Transcript Document

CHROMATOGRAPHY
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
Different affinity of these 2 components to stationary
phase causes the separation.
Kinds of Chromatography
1. Liquid Column Chromatography
2. Gas Liquid 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 O F S IMPLE LIQ UID C O LUMN C HRO MATO G RAPHY
Solvent (m obile or
moving phase)
A+ B+C
Sam ple
(A+B+C)
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
Column
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
Solid P articles
OOOOOO OOOOO (packing materialOOOOOO OOOO stationary phase)
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
Eluant (eluat e)
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOA OOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOB OOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOC OOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
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)
Liquid Solid Chromatography
Normal phase LS
Reverse phase LS
d- d+
Si - O - H
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.
Liquid Solid Chromatography
OH
HEXANE
Si - OH
CH 3
OH
CH 3
C-CH
CH 3
CH 3 - C
CH 3
CH 3
3
Water-Soluble Vitamins
1.
Niacinamide
2.
Pyridoxine
H 3C
N
N
HO
CH 2OH
CH 2OH
CONH 2
3.
Riboflavin
CH 2OH
HOCH
HOCH
HOCH
CH 2
H3C
N
N
H3C
4. T hiamin
O
NH
N
O
H 3C
N
N
NH 2
CH 2
S
N
CH 2CH 2OH
Cl
CH 3
Water-Soluble Vitamins
2
3
Inject
1
0
4
5
10
15
20
Column: u Bondapak C18
Solvent : MeOH
Sam ple: Water-Soluble Vit amins
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.
Types of Chromatography
LIQUID
MOBILE PHASE
Liquid-Liquid
Chromatography (Partition)
FORMAT
STATIONARY PHASE
Normal Phase
Liquid-Solid
Chromatography (Adsorption)
Solid
Liquid
Reverse Phase
Normal Phase
Mobile Phase - Nonpolar
Mobile Phase - Polar
Stationary phase - Polar
Stationary phase - Nonpolar
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).
Mechanism of Ion-Exchange Chromatography of Amino Acids
pH2
SO 3
-
Na
+
H3N
+
COOH
Ion-exchange Resin
SO 3
-
H 3N
Na
+
+
COO
-
pH4.5
Chromatography of Amino Acids
Statio nar y P h ase
Mo bile P h ase
H3 N
-
SO 3 Na+
+
COOH
+
Na
SO 3
OH
-
H3 N
+
COOH
Ex ch an ge Resin
-
SO 3 H3N+
COOH
SO 3
p H3 .5
OH
-
H3 N+
+
-
Na
COO
H
+
-
OH = H2 O
+
Na
SO 3
-
H3 N
+
-
COO
H
+
-
OH = H 2 O
-
SO 3Na+
p H4 .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 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
Schematic Diagram of Liquid Chromatography
Detector
1.
Ultraviolet Detector
200-400nm
254 nm
2.
Reflective Index Detector
Universal Detector
High Performance Liquid Chromatography
High Performance Liquid Chromatography
Retention Time
Time required for the sample to travel from the injection port through
the column to the detector.
Resp o n se
D
B
A
C
5
10
15
Reten tion T ime
20
25
Selectivity
Ratio of Net Retention Time of 2 components.
(Distribution Coefficient)
 
X2
X1
-
X0
X0
Selectivity
–
Selectivity
Response
X
2
X1
X0
1
3
Retention Time
6
Resolution Equation
V2 - V1
R=
1/2(W1 + W2)
Response
V2
V1
W
W2
1
W1
W2
Volumes
Resolution
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
Importance of Theoretical Plates (N)
Theoretical Plate, Selectivity and Height Equivalent
to a Theoretical Plate
2
4
V2
V1
1
3
V0
W2
W1
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
Chromatogram of Orange Juice Compounds
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
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