CHROMATOGRAPHY

Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between 2 different phases. One of these phases is a mobile phase and the other is a stationary phase.

Distribution Coefficient (Equilibrium Distribution )

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

3. Thin-layer 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 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 Particles 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

The 4 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 30 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 N HO CONH 2 3. Riboflavin CH 2OH HOCH HOCH HOCH CH 2 N N N O O NH CH 2OH CH 2OH 2. Pyridoxine H 3C N

4. Thiamin

H 3C H 3C

H 3C N

NH 2 CH 2 N

CH 2CH 2OH Cl CH 3

WATER-SOLUBLE VITAMINS
2

3 Inject 1 4 Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins

10

15

20

LIQUID-LIQUID CHROMATOGRAPHY

ODPN(oxydipropionylnitrile) Normal Phase LLC Reverse Phase LLC

NCCH CH2OCH CH2CN(Normal) 3 2 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.

Types of Chromatography
M B EP A E O IL H S L U IQ ID

F R A OMT

L u -L u iq id iq id C ro ato rap y h m g h (P artitio ) n

L u -S lid iq id o C ro ato rap y h m g h (A so tio ) d rp n

S A IO A Y T T N R P AE H S

L u iq id

S lid o

N rm P ase o al h M b P ase o ile h Nn o o p lar S tatio ary p ase n h P lar o

R erse P ase ev h M b P ase o ile h P lar o S tatio ary p ase n h Nn o o p lar

N rm P ase o al h

R erse P ase ev h

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

pH2
+ +

SO3

Na

H3N

COOH

Ion-exchange Resin

SO3

H3N Na

COO

pH4.5

Chromatography of Amino Acids

Stationary Phase SO 3 Na + N a SO 3 Exchange Resin SO 3 H3N+ CO OH SO 3
-

M obile Phase H3N

+ +

CO H O O H H3 N
+

CO OH

O H H3 N
+ +

pH3.5

N a N a SO 3
+

CO O

O H = H 2O

H3 N
-

CO O SO 3N + a

O H = H 2O

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 OPERATING 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

Detectors 1.
Ultraviolet Detector
200-400nm 254 nm

2.

Reflective Index Detector

Universal Detector

High Performance Liquid Chromatography

High Performance Liquid Chromatography

Chromatogram of Organic Compounds from Fermented Cabbage

Chromatogram of Orange Juice Compounds

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

Rs os ep ne D

1 0

1 5

2 0

2 5

R t nio T e ee t n im

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

X2 X1

X0 X0

Selectivity

R esponse

Selectivity

X1 X0

3 R etention Tim e

RESOLUTION EQUATION
R= V - V1 2 1/2(W1 + W2)

Response

V2

V1

W 2 W 2 Volum es

W1

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

RESOLUTION

EXAMPLES OF THEORETICAL PLATE, SELECTIVITY AND HEIGHT EQUIVALENT TO A THEORETICAL PLATE

2 V2 4

V1 V0

3 W2 V3 W3 W4

W1

V0 = 1.02(Minutes) W1 = 1.0 (Minutes)

V1 = 4.92 V2 = 6.59 V3 = 8.17 V4 = 9.14 W2 =1.0W3 = 1.0 W4 =1.0

GENERAL FACTORS INCREASING RESOLUTION

1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

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