1. CHROMATOGRAPHY

2. 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.

3. 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

4. Kinds of Chromatography
1. Liquid Column Chromatography
2. Gas Liquid Chromatography

5. 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.

6. 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 O F SIMPLE LIQ UID CO LUMN CHRO MATO GRAPHY
A
B
C
Solvent(mobile or
moving phase)
Diagram of Simple Liquid Column Chromatography

7. 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

8. Liquid Solid Chromatography
30 µ
Si - O - H
δ− δ+
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.

9. Liquid Solid Chromatography
Si - OH
HEXANE
OH
C-CH3
CH3
CH3 - C
CH3
CH3
OH
OH
CH3
CH3

10. Water-Soluble Vitamins
1. Niacinamide 2. Pyridoxine
N
CONH2
N
CH2OH
CH2OH
HO
H3C
3. Riboflavin
N
N
NH
N
CH2
HOCH
HOCH
HOCH
CH2OH
O
OH3C
H3C
Cl
N
S
N
NH3C
CH2
NH2
CH3
CH2CH2OH
4. Thiamin

11. Water-Soluble Vitamins
0 5 10 15 20
Column: u Bondapak C18
Solvent: MeOH
Sample: Water-Soluble Vitamins
Inject
1
2
3
4

12. Liquid-Liquid Chromatography
ODPN (oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC
NCCH
3
CH
2
OCH
2
CH
2
CN(Normal)
CH
3
(CH
2
)
16
CH
3
(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.

13. MOBILE PHASE
LIQUID
Liquid-Liquid
Chromatography (Partition)
Liquid-Solid
Chromatography (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

14. 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).

15. Mechanism of Ion-Exchange Chromatography of Amino Acids
SO3
-
SO3
-
Na
+
COO
-
H3N
+
Na
+
COOH
H3N
+
pH2
pH4.5
Ion-exchange Resin

16. 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

17. 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

18. • Polar Solvents
Water > Methanol > Acetonitrile > Ethanol >
Oxydipropionitrile
• Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >
Cyclohexane
Solvents

19. 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

20. Schematic Diagram of Liquid Chromatography

21. Detector
1. Ultraviolet Detector
200-400nm
254 nm
2. Reflective Index Detector
Universal Detector

22. High Performance Liquid Chromatography

23. High Performance Liquid Chromatography

24. 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

25. Selectivity
Ratio of Net Retention Time of 2 components.
(Distribution Coefficient)
X2 - X0
X1 X0-α=

26. Response
Retention Time
X
X
X
1 3 6
2
1
0
– Selectivity
SelectivitySelectivity

27. 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

28. Resolution

29. 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
Y
2
X
Y

30. Importance of Theoretical Plates (N)

31. 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

32. Chromatogram of Orange Juice Compounds

33. 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

34. 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