CHROMATOGRAPHY

Chromatography

Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient of sample components between 2 different phases.

One of these phases is a mobile phase and the other is a stationary phase.

Definition:

 

Different affinity of these 2 components to stationary phase causes the separation.

Concentration of component A in stationary phase

Concentration of component A in mobile phase

Distribution Coefficient

Kinds of Chromatography

1. Liquid Column Chromatography

 

2. Gas Liquid Chromatography

 

Liquid Column Chromatography

A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.

With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.

A + B + C

OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO

OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO

Sample (A+B+C)

Column

Solid Particles(packing material- stationary phase)

Eluant (eluate)

DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY

A

B

C

Solvent(mobile or moving phase)

Diagram of Simple Liquid Column Chromatography

Basic liquid chromatography modes are named according to the mechanism involved:

 1. Liquid/Solid Chromatography (adsorption chromatography)

A. Normal Phase LSC

B. Reverse Phase LSC

 2. Liquid/Liquid Chromatography (partition chromatography)

A. Normal Phase LLC

B. Reverse Phase LLC

 3. Ion Exchange Chromatography

 4. Gel Permeation Chromatography (exclusion chromatography)

Four Basic Liquid Chromatography

Liquid Solid Chromatography

Si - O - H

Normal phase LS Reverse phase LS

Silica Gel

The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.

Liquid Solid Chromatography

Si - OH

HEXANE

OH

C-CH3

CH3

CH3 - C

CH3

CH3

OH

OH

CH3

CH3

Water-Soluble Vitamins

1. Niacinamide 2. Pyridoxine

N

CONH2

N

CH2OH

CH2OH

HO

H3C

3. Riboflavin N

NNH

N

CH2

HOCH

HOCH

HOCH

CH2OH

O

OH3C

H3C

ClN

S

N

NH3C

CH2

NH2

CH3

CH2CH2OH

4. Thiamin

Water-Soluble Vitamins

0 5 10 15 20

Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins

Inject1

2

3

4

Liquid-Liquid Chromatography

ODPN (oxydipropionylnitrile)

Normal Phase LLC Reverse Phase LLC

NCCH3

CH2

OCH2

CH2

CN(Normal)

CH3

(CH2

)16

CH3

(Reverse)

The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase. Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.

MOBILE PHASELIQUID

Liquid-LiquidChromatography (Partition)

Liquid-SolidChromatography (Adsorption)

Liquid Solid

Normal Phase Reverse Phase Normal Phase Reverse Phase

Mobile Phase -

Nonpolar

Stationary phase - Polar

Mobile Phase - Polar

Stationary phase -

Nonpolar

FORMAT

STATIONARY PHASE

Types of Chromatography

Ion-Exchange Chromatography

SO3- Na+

Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).

Mechanism of Ion-Exchange Chromatography of Amino Acids

SO3-

SO3-

Na+

COO-

H3N+

Na+

COOHH3N

+

pH2

pH4.5

Ion-exchange Resin

H3N

+

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

H3N+

COOH

OH

COOH

COOH

H3N+

H3N+

OH

COO-

Na+

H3N+

COO-

Na+

Na+

H+ OH

- = H2O

H+ OH

- = H2O

Na+

Na+

pH3.5

Mobile PhaseStationary Phase

Exchange Resin

pH4.5

Chromatography of Amino Acids

Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules.

Gel-Permeation Chromatography

• Polar Solvents

Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile

 • Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane

Solvents

Sample Type LC Mode Positional isomers LSC or LLC

Moderate Polarity Molecules LSC or LLC

Compounds with Similar Functionality LSC or LLC

Ionizable Species IEC

Compounds with Differing Solubility LLC

Mixture of Varying Sized Molecules GCC

Selecting an Operation Mode

Schematic Diagram of Liquid Chromatography

Detector

1. Ultraviolet Detector

200-400nm 254 nm

2. Reflective Index Detector

Universal Detector

High Performance Liquid Chromatography

High Performance Liquid Chromatography

Retention Time

Time required for the sample to travel from the injection port through the column to the detector.

Response

Retention Time

5 10 15 20 25

A

B

C

D

Selectivity

Ratio of Net Retention Time of 2 components.

(Distribution Coefficient) X2 - X0

X1 X0-

Response

Retention Time

X

X

X

1 3 6

2

1

0

– Selectivity

SelectivitySelectivity

Resolution Equation

V - V

1/2(W + W )

2

2

1

1

R =

Response

Volumes

W W

W W

V

V1

1 2

2

21

Resolution

Height Equivalent to a Theoretical Plate

Length of a column necessary for the attainment of compound distribution equilibrium measure the efficiency of the column.

Theoretical plates (N) = 16 ( )X

Y2

X

Y

Importance of Theoretical Plates (N)

Theoretical Plate, Selectivity and Height Equivalent to a Theoretical Plate

1

2

3

4V

V

V

V

W W W W

2

1

0

1

2

4

3 4

3

V

V0 = 1.0 (Minutes) V1 = 5.0, V2 = 7.0, V3 = 11.0, V4 = 13.0

W1 = 1.0, W2 =1.0, W3 = 1.0, W4 =1.0

Chromatogram of Orange Juice Compounds

General Factors Increasing Resolution

• Increase column length• Decrease column diameter• Decrease flow-rate• Pack column uniformly• Use uniform stationary phase (packing material)• Decrease sample size• Select proper stationary phase• Select proper mobile phase• Use proper pressure• Use gradient elution

LC Application in Food System

Carbohydrates

Amino acids, proteins

Vitamins, A, D, E, K

Nucleosides (purines and pyrimidines)

Fatty acids, fats

Aflatoxins

Antioxidants

Contaminants of packaging materials

Carotenoids, chlorophylls

Saccharines