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HPLCHIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Submitted To:Sayema KhanumSenior LecturerDepartment Of Pharmacy,SEU.

Submitted By:MD. Rashed MahmudNilufa Haque ChaityNusrat JahanSarmin Akter

HPLCHPLC is an abbreviation for-High Performance Liquid

Chromatography(It has also been referred to as High Pressure LC)

HPLC is a separation technique that involves: the injection of a small volume of liquid sample into a tube

packed with tiny particles (3 to 5 micron (µm) in diameter called the stationary phase)

where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump.

These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.

These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector liquidis chromatogramcalled

a liquid chromatogram.

Stationary phases The substance on which adsorption of the analyte (the

substance to be separated during chromatography) takes place. It can be a solid, a gel, or a solid liquid combination.

Reverse phase -β, β '-Oxydipropionitrile

-Carbowax.

-Glycols (ethylene, diethylene),

-Cyanoethylsilicone Normal phase

-Squalane

-Zipax-HCP

-Cyanoethylsilicone

Mobile Phase The mobile phase carries the sample molecules along

whereas the stationary phase interacts with them, the stronger the interaction the slower their movement and this differential interaction causes them to separate out and elute at different times.

Normal phase chromatography

-hexane

-heptane

-aromatic solvents(e.g. benzene, xylene) Reverse phase chromatography

-Water and alcohol-water mixtures

-acetonitrile and

-acetonitrile-water mixtures

Parameters used in HPLC

1.Theoritical Plate2.Retention Time3.Retention Volume4.Seperation Factor6.Resolution 5.Tailing Factor etc.

1.Theoritical Plate

A theoretical plate in many separation Processes, is a hypothetical zone or stage in which two phases, such as the liquid and vapor phases of a substance, establish an equilibrium with each other.

Such equilibrium stagesmay also be referred to as an equilibrium stage, ideal stage, or a theoretical tray.

The performance of many separation processes depends on having a series of equilibrium stages and is enhanced by providing more such stages.

In other words, having more theoretical plates increases the efficacy of the separation process be it either a distillation, absorption, chromatographic, Adsorption or similar process.

2.Retention time (RT) In a chromatogram, different peaks correspond to different components of the separated mixture.

Time elapsed between sample introduction and maximum of response, it is the characteristic time it takes for a particular analyte to pass through the system.

Time taken for the analyte to travel from the column inlet to the point of detection(maximum peak)

Retention time (RT)

Different compounds have different retention times. For a particular compound, the retention time will vary depending on:

the pressure used (because that affects the flow rate of the solvent)

the nature of the stationary phase (not only what material it is made of, but also particle size)

the exact composition of the solvent the temperature of the column That means that conditions have to be carefully

controlled if you are using retention times as a way of identifying compounds.

 

3.Retention volume:

Retention volume is the volume of carrier gas required to elute 50% of the component from the column. It is the product of retention time and flow rate.

Retention volume = Retention time × flow rate.

4.Seperation factor:

Separation factor is the ratio of partition coefficient of the two components to be separated.

S=Ka/Kb=(tb-to)/(ta-to)

Where ,

to = Retention time of unretained substance

Ka, Kb = Partition coefficients of a, b

ta, tb = Retention time of substance a, b

If there is a big difference in partition coefficient between 2 compounds, the peaks are far apart and the separation factor is more. If the partition coefficient of 2 compounds are similar, then the peaks are closer and the separation factor is less.

5.Resolution:Resolution is the measure of extent of separation of 2

components and the base line separation achieved.

Rs = 2 (Rt1-Rt2) / w1+w2

6.Tailing Factor

The tailing factor is a measure of peak tailing. It is defined as the distance from the front slope of the peak to the back slope divided by twice the distance from the center line of the peak to the front slope, with all measurements made at 5% of the maximum peak height. The tailing factor of a peak will typically be similar to the asymmetry factor for the same peak, but the two values cannot be directly converted. 

Application:

Chemical Separations The extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase.

Purification Purification is defined as the process of separating or extracting the target compound from a mixture of compounds.Each compound showed a characteristic peak under certain chromatographic conditions.

Identification Generally assay of compounds are carried using HPLC. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph. The identifying peak should have a reasonable retention time and should be well separated from extraneous peaks at the detection levels which the assay will be performed.

Pharmaceutical applications Tablet dissolution study of pharmaceutical dosages form. Shelf-life determinations of pharmaceutical products Identification of active ingredients of dosage forms  Pharmaceutical quality control Environmental applications  Detection of phenolic compounds in drinking Water Identification of diphenhydramine in sedimented samples  Bio-monitoring of pollutant  Forensics  Quantification of the drug in biological samples.  Identification of anabolic steroids in serum, urine, sweat, and hair Forensic analysis of textile dyes. Clinical Quantification of ions in human urine analysis of antibiotics in blood

plasma. Estimation of bilirubin and bilivirdin in blood plasma in case of hepatic

disorders. Detection of endogenous neuropeptides in extracellular fluids of brain.

 

 

Advantages of HPLC1. Separations fast and efficient (high resolution power)

2. Continuous monitoring of the column effluent

3. It can be applied to the separation and analysis of very complex mixtures

4. Accurate quantitative measurements.

5. Repetitive and reproducible analysis using the same column.

6. Adsorption, partition, ion exchange and exclusion column separations are excellently made.

7. HPLC is more versatile than GLC in some respects, because it has the advantage of not being restricted to volatile and thermally stable solute and the choice of mobile and stationary phases is much wider in HPLC

8. Both aqueous and non aqueous samples can be analyzed with little or no sample pre treatment

9. A variety of solvents and column packing are available, providing a high degree of selectivity for specific analyses.

10. It provides a means for determination of multiple components in a single analysis.

Disadvantages of HPLC

Cost Complexity-There is complexity of usage as well as low

sensitivity for some compounds. Low sensitivity for some compounds Irreversibly adsorbed compounds notdetected Coelution difficult to detect

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