HIGH PERFORMANCE LIQUID CHROMATOGRAPHY workflow, types of hplc, normal phase HPLC, reverse phase HPLC,types of column, advantages pf HPLC over other liquid chromatography, parameters of HPLC. SOURCE: ARPAN YOUTUBE

1. HPLC: HIGH
PERFORMANCE
LIQUID
CHROMATOGRAPH
Y

3. PRINCIPLE :
Before HPLC lets see the principle behind liquid chromatography
Liquid chromatography involves :
The placement (injection) of small volume of liquid sample into a tube
packed with porous particles(stationary phase) where individual components
of the sample are transported along the packed tube(column) by a liquid
moved by gravity.
The main principle behind separation is adsorption that is separation of two
or more components based on their polarity.
The components are introduced in the column, the components travel
according to their relative affinity. The components which has great affinity
towards adsorbent travels slower.

12. 1. Absorption chromatography.
In the absorbstion chromatography the solute molecules bond directly to the surface of the stationary
phase.
The component which has more affinity towards the mobile phase elutes first & cpmponents which
has lesser affinity towards stationary phase elutes later.
2. Ion – exchange chromatography
The process that allows the separation of ions
and polar solutes based on their charge.
Retention is based on the attraction between
solute ions and charged sites bound to the
stationary phase. Ions of the same charge are
excluded.

13. OPERATION:
Switch on the instrument
Check system set up
Prime the pump
Prepare the column
Set-up software(for system flushing)
Software setup (for run)
Sample injection
Chromatograph data aquisition

14. PARAMETERS OF HPLC
 Retention time(RT) in the chromatogram different peaks correspond to different components
of separated mixture. That is a characteristic time taken for any particular analyte to pass
through the system.
 Retention volume : it is the volume of carrier gas required to elute 50% of the component
from the column. It is the product of the retention time and flow rate.
 Separation factor : ratio of partition coefficient of the two components to be separated.
S=Ka/Kb=(tb-to)/(ta-to)
 Resolution: measure of extent of separation of 2 components and the baseline separation
achieved.
 Efficiency: efficiency of a column is expressed by the theoretical plates.

16. APPLICATIONS:
Pharmaceutical
 Tablet dissolution of pharmaceutical dosages
 Shelf life determination of pharmaceutical pruducts.
 Identification of counterfeit drug products
 Pharmaceutical quality control

17. APPLICATIONS:
Environmental
 Phenols in drinking water
 Identification of diphenylhydramine in sediment samples.
 Estrogens in coastal water- the sewage source
 Assesment of TNT toxicity in sediment.

18. APPLICATIONS:
Clinical
 Quantification of DEET in human urine.
 Analysis of antibiotics.
 Increased urinary of aquaporin 2 in patients with liver
 Detection of endogenous neuropeptides in brain extracellular fluids.

19. APPLICATIONS:
Food and flavour
 Ensuring soft drink consistency and quality
 Analysis od vicinal diketones in beer
 Sugar analysis in fruit juices
 Polycyclic aromatic hydrocarbons in fruits and vegetables.