CHROMATOGRAPHY

THEORY & PRACTICE INTRODUCTION

PROF. DERICK CARBOO

DEPARTMENT OF CHEMISTRY UNIVERSITY OF GHANA, LEGON O244682712

OUT LINE
1. 2. 3. 4.

What is chromatography? History of chromatography Nomenclature for chromatography Classification of chromatography

Classification based on mechanism of interaction of solute with stationary phase Classification based on development technique Classification based on physical state of mobile phase Retention time
Capacity factor Selectivity factor

5.

The chromatogram

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6. Theories of chromatography
1) Plate theory (Theoretical plate theory)
Plate height (HETP) Resolution Theory of band broadening

2) The rate theory

The van-Deemter equation Van Deemter plots Resolution

7. Liquid chromatography
Stationary phase materials Eluotropic series and Eluents Gradient and isochratic elution techniques
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8. High performance liquid chromatography (HPLC)

Characteristics of a HPLC system Essential components of HPLC Pump & Injection systems Eluent Column
Stationary phase : Types Normal phase Bonded phase Reversed phase

Detectors Spectrophotometric Refractometric Conductometric

9. Choice of Mode of HPLC 10. Ion Chromatography; Reversed Phase Chromatography, Bonded Phase Chromatography etc,
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11. Gas chromatography

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Definitions: Chromatography

is the collective term for a set of techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the components in the mixture based on differential partitioning between the mobile and stationary phases. Subtle differences in the partition coefficient of compounds results in differential retention on the stationary phase and thus changing the separation.

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 In chromatography two or more solutes

distribute themselves between two immiscible phases, such as: liquid/liquid system liquid/solid system gas/liquid system gas/solid systems During chromatography the various solutes show different affinities for the individual phases.
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History of chromatography
The Russian botanist Mikhail Tswett has been credited with the invention of chromatography. He used the technique to separate plant pigments (e.g. chlorophylls and xanthophylls) by passing solution of them through glass columns packed with finely divided CaCO3 . Chromatography became developed substantially as a result of the work of A.J.P. Martin and R.L.M. Synge during the 1940s and 1950s. They established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several types of chromatography method: paper chromatography, gas chromatography, and what would become known as high performance liquid chromatography. They were awarded the Nobel prize in Chemistry in 1950 for their work the development of modern chromatography.

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 Nomenclature Ref: IUPAC Recommendation: Pure & Appl, Chem., Vol. 65, No. 4, pp. 819 - 872, 1993.

1. Stationary Phase

The stationary phase is one of the two phases forming a chromatographic system. It may be a solid, a gel or a liquid. If a liquid, it may be distributed on a solid support. This solid support may or may not contribute to the separation process. The liquid may also be chemically bonded to the solid (Bonded Phase) or immobilized onto it (Immobilized Phase).
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2. Mobile Phase A fluid which percolates through or along the stationary bed, in a definite direction. It may be a liquid (LC) or a gas (GC) or a supercritical fluid (S-F C). In GC the expression Carrier Gas may be used for the mobile phase. In elution chromatography the expression Eluent is also used for the mobile phase.
3. Eluate (Effluent) The mobile phase leaving the column.
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4. Elute (verb): To chromatograph by elution chromatography. 5. Chromatograph (verb): To separate by chromatography 6. Chromatograph (noun): The assembly of apparatus for carrying out chromatographic separation. 7. Chromatogram : A graphical presentation of detector response (on the ordinate) versus retention time or effluent volume In planar chromatography "chromatogram" may refer to the paper or layer with the separated zones.
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7. Retention time : The time between sample injection and an analyte peak reaching a detector at the end of the column. 8. Solute A term referring to the sample components in partition chromatography.

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CLASSIFICATION OF CHROMATOGRAPHY 1. according to Mechanisms of interaction of solute with stationary phase:

Adsorption chromatography Partition Chromatography Ion-exchange chromatography Size exclusion chromatography Affinity chromatography

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Adsorption chromatography : Separation is based

mainly on differences between the adsorption affinities of the sample components for the surface of the solid stationary phase. Here solute is adsorbed on the surface of the solid stationary phase. The mobile phase is either liquid or gas. Equilibrium is established between the mobile phase and stationary phase .
The solute with the higher adsorption affinity

remains longer on the stationary phase.

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Partition Chromatography :

In Liquid Chromatography: Separation is based on differences between the solubilities of the components in the mobile phase and liquid stationary phase or In Gas Chromatography (specifically, GLC): Separation is based mainly on differences between the solubilities of the sample components in the liquid stationary phase. So the solute with the higher solubility in the stationary phase remain longer on the column.
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Ion-Exchange Chromatography Here the stationary phase is a resin with such functional groups as -COO- , -SO3-, that can exchange cations (cation exchanger) or, the resin may contain -NR3+ and can exchange anions (anion exchanger). The functional groups are covalently bonded to the resin and the counter ions are attached by electrostatic force. Separation is based mainly on differences in the ion exchange affinities of the sample components

Modern ion-exchange chromatography on small particle, high efficiency columns, and usually utilizing conductometric or spectroscopic detectors is often referred to as Ion Chromatography (IC).
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Size exclusion chromatography(SEC)

separates molecules according to their size (or

more accurately according to their hydrodynamic diameter or hydrodynamic volume) on porous gel (stationary phase).
Smaller molecules are able to enter the pores

of the stationary phase and, therefore, take longer to elute, whereas larger molecules are excluded from the pores and elute faster.
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 SEC can be carried out

using two different

techniques: a) Gel filtration chromatography when the mobile phase is an aqueous solution. b) Gel permeation chromatography when an organic solvent is used as a mobile phase.
SEC is usually applied to separation of large

molecules or macromolecular complexes such as proteins and industrial polymers

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Affinity chromatography

Employs a selective non-covalent interaction between one kind of solute molecule and a second that is covalently attached (immobilized) to the stationary phase.
The method is most selective kind of chromatography Example: the immobilized molecule (or ligand) may be an antibody to particular protein. When a mixture of many proteins is passed through the column, only the one protein that reacts with the antibody is bound to the column. The desired protein is dislodged from the column after all the others have been washed away.
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Affinity chromatography is used in the separation of biochemical mixtures, biomolecules, purification of proteins etc. Ligand availability and the cost of the specialized media are usually prohibitive at large-scale.

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2. Classification based on the method of Development of the chromatogram : COLUMN or PLANAR Chromatography 1. Column Chromatography: is a separation technique in which the stationary phase is held within a tube called column. In packed column the particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube. In open tubular column the stationary phase is concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube . The mobile phase can be liquid (LC) or gas (GC) or super critical fluid (SFC)
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2. Planar chromatography
is

a separation technique in which the stationary phase is present as or on a (two-dimensional) plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary phase (paper chromatography), or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography). The mobile phase moves through the stationary phase by capillary action or by gravity. Different compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.
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Diagram 1 shows begin of tlc experiment

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Diagram 2 shows the plate after the solvent has moved about half way up it.

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3. Classification based on type of mobile phase In Liquid chromatography the mobile phase is always liquid But the stationary phase can be liquid or solid, and the stationary phase can be held in a column as in column liquid operation or on a plate as in TLC or paper chromatography. In liquid-bonded phase chromatography: the liquid stationary phase is chemically bonded to a solid support. Usually the silanol group (Si-OH) of the silica gel is reacted with dimethyl alkyl chloro silane, (Cl-Si(CH3)2R. R can be varied but usually is octadecyl (C18H37)
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Gas chromatography (GC): Here the mobile phase is a gas (carrier gas). The stationary phase is a solid matrix inside a larger metal tube (a packed column). Or solid matrix adhered to the inside of a small-diameter glass tube (a capillary column or open tubular column) Liquid stationary phase adsorbed or covalently bound onto a solid matrix and packed or adhered to the inside of the column (GLC)

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Supercritical Fluid Chromatography (SFC)

Here the mobile phase is a supercritical fluid. This is a fluid with properties between a gas and a liquid. Typical example is carbon dioxide in its supercritical state. SFC uses all the common instruments employed by GC and LC in addition to those required to generate and maintain the supercritical fluid. SFC is best suited for separating compounds that are not volatile , or non-polar or that are thermally unstable. It is intermediate between GC and LC. Other advantages is that removal of the mobile phase is very easy and environmental friendly.

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Supercritical Fluid
The critical point (CP) marks the end of the vapor liquid coexistence curve. A fluid is termed supercritical when the temperature and pressure are higher than the corresponding critical values. Above the critical temperature, there is no phase transition in that the fluid cannot undergo a transition to a liquid phase, regardless of the applied pressure. A supercritical fluid (SCF) is characterized by physical and thermal properties that are between those of the pure liquid and gas. The fluid density is a strong function of the temperature and pressure. The diffusivity of SFC is much higher than for a liquid and SCF readily penetrates porous and fibrous solids. Consequently, SCF can offer good catalytic activity.

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Supercritical Fluids

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