PRESENTATION ON :-

Advance separation technology: high performance liquidchromatography(HPLC)

PRESENTED BY-MD:Jabiur Rahaman

Daffodil International University,Bangladesh

Dept. Of Pharmacy

ID:121-29-404

Introduction :-HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution.

Preparative chromatography

•

Analytical chromatography

TYPES OF PHASES :-

Mobile Phase

• •

Stationary

Phase

• •

Mobile Phase Stationary Phase

Solvent Bonded Phase

Separation is based on the analyte’s relative solubility between two liquid phases

Partitioning :-

HPLC - Modes

Normal

Phase.

Reverse

Phase.

Normal Phase.- Polar stationary phase and non-polar solvent.

Reverse Phase.- Non-polar stationary phase and a polar solvent.

PICTURE OF HPLC SYSTEM :- 1) Solvent reservoirs, 2) Solvent degasser, 3) Gradient valve, 4) Mixing vessel for

delivery of the mobile phase,

5) High-pressure pump,

6) Switching valve in "inject position" Switching valve in "load position",

7) Sample injection loop,

8) Pre-column (guard column),

9) Analytical column, 10) Detector (i.e. IR,

UV), 11) Data acquisition, 12) Waste or fraction

collector.

PICTURE OF HPLC EQUIPMENT :-

FOUR TYPES OF HIGH PERFOMANCE LIQUID CHROMATOGRAPHY :-

1. PARTITION CHROMOTOGRAPHY

•

2. ION EXCHANGE CHROMATOGRAPHY

•

3. Size exclusion chromatography

4. AFFINITY CHROMOTAGRAPY

1. PARTITION CHROMOTOGRAPHY :-

Partition chromatography is process of separation whereby the components of the mixture get distributed into two liquid phases due to differences in partition coefficients during the flow of mobile phase in the chromatography column.Here the molecules get preferential separation in between two phases. i.e. both stationary phase and mobile phase are liquid in nature. So molecules get dispersed into either phases preferentially. Polar molecules get partitioned into polar phase and vice-verse. This mode of partition chromatographyapplies to Liquid-liquid, liquid-gas chromatography and not to solid-gas chromatography. Because partition is the phenomenon in between a liquid and liquid or liquid and gas or gas and gas. But not in solid involvement.

Partition Chromatography:-

Most widely used Bonded-phase Chromatography Silica Stationary Phase: OH OH OH OH O O O Si Si Si Si Siloxanes: O CH3

Si O Si R R= C8, C18

O CH3

Operation in partition

chromotography

2. ION EXCHANGE CHROMATOGRAPHY

Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their charge. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the individually separated components are called analytes. It is often used in protein purification, water analysis, and quality control.

Basis for molecular separation :-

Ion Exchange

Gel Filtration

Affinity

Charge

Size

Conformation

Components of ion exchange :-

A charge solid phase or matrix

Liquid phase contains molecules of different charges

Solutions (eluant) of different charges to influence interactions between liquid and solid phases

Operation in ion Exchange

http://en.wikipedia.org/wiki/File:Metrohm_850.jpg

3. Size exclusion chromatography :-

Size Exclusion Chromatography (SEC) is the separation technique based on the molecular size of the components. Separation is achieved by the differential exclusion from the pores of the packing material, of the sample molecules as they pass through a bed of porous particles. The principle feature of SEC is its gentle non-adsorptive interaction with the sample, enabling high retention of biomolecular activity.

SEC (continued) :-

Specific pore sizes.average residence time in the pores depends on the effective size of the analyte molecules› larger molecules› smaller molecules› intermediate size molecules

Operation in size exclusion

http://en.wikipedia.org/wiki/File:Size_exclusion.JPG

4. AFFINITY CHROMOTAGRAPY :-

This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solutes containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH..

Affinity Chromatography

Surface bound with

Epoxy, aldehyde or aryl ester groups

Metal Interaction Chromatography

Surface bound with

Iminodiacetic acid + Ni2+/Zn2+/Co2+

Affinity Chromatography

Operation in Affinity

Chromotagraphy

ACCESSORIES FOR HPLC EQUIPMENT :-

HPLC COLOUMS:-The column is one of the most important components of the HPLC chromatograph because the separation of the sample components is achieved when those components pass through the column. The High performance liquid chromatography apparatus is made out of stainless steel tubes with a diameter of 3 to 5mm and a length ranging 10 -30cmNormally, columns are filled with silametica gel because its particle shape, surface properties, and pore structure help to get a good separation. Silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior is generally predictable and reproducible.

HPLC COLOUMN :-

Pump

Injector

ColumnDetector

Gradient Controller •

Instrumentation :-

Liquid Chromatographic Column

Smooth-bore stainless steel or heavy-walled glass tubing

Hundreds of packed columns differing in size and packing are available from manufacturers ($200-$500)

Add columns together to increase length

Sample Injection Systems

For injecting the solvent through the column Minimize possible flow disturbances Limiting factor in precision of liquid

chromatographic measurement Volumes must be small .1-500 L Sampling loops

› interchangeable loops (5-500 L at pressures up to 7000 psi)

Detector

Mostly optical Equipped with a flow cell Focus light beam at the

center for maximum energy transmission

Cell ensures that the separated bands do not widen

Detectors :-UV

•

Fluorescence

•

Electrochemical

Mass Spectrometric

Mobile Phases

A and B

HPLC Pump

syringe

6-port valveSampl

e loopHPLC column

DetectorMP waste

HPLC-UV:-

Chromatograms :-

Restek® ULTRA C-18 and CN Columns (250mm x 4.6mm, 5µ), Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.

Chromatograms :-

A Supelcosil LC-PAH Columns B

Conditions: A: 150mm x 4.6mm, 5µ.Flow Rate: 1.5 mL/min

Conditions: B: 50mm x 4.6mm, 3µ.Flow Rate: 3.0 mL/min

USES OF HPLC :-

1.This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.

2. In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.

THANK YOU

ANY QUESTIONS ???