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)

Solid articles (packing materialstationary phase)

luant (eluate)

OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO

olumn

A+B+C

Sample ( +B+ )

OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOO A OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO BOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO COOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO

FOUR A IC LIQUID CHROMATOGRAPHY

The 4 basic liquid chromatography modes are named according to the mechanism involved:

1. Liquid/Solid hromatography (adsorption chromatography) A. Normal Phase L C . Reverse Phase L C 2. Liquid/Liquid hromatography (partition chromatography) A. Normal Phase LLC . Reverse Phase LLC 3. Ion xchange hromatography 4. el ermeation hromatography (exclusion chromatography)

LIQUID OLID CHROMATOGRAPHY

ormal phase L Reverse phase L HH i-O-H Q ilica Gel

The separation mechanism in L C is based on the competition of the components of the mixture sample for the active sites on an absorbent such as ilica Gel.

LIQUID OLID CHROMATOGRAPHY

OH

H XA

i - OH

CH 3

OH

CH 3 C-CH 3 CH 3

CH 3 - C
CH 3

CH 3

WATER- OLU LE VITAMIN

1. Niacinamide 2. Pyridoxine

H3C
N

N CH2OH CH2OH

HO
CONH2
3. Riboflavin

H3C H3C

CH2OH HOCH HOCH HOCH CH2 N N N O

4. Thiamin

O NH

H3C N

NH2 N CH2

CH2CH2OH Cl CH3

WATER- OLU LE VITAMIN

I j

8 M O W V

LIQUID-LIQUID CHROMATOGRAPHY

ODPN(oxydipropionylnitrile) Normal Phase LLC Reverse Phase LLC

NCCH3 CH2 OCH2 CH2 CN(Normal) CH3 (CH2 ) 16 CH3 (Reverse)

The stationary solid surface is coated with a 2nd liquid (the tationary 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 OBI S I ID

ORM AT

iq u id - iq u id h ro m a to g ra p h y ( a rtitio n )

iq u id -S o lid h ro m a to g ra p h y ( d s o rp tio n )

S T A T IO N A R Y PH AS

iq u id

S o lid

o rm a l M o b ile

hase

e v e rse

hase

o rm a l

hase

e v e rse

hase

hase o n p o la r S ta tio n a ry p h a s e o la r

M o b ile h a s e o la r S ta tio n a ry p h a s e o n p o la r

ION-E CHANGE CHROMATOGRAPHY

SO N

Sep r tion in Ion-exch nge Chrom togr phy is b sed on the competition of different ionic compounds of the s mple for the ctive sites on the ion-exch nge resin (column-p cking).

MECHANI M OF ION-E CHANGE CHROMATOGRAPHY OF AMINO ACIDS

pH2
-

SO 3

Na

H3N COOH

Ion-exchange Resin

SO 3

H3N Na

COO

pH4.

Chromatography of Amino Acids

Stationary Phase
3

Mobile Phase H 3N Na SO
3

SO

Na

H 3N

Exchange Resin
3

SO

H 3N

COOH SO
3

Na Na SO
3

COO

COO SO 3 Na
-

H 3N

OH

H 3N

COOH OH COOH pH3. OH OH

-

=H

2O

=H

2O

pH4.

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 SolubilityLLC Mixture of Varying Sized Molecules GCC LC Mode LSC or LLC LSC or LLC LSC or LLC IEC

Schematic Diagram of Liquid Chromatography

Detectors 1.
Ultraviolet Detector
200200-400nm 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 in ection port through the column to the detector.

Response D

Retention Time

C 1 2 2

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

E!

Selectivity

Selectivity

R esponse

3 R e te n tio n T im e

RESOLUTION EQUATION
R
2

1/2(W1 + W2)

W1 W1

W2 W2

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 (

)2

RESOLUTION

E AMPLES OF THEORETICAL PLATE, SELECTIVITY AND HEIGHT EQUIVALENT TO A THEORETICAL PLATE

V0 = .0 (Minutes) W = .0 (Minutes)

V = .9

W = .0W = .0

V = 6. 9 V = . 7 V = 9. W = .0

  

 

GENERAL FACTORS INCREASING RESOLUTION

1.

2. 3. 4. . 6. 7. . 9. 1 .

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