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Supervised by :
Dr. Noel Rahul Shaw
Assistant Professor
Department of Quality Assurance
Presented by :
Lakshay Tayal
B.Pharm VII Sem
B.Pharm VII Sem (2020-2021)
HPLC
Affiliated to
Jai Narain Vyas University, jodhpur
3. Introduction
• Chromatography is a laboratory technique used for separation of mixture.
• HPLC is a type of Chromatography which shows high Performance
• HPLC is a process of separation of mixture containing two or more
components under high pressure by passing sample through a column
containing stationary solid bed by means of pressurized flow of liquid mobile
phase.
• The pressure used is 1000- 5000 psi.
• The Principle of separation of component of mixture depends upon their
relative affinity towards St. phase and m.phase or depends upon adsorption/
partition coefficient or depend upon charge or molecular size of mixture. 3
4. Types of HPLC
According to Phases:
• Liquid-solid chromatography or adsorption Chromatography
• Liquid-liquid chromatography or Partition Chromatography
• Ion exchange chromatography or Separation base on charge
• Size exclusion chromatography or Separation base on molecular size
of particle.
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5. Types of HPLC
On the basis of modes:
1. Normal phase HPLC
• Stationary phase: High polar rigid silica, or silica based
compositions.( Hydrophilic, Polar)
• Mobile phase: Relatively non polar solvent, hexane, heptane etc.
2. Reverse phase HPLC ( Often used )
• Stationary phase: Bonded hydrocarbons (C18, C8 etc.)
• Mobile phase: Polar solvents or mixtures such as methanol-water or
acetonitrile -water.
• The most polar component is eluted first.
• It is useful for polar sample analysis (organic compounds)
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9. Parts of HPLC
• Degasser: It is used to remove the air from solvent.
• Reservoir: These are glass or stainless steel containers capable of holding mobile
phase.
Pump: The pump provide a steady high pressure to the sample solution/mobile
phase flowing inside the column. Eg . Reciprocating pump/ constant flow pump
. Displacement pump/ syringe pump
Ideal pump property:
- Ability to generate high pressure
-Accurate control of flow
- Corrosion resistant
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10. Injector: It is used to inject the sample into the continuously flowing mobile phase stream that carries the
sample into the HPLC column.
Column: They are constructed of stainless steel for highest pressure resistance.
• - Length (10-30 cm)
• - Internal diameter (4-10 mm)
• - Particles size (3-10 µm);
Detector: It is used to detect the separated compound bands as they elute from the HPLC column.
Generally UV detector is used in it.
• Characteristics of an ideal detector:
- Adequate sensitivity
- Good stability and reproducibility
- Gives linear response to analysts
- Short response time
Waste collector: The mobile phase/ sample solution exits from the detector and is collected in the waste
chamber where it is either collected or thrown, as desired.
Display unit: A device that records the electrical response of a detector on a computer screen in the form of
a chromatogram.
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11. Applications
• Widely applicable to numerous fields of study; both academic and
industrial work.
• Separate, identify and quantify the active compounds.
• Qualitative and quantitative determination of sample.
• Separation of non-volatiles:
Amino acids, proteins, carbohydrates, pharmaceuticals, pesticides,
pigments, antibiotics, steroids, vitamins, and various other organic and
inorganic substances.
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12. Applications
Food and Flavor Analysis:
• Ensuring the quality of drinking water.
• Sugar analysis in fruit juices
• Analysis of compounds in vegetables.
• Trace analysis of agricultural crops
Application in Clinical Tests :
1.Urine Analysis
2.Antibiotic analysis in blood
3.Identification of Steroids in blood and urine
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13. Applications
In drug manufacturing stage:
• Identification tests of the drug product or its ingredients
• Assay/ Content Uniformity test
• Dissolution test
• Drug Impurity testing
• Drug Stability
• Cleaning Validation/testing of the manufacturing equipments such as blender, tablet
press, etc.
• In process quality control testing
•Data manipulation can be prevented 13
14. Advantages and Disadvantages
• Speed (analysis can be accomplished in 20 min. or less)
• Greater sensitivity (various detectors can be employed)
• Improved resolution (wide variety of stationary phases)
• Reusable columns (expensive columns but can be used for many analysis)
• Used for sepration of sample which tend to decompose at higher Temp
• Disadvantages:
1. It is Costly
2. It is complex
3. Low sensitivity for some compounds
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