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.

Distribution Coefficient
Definition: Concentration of component A in stationary phase Concentration of component A in mobile phase

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

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.

Diagram of Simple Liquid Column Chromatography

DIAGRAM OF SI MPLE LIQUI D COLUMN CHROMATOGRAPHY Solvent(mobile or moving phase) A+ B+C Sample (A+ B+C) OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO A OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOB OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOC OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO

OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO Column OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO Solid Partic les OOOOOOOOOOO (packing materialOOOOOOOOOO stationary phase) OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO Eluant (eluate)

Four Basic Liquid 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)

Liquid Solid Chromatography

Normal phase LS Reverse phase LS d- d+ Si - O - H

30 m

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

OH

HEXANE

Si - OH

CH 3

OH

CH 3 C-CH CH 3
3

CH 3 - C
CH 3

CH 3

Water-Soluble Vitamins

1.

Niacinamide
N

2.

Pyridoxine H3C HO

N CH2OH CH2OH

CONH 2

3.

Riboflavin CH2OH HOCH HOCH HOCH CH2 H3C N N

H3C N O

4. Thiamin

O NH

H3C N

NH 2 CH2 N

CH2CH2OH Cl CH3

Water-Soluble Vitamins
2

3 Inject 1 4

10

15

20

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

Liquid-Liquid Chromatography
ODPN (oxydipropionylnitrile)
Normal Phase LLC Reverse Phase LLC

NCCH CH OCH CH CN(Normal) 3 2 2 2 CH (CH ) CH (Reverse) 3 2 16 3

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.

Types of Chromatography
MOBILE PHASE LIQUID

FORMAT

Liquid-Liquid Chromatography (Partition)

Liquid-Solid Chromatography (Adsorption)

STATIONARY PHASE

Liquid

Solid

Normal Phase Mobile Phase - Nonpolar Stationary phase - Polar

Reverse Phase Mobile Phase - Polar

Normal Phase

Reverse Phase

Stationary phase - Nonpolar

Ion-Exchange Chromatography

SO 3 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

pH2
+ +

SO 3

Na

H3N

COOH

Ion-exchange Resin

SO 3

H 3N Na
+

COO

pH4.5

Chromatography of Amino Acids

Station ary Phase SO3 Na+
-

Mobile Ph ase H3 N Na
+ +

COOH OH H3 N
+

SO3

COOH Exc hange Resin SO3 H3N+ COOH SO3

-

pH3. 5 OH

H3 N
+

Na Na SO3
+

COO

OH = H2 O

H3 N
-

COO SO3Na+

OH = H2 O

pH4. 5

Gel-Permeation Chromatography

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.

Solvents
Polar Solvents Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile Non-polar Solvents

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

Selecting an Operation Mode

Sample Type
Positional isomers Moderate Polarity Molecules Compounds with Similar Functionality Ionizable Species Compounds with Differing Solubility Mixture of Varying Sized Molecules

LC Mode
LSC or LLC LSC or LLC LSC or LLC IEC LLC GCC

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.
Res pons e D

10

15

20

25

Retention Time

Selectivity
Ratio of Net Retention Time of 2 components.
(Distribution Coefficient)

X2 X1

X0 X0

Selectivity
Selectivity

Response X

X1 X0

3 Retention Time

Resolution Equation
R= V - V1 2 1/2(W1 + W2)

Res pons e

V2

V1

W1 W1 Volumes

W 2 W 2

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.

X 2 Theoretical plates (N) = 16 ( ) Y

X

Importance of Theoretical Plates (N)

Theoretical Plate, Selectivity and Height Equivalent to a Theoretical Plate

2 V2 4

V1 V0

1 3 W1 V3 W2 W3 W4

V4

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