High performance liquid chromatography (HPLC) (1).pdf

High performance liquid chromatography (HPLC) (1).pdf

1. High performance liquid
chromatography (HPLC)
Prepared by : YoumnaHawas

2. Introduction:
•HPLC stands for “ high performance liquid chromatography “ or “high
pressure liquid chromatography “ or “ high priced liquid
chromatography “

3. Definition :
•High performance liquid chromatography is a form of liquid
chromatography used to separate compounds dissolved in a solution.
•HPLC is characterized by using high pressure to push a mobile phase
solution through a column of stationary phase allowing separation of
mixture

4. Principle of HPLC :
•A separation column separates the stationary and mobile phases during
purification.
•The mobile phase is a solvent or solvent combination that is pushed
through the separation column under high pressure.
•The sample is loaded into the mobile flow from the pump to the separation
column using a syringe through a valve
•As a result, owing to interactions with the stationary phase, the
components of a mixture migrate through the column at different speeds.
•Individual compounds are identified by an appropriate detector after
exiting the column and transmitted as a signal to the computer’s HPLC
software.

6. Parts of HPLC:
•Solvent Reservoir : A glass reservoir holds the mobile stage
ingredient.
•Pump: The pump is located in the upper stream of the liquid
chromatographic column and pumps eluent into the system from the
solvent reservoir.
•Injector: Next to the pump, there is an injector. The easiest way is to
use a syringe to insert the sample into the eluent flow.
•Column: The separation takes place within the column. Instead of
glass columns, contemporary columns are frequently manufactured in
a stainless steel housing.

7. Parts of HPlC:
•Detector :The separation of analytestakes place inside the column,
and the separation is seen using a detector. When no analyteis
present, the eluent has a constant composition. While the presence
of analytealters the eluent’s composition. These differences are
measured by the detector. This disparity is measured using an
electrical signal.
•Data Collection

8. Applications of HPLC:
•Analysis of drugs
•Separation and purification of biopolymers such as enzymes or
nucleic acids
•Water purification
•Product purity and quality control of industrial products and fine
chemicals
•Impurity detection in the pharmaceutical industry.

9. Advantages :
•Speed
•Efficiency
•Accuracy

10. Disadvantages :
•Cost: Despite its advantages, HPLC can be costly, requiring large
quantities of expensive organics.
•Complexity
•low sensitivity : HPLC have low sensitivity for certain compounds,
and some cannot be detected as they are irreversibly adsorbed.
•Volatile substances are better separated by gas chromatography.