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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. 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.

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. Diagram of Simple Liquid Column Chromatography
DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY
Solvent(mobile or
moving phase)
OOOOOOOOOOO
A+B+C Sample OOOOOOOOOOO
(A+B+C) OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO A
OOOOO OOOO
OOOOOOOOOO Column OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO Solid Particles OOOOOOOOOO
OOOOOOOOOOO (packing material- OOOOOB OOOO
OOOOOOOOOO stationary phase) OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOC OOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
Eluant (eluate)

7. 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)

8. Liquid Solid Chromatography
Normal phase LS
Reverse phase LS
δ− δ+
Si - O - H
30 µ 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
OH HEXANE
Si - OH
OH CH3
CH3
CH3 - C C-CH3
CH3
CH3
CH3

10. Water-Soluble Vitamins
1. Niacinamide 2. Pyridoxine
H 3C N
N
HO CH 2OH
CONH 2 CH 2OH
3. Riboflavin
CH 2OH
HOCH
HOCH 4. Thiamin
HOCH
CH 2
H 3C N N O H 3C N NH 2 S CH 2CH 2OH
NH Cl
H 3C N N N
CH 2 CH 3
O

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

12. 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.

13. Types of Chromatography
LIQUID
MOBILE PHASE
Liquid-Liquid Liquid-Solid
FORMAT Chromatography (Partition) Chromatography (Adsorption)
Liquid Solid
STATIONARY PHASE
Normal Phase Reverse Phase Normal Phase Reverse Phase
Mobile Phase - Nonpolar Mobile Phase - Polar
Stationary phase - Polar Stationary phase - Nonpolar

14. 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).

15. Mechanism of Ion-Exchange Chromatography of Amino Acids
pH2
- + +
SO 3 Na H3N
COOH
Ion-exchange Resin
- +
SO 3 H 3N
-
COO pH4.5
+
Na

16. Chromatography of Amino Acids
Stationary Phase Mobile Phase
+
H3 N
-
SO3 Na+
COOH
+
Na
OH
- +
SO3 H3 N
COOH
Exchange Resin
-
SO3 H3N+
COOH
pH3.5
OH
-
SO3 H 3 N+
+ - + -
Na COO H OH = H 2 O
+
Na
-
SO3 H3 N
+
- + -
COO H OH = H 2 O
-
SO3Na+
pH4.5

17. 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.

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

19. 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

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
D
B
A
C
5 10 15 20 25
Retention Time

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

26. Selectivity
– Selectivity
Response
X 2
X1
X0
1 3 6
Retention Time

27. Resolution Equation
V - V1
2
R=
1/2(W1 + W2)
Response
V2
V1
W W
1 2
W1 W2
Volumes

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

30. Importance of Theoretical Plates (N)

31. Theoretical Plate, Selectivity and Height Equivalent
to a Theoretical Plate
2
V2 4
V1 1
3
V0
W1 W2 W3 W4
V3
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

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