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HPLC Introduction & Application in Biotechnology

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Elahe R

on 21 May 2015

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Transcript of HPLC Introduction & Application in Biotechnology

Presented By:
Zahra Foroud
Sara Gholami
Elahe Rezagholizade
History of HPLC
Mobile Phase
This is a solvent which contains the analyte

Mobile Phase
Solvents used to create the necessary polarity for the separation being done
This solvent carries the sample through the stationary phase.
pumps the mobile phase through the stationary phase
Provides a pressure 150X higher than that of the atmosphere
Injection Port
Used when single sample is being analyzed
The Detector
Identifies the components of the mobile phase and their concentrations
The Detector generates a chromatogram and displays it on the computer system attached to it.
A Schematic of an HPLC Instrument
The Column
Retrieved from:

Stationary Phase
A substance within the column with varying affinity for the mobile phase
Comprises of a substance, which is known to provide the ideal obstacle course for the sample being analyzed
Features of a detector
sensitivity toward solute over mobile phase
Low cell volumes to minimize memory effects
Low detection limits
Large linear dynamic range
Low detector noise
HPLC is a form of liquid chromatography used to separate components dissolved in solution and also to identify and quantify each component.

High performance liquid chromatography is formerly known as high pressure liquid chromatography.
Compounds are separated by injecting a sample mixture onto the column. The different components in the mixture are at differentiates due to differences in partition behavior between the mobile phase and the stationary phase.

The HPLC instrument consists of a reservoir of mobile phase, a pump, an injector, a separation column and a detector.
History of HPLC
History of HPLC
In the early 1900’s, a Russian botanist Mikhail S. Tswett discovered a separation technique involving the passage of a mixture of plant pigments to be separated through a column of finely powdered adsorbent.

He poured the sample into an open glass column containing alumina and calcium (stationary phase) along with a pure solvent (mobile phase).
History of HPLC
As the sample moved down the column, the coloured bands appeared correlating to the sample component.

These bands are separated as some of the components were moving faster than the others. The mixture was separated based on the strength of each compound’s chemical attraction to the stationary phase.
History of HPLC
Tswett coined the name chromatography from the Greek words “chroma” meaning colour and “graph” meaning writing

The basic principles of HPLC was established in 1960 but the actual HPLC method was developed in the 1970’s.

History of HPLC
In 1990 the development of micro columns , detectors combined with integrated data acquisition, storage and retrieval capabilities has increased the speed and efficiency of HPLC instruments.
Separation Modes of HPLC
There are 5 major separation modes that are used seperate most compounds. Normally it is described either by the nature stationary and mobile phase or the separation process.

Separation Modes of HPLC
Normal Phase
Separates analyte based on their polarity where the stationary phase is strongly polar which retains a polar analyte and the mobile phase less polar/ non- polar
Increasing the polarity of the analyte increases the retention time (adsorption strength)hence increasing the polarity of mobile phase decreases the retention time
sometimes referred to as Adsorption
Separation Modes of HPLC
• - where the stationary phase is non-polar and mobile phase is aqueous, moderately polar
• - separates molecules in solution based on their hydrophobicity
• - more non- polar substance have a longer retention 

Separation Modes of HPLC
Size Exclusion
Separation Modes of HPLC
separation  of organic and inorganic ions by their partitioning onto ionic stationary phase bonded to a solid support

buffers are used as the mobile phase 

exclusively used for ionizable samples
Reversed phase
separates particles based on their size
- column are filled with particular pore size serving to filter the sample
- large molecules wash through the column faster than the smaller ones  
- useful for the separation of biopolymers such as protein and polysaccharides 

Separation Modes of HPLC

In between normal and reversed

separation of moderately polar analyte using adsorption onto a pure stationary phase
Ion exchange
Factors influencing HPLC performance
Internal Diameter of the column

Pump Pressure

Sample size

Polarity of sample, solvent and column


Efficiency Factor

Retention Factor
Advantages and Disadvantages of HPLC

High resolution

Efficient and highly selective

Non- destructive of sample

Provide accurate, precise reproducible results

Analysis the compounds that are temp-sensitive & non-voliate

Cost - expensive technique


Low sensitivity for some compounds

Application of HPLC in Biotechnology
Medicinal Chemistry Application of HPLC
Quantifying (separate, compounds /mixture)
Determination of pharmaceutical dosage form, such as Paracetamol determination in panadol tablet
Drug determination from biological fluids, such as blood glucose level

Purification of the active drug molecules
Isolation of plant stem cell active compound meristem.
Identify various characteristics, such as plant stem cell.

Quality/Efficacies of the drug discovered ( how to package the drug, the shelf life of the drug, how to package the drug, how much of the drug is absorb in the body)
strong vitality, anti-oxidation, anti-inflammation, immune enhancement, anti-cancer and anti-aging properties.

Medicinal Chemistry Application of HPLC
Thanks for Listening

Any Question?
HPLC Can Be Used
Detecting the levels of substances in blood (Medical)
Separation of the components of complex biological mixtures (Scientific)
During the production process of biological substances and drugs etc.
And basically, any other process that requires accurate separation, purification and identification.

HPLC is probably the best instrumental technique when it comes to effective separation, purification & identification of substances that are in a complex mixture.
Full transcript