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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 SIMPLE LIQUID C OLUMN C HROMATOGRAPHY
S olvent(mobile or
moving phas e)
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
A
OOOOO
OOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
Column
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO S olid Particles
B OOOO
OOOOOOOOOOO
(packing material- OOOOO
OOOOOOOOOO s tationary phas e) OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
C OOOO
OOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
A+ B + C

S ample
(A+B+C)

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

Si - O - H

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 3
CH 3

CH 3 - C
CH 3

CH 3

Water-Soluble Vitamins
1.

Niacinamide

2.

Pyridoxine
H3C

N
HO
CONH 2
3.

H 3C
H 3C

CH 2OH

Riboflavin
CH 2OH
HOCH
HOCH
HOCH
CH 2
N
N

4. Thiamin

O
NH

N
O

CH 2OH

H3C

N
N

NH 2
CH 2

S
N

CH 2CH 2OH
Cl
CH 3

Water-Soluble Vitamins
2

3
Inject
1

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
LIQUID

MOBILE PHASE

Liquid-Liquid
Chromatography (Partition)

FORMAT

STATIONARY PHASE

Normal Phase

Liquid-Solid
Chromatography (Adsorption)

Solid

Liquid

Reverse Phase

Normal Phase

Mobile Phase - Nonpolar

Mobile Phase - Polar

Stationary phase - Polar

Stationary phase - Nonpolar

Reverse Phase

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


Stationary Phase

Mobile Phase
H3 N

SO 3 Na+

COOH
+

Na
SO 3

OH
H3 N

COOH
Exchange Resin
-

SO 3 H3N+
COOH
SO3

pH3.5

OH

H 3 N+
+

Na

COO

OH = H 2 O

Na
SO 3

H3 N

+
-

COO

OH = H 2 O

SO 3Na+
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

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

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
D

10

15

Retention Time

20

25

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
V2 - V

R=

1/2(W + W2)
1

Response

V2

V1

W2

W1

W2
Volumes

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
Theoretical plates (N) = 16 ( )2
Y

Importance of Theoretical Plates (N)

Theoretical Plate, Selectivity and Height Equivalent


to a Theoretical Plate

2
4

V2

V1

V0
W2

W1

W3

W4

V3

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