TOPIC: HPLC and The Basic Terminologies Of Chromatography.

By

Sayyad AliReg.# FA11-R60-002/ATD

03/12/15 To Prof. Jamshed Iqbal 1

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC)

03/12/15 To Prof. Jamshed Iqbal 2

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

• HPLC stands for “High-performance liquid chromatography”(sometimes referred to as High-pressure liquid chromatography).

• High performance liquid chromatography is a powerful tool in analysis, it yields high performance and high speed compared to traditional columns chromatography because of the forcibly pumped mobile phase.

• HPLC is a chromatographic technique that can separate a mixture of compounds

• It is used in applied science and chemistry to identify, quantify and purify the individual components of a mixture.

03/12/15 To Prof. Jamshed Iqbal 3

• Chromatography is a physical method in which separation of components takes place between two phases a stationary phase and a mobile phase

• Stationary phase : 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

• Mobile phase : solvent which carries the analyte (a liquid or a gas)

03/12/15 To Prof. Jamshed Iqbal 4

Chromatographic techniques are divided into different types based on: 1.The type of chromatographic bed used, i.e. column chromatography (gas chromatography) and planar chromatography (paper and thin layer).2.The physical state of mobile phase i.e. gas chromatography and liquid chromatography3.The separation mechanism i.e. ion-exchange and affinity chromatography.

HPLC is a type of liquid chromatography where the sample is forced through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles by a liquid (mobile phase) at high pressure.

03/12/15 To Prof. Jamshed Iqbal 5

PRINCILPE

Liquid chromatography is a separation technique that involves:

•Putting or injecting of a small volume of liquid sample into a tube packed with porous particles (stationary phase).

•Where individual components of the sample are transported along the packed tube (column) by a liquid moved under the effect of gravity.

The main principle of separation is adsorption .

To understand the principle of HPLC , we must first look at the principle behind liquid chromatography

03/12/15 To Prof. Jamshed Iqbal 6

• When a mixture of components are introduced into the column. various chemical and physical or both types interactions take place between the sample molecules and the particles of the column packing .

• They travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the adsorbent, travels slower.

• The component which has less affinity towards the stationary phase travels faster.

• Since no two components have the same affinity towards the stationary phase, the components are separated

03/12/15 To Prof. Jamshed Iqbal 7

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. The output from the detector is called a chromatogram

In principle, LC and HPLC work the same way except the speed , efficiency and sensitivity of operation .

03/12/15 To Prof. Jamshed Iqbal 8

TYPES OF HPLCBASED ON MODE OF SEPERATION

1.Normal phase chromatography:stationary phase is polar (hydrophilic) and mobile face is non-polar (hydrophobic).

2.Reverse phase chromatography:stationary face is non-polar (hydrophobic) and mobile face is polar (hydrophilic).

•Polar-Polar bonds and Non Polar-Non Polar bonds have more affinity than Polar-Non Polar bonds.

•Reverse phase chromatography is more commonly used as drugs are usually hydrophilic

03/12/15 To Prof. Jamshed Iqbal 9

BASED ON PRINCIPLE OF SEPERATION

Absorption Chromatography• In the Absorption Chromatography solute molecules bond directly to the surface of the stationary phase

• The component which has more affinity towards mobile phase elutes first & the component which has less affinity towards stationary phase elutes later.

No two components have same affinity towards mobile phase & stationary phase.

03/12/15 To Prof. Jamshed Iqbal 10

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.

•Retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Ions of the same charge are excluded.

•The use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.

03/12/15 To Prof. Jamshed Iqbal 11

03/12/15 To Prof. Jamshed Iqbal 12

03/12/15 To Prof. Jamshed Iqbal 13

Selectivity and retention can be adjusted by changing the pH of the mobile phase. This occurs because such a change in pH modifies the character of both the ion exchange medium and the acid–base equilibrium as well as the degree of ionization of the sample.

Affinity Chromatography

•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.

•It is a liquid chromatographic technique that uses a biologically related agent as a stationary phase for the purification or analysis of sample components.

•The retention of solutes in this method is based on the specific reversible interactions that occur in many biological systems, such as the binding of an enzyme with a substrate or of an antibody with an antigen.

•These interactions are exploited in affinity chromatography by placing one of a pair of interacting molecules onto or within a solid support and using this immobilized agent as a stationary phase. This immobilized agent is known as the affinity ligand and is what gives an affinity column the ability to bind to particular compounds in a sample.

03/12/15 To Prof. Jamshed Iqbal 14

03/12/15 To Prof. Jamshed Iqbal 15

Steric hindrance: It refers to the loss of ligand activity due to the presence of a nearby support or neighboring ligands that interfere with solute binding. This effect can be avoided through the use of a spacer arm or by using stationary supports that contain a relatively less dense attachment of the ligand.

03/12/15 To Prof. Jamshed Iqbal 16

BASIC TERMINOLOGIES OF CHROMATOGRAPHY.

DISTRIBUTION

03/12/15 To Prof. Jamshed Iqbal 17

03/12/15 To Prof. Jamshed Iqbal 18

Movement Through the Column

03/12/15 To Prof. Jamshed Iqbal 19

a)

To Prof. Jamshed Iqbal 20

b)

03/12/15 To Prof. Jamshed Iqbal 21

Continue…

03/12/15 To Prof. Jamshed Iqbal 22

Resolution

03/12/15 To Prof. Jamshed Iqbal 23

Theoretical plates: •As the column efficiency is related to the “number of theoretical plates” or “N”. The value of “N” is related to the column length “L” and the size or height “H” of an individual plate by the following equation:

N = L/H •The value of “L” is usually measured in cm, and can vary from just a few centimeters for HPLC columns to many meters for GC columns. •The values of N are usually large, in the thousands or even millions, giving values of H. •The size of H affect the efficiency of the column and therefore it is important to understand what affects the value of H.•The value of H depends primarily on four factors,

1) the velocity of the mobile phase, 2) multipath diffusion, 3) the diffusion of the compound in the mobile phase, and 4) the transfer of the compound between the stationary phase and the

mobile phase.

03/12/15 To Prof. Jamshed Iqbal 24

Linear Velocity, u

Pla

te H

eigh

t, H

Multipath Term, A

Mass Transfer (both), Cu

Longitudinal diffusion, B/u

A + B/u + Cu

A typical Van Deemter plot

03/12/15 To Prof. Jamshed Iqbal 25

The Multipath Term or constant A:•This factor is also called Eddy diffusion, but more modern textbooks use the term multipath. •In packed columns, peak-broadening is the result of a number of factors. •As molecules of the analyte move through the column, they take many different paths around the packed particles. • Some of these paths are longer than others so as the molecules move through the column, thus they tend to spread out. •The amount of spreading is affected by the nature of the column material and how well the column is packed. •This factor is generally proportional to the particle size of the packing material.

1 2

FlowDirection

Pathways of two moleculesduring elution. Distance traveledby molecule 1 is longer thanthat traveled by molecule 2, thus molecule 1 will take longer toelute.

03/12/15 To Prof. Jamshed Iqbal 26

The Longitudinal Diffusion Term B/u (m2/sec) •Longitudinal diffusion also contributes to peak broadening. •In this process, analytes diffuse from areas of high concentration to more dilute area in front of and behind the moving band.

•The concentration of the analyte is less at the edges of the band than at the center.•Hence analyte diffuses out from the center to the edges, causing band broadening. •If the velocity of the mobile phase is high then the analyte spends less time on the column, decreases the effects of longitudinal diffusion.

Flow

Flow

Molecules diffuse from areas of highconcentration to areas of low concentration.

Over time….

03/12/15 To Prof. Jamshed Iqbal 27

The Mass Transfer Terms Cu:

•The analyte takes certain amount of time to reach equilibrium between the stationary and mobile phase.

•If the velocity of the mobile phase is high and the analyte has a strong attraction for the stationary phase, then the analyte in the mobile phase will move ahead of that in the stationary phase.

•Like the longitudinal term, the mass transfer term is based on diffusion. However, longitudinal diffusion takes place parallel to the direction of flow, and therefore is inversely related to the mobile phase flow rate, while mass transfer diffusion takes place perpendicular to the flow rate. •As a result, the faster the mobile phase moves, the less time there is for equilibrium between the phases and the mass transfer effect.

• peak broadening is directly related to mobile phase velocity or flow rate.

03/12/15 To Prof. Jamshed Iqbal 28