition, ion exchange, or size exclusion, depending on the type of stationary phase and the chemical nature of analytes. --- 3. Major Components of HPLC System An HPLC instrument consists of several integrated components that work in harmony to achieve precise and efficient separations. 3.1. Solvent Reservoirs The solvents or mobile phase are stored in reservoirs made of glass or stainless steel. The composition of the mobile phase depends on the type of separation — for example, polar or nonpolar solvents for normal-phase or reverse-phase chromatography, respectively. Solvents are filtered and

1. Anticoagulant
● When Blood is collected, it clots after sometime. The
anticoagulants are the chemical agents which prevent the
clotting of Blood when mixed with Blood in proper
proportion.
● Purpose of using anticoagulants:
- For study of various constituents of Blood components.
- Study of coagulation(clotting of Blood).
- Preservation of Blood in Blood Bank.
● Properties of anticoagulant
- It must be soluble in Blood.
- It must be keep the blood in fluid condition,
- It must not be bring haemolysis of Blood cells.
- It must not be change the size of RBC.
- It must minimize destruction of leukocytes.
- It must minimize aggregation of platelets.

2. Coagulation(clotting)
mechanism
Thromboplastin
Calcium

4. Classification
of
anticoagula
nt
1.Calcium
chelator
Bind with Calcium
● Oxalates
It is following forms:-
-Ammonium Oxalate
-Potassium Oxalate
- Double Oxalate
● EDTA
(Ethylenediaminetetraacetiacid)
● Tri-sodium citrate etc.
2. Non-calcium chelator
Do not bind with
Calcium
● Heparin
● Warfarin etc.
Most of the anticoagulants used in laboratory act by binding with calcium
and prevent clotting of blood since calcium ion is essential for many of the
steps in coagulation mechanism.

5. Oxalate
s
● Ammonium oxalate:-
This is used at a concentration of 2 mg for 1 ml of
Blood.
This anticoagulant causes swelling of the RBC therefore, it is not
recommended for use with blood for PCV
,ESR and cell
morphology.
● Potassium oxalate:-
It is used at a concentration of 2 mg for 1 ml of Blood.
This anticoagulant is most often used for chemical analysis. It
causes shrinkage of RBC therefore it is also is not recommended for
the study of PCV
,ESR and other cell morphology.
Double oxalate:-
Ammonium oxalate and Potassium oxalate are combined
together to balance the swelling effect of Ammonium oxalate and
the shrinking effect of Potassium oxalate on the red cells. It is the
mixture of 3 parts of Ammonium oxalate and 2 parts of
Potassium oxalate which is prepared as follows:-
o Ammonium
oxalate
o Potassium oxalate
2.4
gram
1.6

6. E.D.T.A.(Ethylenediamine tetra-acetic
acid)
● It is the most commonly used anticoagulant in
Haematology lab because E.D.T.A. is the most
powerful calcium chelating (binding) agent we have.
It gives the best preservation to the cell morphology
therefore E.D.T.A. is the preferred anticoagulant
for all cell count and blood smear preparation.
E.D.T.A. is used in two different forms:-
● Di-sodium E.D.T.A. salt (Versene)
● Di-potassium E.D.T.A. salt (Sequestrene)

7. Preparation of E.D.T.A vials (bulb) in your
laboratory
4 gm/dl of this solution is used to prepare EDTA
bulbs.
20µl of this solution is dried per bottle which contains 0.8 mg
of dry chemical and prevents coagulation of 1 to 2 ml blood.
Required materials
1. Physical balance
2. EDTA powder
3. Distilled water
4. Conical flask
5. Measuring cylinder
6. Micropipette (20µl capacity)
7. Spatula
8. Hot air oven
9. Vials

8. e
1. Keep all the required materials in the working table.
2. Put the physical balance in plane surface and balanced it properly.
3. Put the equal sizes of paper pieces in both sides of pan and balance
it again.
4. Put 4 gm weight in one side of the pan.
5. Put EDTA powder on the other side of the pan slowly till it
shows equals to the 4 gm weight.
6. Put the EDTA powder in the conical flask
7. Measure100 ml of distilled water by using measuring cylinder,
pour into the conical flask containing EDTA powder and mix
well.( Heat can be applied for proper mixing of the solution).
8. Pipette 20µl of anticoagulant solution in each clear and dry vials.
9. Put the vials containing anticoagulant solution in the hot air oven
at the temperature of 60oC for 30 minutes.
10. After drying EDTA powder remain attached at the bottom of
the vials. Store the EDTA vials by putting lid over it and keep in
safe place which can be used for 1-2 ml of blood sample.

9. ● Calculation
4 gm% EDTA solution
in 100 ml of distilled water (D.W.)= 4 gm of EDTA
in (100 x 1000)µl ofD.W.= (4 x 1000) mg of
EDTA
4000
1 ,, ,, = —— mg
100000
4
20 µl ,, ,, = —— x 20 mg
100
= 0.8 mg of
EDTA.

10. Excess
of
EDTA
● Excess of EDTA affects both red blood
cells and leukocytes causing shrinkage
and degenerative changes.
● Excess of EDTA ( in case of 2 mg/ml)
may
cause
significant
volume(PCV)
and
decrease
increas
e
in packed
cell in
mean cell
haemoglobin concentration (MCHC).
● Platelets swell and disintegrate due to
the excess of EDTA and artificially high
platelets count may be obtained due to
disintegrated platelets.

11. Tri-sodium
citrate
● It is used as a liquid form.
● It binds with calcium.
● The concentration of Tri- sodium citrate is used as
3.8%.
● For PT (Prothrombin Time) a kind of coagulation test,
3.8% Tri-Sodium citrate is used at the ratio of 1:9 i.e. 1
part of anticoagulant and 9 part of Blood.( 200 µl of
3.8% Tri-Sodium citrate and 1.8 ml of Blood is used.)
● Fro ESR (Erythrocyte Sedimentation Rate)
by Westerngreen method
1 part of 3.8% Tri-Sodium citrate is mixed with 4 part
of Blood that is the ratio of 1:4.( 400 µl of 3.8% Tri-
Sodium citrate and 1.6 ml of Blood is mixed).
It do not preserve the cell morphology.

12. Heparin
● It is a natural anticoagulant and
is normally present in the blood in
small amount and highly acidic. This is
the best anticoagulant for open heart
surgery and it causes minimum
haemolysis.
● It is very expensive.
● It produce black back ground in
the smear so it is not use for
smear preparation.

13. ● Anticoagulant used in Blood Bank
● ACD (Acid Citrate Dextrose)
● CPD ( Citrate Phosphate Dextrose )
● CPDA (Citrate Phosphate Dextrose
Adenine)

14. THANK YOU