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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
Kinds of 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.
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)
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.
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.
4. Thiamin
O NH
H3C N
NH 2 CH2 N
CH2CH2OH Cl CH3
Water-Soluble Vitamins
2
3 Inject 1 4
10
15
20
Liquid-Liquid Chromatography
ODPN (oxydipropionylnitrile)
Normal Phase LLC Reverse Phase LLC
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
STATIONARY PHASE
Liquid
Solid
Normal Phase
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).
pH2
+ +
SO 3
Na
H3N
COOH
Ion-exchange Resin
SO 3
H 3N Na
+
COO
pH4.5
Mobile Ph ase H3 N Na
+ +
COOH OH H3 N
+
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
LC Mode
LSC or LLC LSC or LLC LSC or LLC IEC LLC GCC
Detector
1. Ultraviolet Detector
200-400nm 254 nm
2.
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
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