Chromatography is a laboratory technique used to separate mixtures by dissolving them in a mobile phase that carries the mixture through a structure containing a stationary phase. There are 12 types of chromatography depending on the physical state of the mobile phase, shape of the bed, and separation mechanism. High-performance liquid chromatography (HPLC) is commonly used and separates mixtures by polarity, molecular size, or ionic charge using a pump, injector, analytical column, and detector. HPLC is used in pharmaceutical, industrial, and research applications such as drug analysis, quality control, and quantification of compounds.

1. Sohaib Umar
Ammar Hassan Jiskani
Muhammad Umar

2. o Chromatography
o Types of Chromatography
o HPLC and its Types
o Instrumentation
o Working
o Chromatogram
o Application and Uses

3. o Chromatography is a laboratory technique for the
separation of a mixture. The mixture is dissolved in a
fluid called the mobile phase, which carries it through a
structure holding another material called the stationary
phase.

4. o Chromatography is derived from Greek words Chroma
means “color” and graphein means “to write”.
o It was first employed in Russia by the Italian-born
scientist Mikhail Tsvet in 1900.
o Nobel prize in chemistry in 1952 was awarded to
Archer Martin and Richard Synge.

5. o Analyte:
The substance to be separated during chromatography.
o Stationary phase:
The substance on which adsorption of analyte takes place.
o Mobile Phase:
It is the solvent which carries the analyte.

6. o There are around 12 different types of
chromatographic technique depending upon:
o The physical state of the mobile phase.
o The shape of chromatographic bed.
o The separation mechanism.

7. o High-performance liquid chromatography
(HPLC) is an analytical technique to separate, identify
and quantify each component of a mixture.
o It is a modern application of liquid chromatography
which guarantees a high sensitivity.

8. o There are some variants of HPLC, depending upon
the phase system (stationary) in the process:
1. Normal Phase HPLC
2. Reverse Phase HPLC
3. Size-exclusion HPLC
4. Ion exchange HPLC

9. It separates the analyte on the basis of polarity.

10. It is reversible of normal phase HPLC.

11. The column is filled with material having precisely
controlled pore sizes and particles are separated according to
their molecular size.

12. The stationary phase has an ionically charged surface of
opposite charge to the sample ions.

13. o HPLC can be categorized into 2 parts.
o Components of HPLC
o Working of HPLC

14. o It consist of following components :
o Solvent reservoir
o Degasser
o Solvent mixing valve
o HPLC pump
o Pre column or guard column
o Sample injector
o Analytical column
o Detector

15. Solvent reservoir
It is used to store the mobile phase
Types of solvent reservoir:
o Binary system
o Quaternary system
o High grade solvent are used.

16. o Degasser
o Its is used to remove the gasses which are dissolve in
our mobile phase or in our solvent
o Degassing is done by using vacuum pump

17. o Solvent mixing valve
o Used for the mixing of solvent from the reservoir
o All the solvent run through a pipe
o Diameter of pipe is 1/8 inch.
o Made of PTFA

18. o HPLC PUMP
o Types of Pump:
o Constant Pressure Pump
o Constant Flow Rate Pump
o 1ml/min flow rate in HPLC
o

19. o Back pressure in column
o Types of Back Pressure
o Low back pressure
o High back pressure

20. o Guard column or Pre column
o Used to protect analytical column
o Any type of impurity will be remove by pre column.
o Low cost
o Size of pre column
o Diameter 4-5mm

21. o Sample Injector
o Three types
o Septum injector
o Stop flow septum less injector
o Micro volume sampling

22. o Analytical Column
o Length
o Internal Diameter
o External Diameter
o C18 Column
o Resist High Pressure

23. o Detector
o Various detectors are used
o Refractive Index Detector
o Conductivity Detector
o UV Visible Detector
o Mass Detector

24. Working of HPLC

26. o Identification of the Compound
o Concentration of the Compound

32. o Output of chromatography
o Retention time tr
o Area under the peak

34. o Pharmaceuticals Uses
o Industrial Uses
o Research Uses

35. ◦ To control drug stability.
◦ Tablet dissolution study of pharmaceutical dosages form.
◦ Pharmaceutical quality control.

36. ◦ Detection of phenolic compounds in drinking water.
◦ Bio-monitoring of pollutants.

37. ◦ Quantification of drugs in biological samples.
◦ Identification of steroids in blood, urine etc.
◦ Forensic analysis of textile dyes.
◦ Determination of cocaine and other drugs of abuse in
blood, urine etc.

38. ◦ Measurement of Quality of soft drinks and water.
◦ Sugar analysis in fruit juices.
◦ Analysis of polycyclic compounds in vegetables.
◦ Preservative analysis.
o U.S. Food and Drug Administration (FDA)

39. ◦ Urine analysis, antibiotics analysis in blood.
◦ Analysis of bilirubin, biliverdin in hepatic disorders.
◦ Detection of endogenous Neuropeptides in extracellular fluid
of brain etc.
◦ Uses in proteomics

40. o To test for the presence of a specific compound
o To analyze all of the compounds in a complex mixture
o To separate and purify compound(s) of interest
o Monitor chemical reactions
o Quantification