Hplc

This is a PPT for HPLC which I made for presenting my assigned topic for Practice School during 7 sem of my graduation . Hope it is useful for you guys :)

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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

2. Presentation Plan
•Introduction
•Types of HPLC
•Flow Scheme of HPLC
•Instrumentation
•Advantages and Disadvantages
•Applications
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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.
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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, heptaneetc.
2. Reverse phase HPLC ( Often used )
•Stationary phase: Bonded hydrocarbons (C
18, C
8 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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7. A flow scheme of HPLC showing operation mode of
its parts
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8. Instrumentation of HPLC
•Degasser
•Reservoir
•Pump
•Injector
•Column
•Detector
•Waste collector
•Display unit
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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
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14. Advantagesand 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 seprationof 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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15. Reference
•https://lab-training.com/hplc/
•https://microbenotes.com/high-performance-liquid-chromatography-
hplc/Accessed on 15/01/21
•https://lab-training.com/wp-content/uploads/2014/11/HPLC-E-Book.pdf
•https://laboratoryinfo.com/hplc/ Accessed on 18/01/21
•Introduction to HPLC by Lab Training.com (Saurabh Arora )
•Handbook of HPLC New York: M. Dekker, ©1998 by Elena Katz
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