Chromatography
General
Chromatographic Process
Chromatographic Systems
Chromatographic Techniques
TLC/PC
PC-Paper Chrom
HPLC
GC/SFC
Chromatography – Separation Mechanism
 Adsorption
 Partition
 Ion - Exchange & Ion - Interaction
 Size Exclusion
 Affinity (antibody-antigen interactions;
chemical interaction; attraction)
 Complexation - Chelation
 Ion – exclusion (Separation of weak acids)
Sorption problems
ADsorption
ABsorption
Different sorptions explained
Chromatograhy – Mechanism of Separation
Adsorption
Partition
Ion exchange
Chromatography – Mechanism of Separation
Affinity
Size Exclusion
Chromatogram – Basic Parameter
tR = retention time
tm = dead time
W1/2
1/2H
unretained
H
Chromatographic Theories
 Adjusted retention time:
tR’ = tR – tM
 Plate theory – distillation – plate
number
N = 5.54[(tR – tM)/w1/2]2
 Plate height H = L/N
 This theory did not include interaction of analytes
with stationery phase
Chromatography – Peak Broadening
Chromatographic Theories
 Rate Theory – kinetic factors – van
Deemter
H = B/u + Cu (+ A)
Where: u – velocity of mobile phase
B – effect of molecular diffusion
C – Resistance to mass transfer
A – Spreading related to different
distance traveled by molecules
in packed columns
Chromatography – Packing Effect on Broadening
Chromatography - Equilibrium
Amobile
Astationary
Van Deemter factors:
Molecular diffusion (B) – in mobile
phase
 proportional to time analyte spends in
a column
 affected by diffusion coefficient of
analyte in mobile phase
 affected by temperature and pressure
 not important in LC – low diffusion
coefficient
 inversely affected by mobile phase
velocity
Van Deemter factors:
Resistance to mass transfer (C):
 Mass transfer in mobile and stationary
phase
 Lack of equilibrium – moving phase
 Affected by thickness of liquid phase
 Affected inversely by the diameter of
particles or inner diameter of capillary
column
 Lower at higher temperatures (viscosity)
Van Deemter factors:
Conclusions:
 Minimum value for H is achieved
when:
 stationery phase thickness is minimal
 column packed with the smallest particles
 capillary columns have the smallest
internal diameter
 mobile and stationary phases have low
viscosity and high diffusion coefficient
Plate height (cm)
Chromatography – van Deemter Plot
H
Cu
Mass transfer
A
Multipath effect
B/u Diffusion (Longitudinal)
Mobile phase velocity
Chromatography - Resolution
R = 2(tR1 – tR2)/Wb1 – Wb2
tR1 tR2
DtR
Response
100%
Baseline resolution
for Gaussian
shape peaks = 1.5
Wb1
Wb2
Chromatography - Resolution
 Resolution equation where separation
parameters are included:
Rs = ½ x (a-1/a+1) x k’2/1+k’2x (L/h)1/2
Where: a – selectivity factor (separation) a = tR1/tR1
k’ – migration term, capacity factor;
k’ = ms/mm
L – column length
h – plate height
Chromatography - Resolution
Chromatography
 Qualitative Analysis
 Retention data – RT; Rf; RRT; Kovacs
Index
 Quantitative Analysis
 Peak area and height usually proportional
to the amount of component
 Calibration
 Internal Standard method
 External Standard method
 Area Normalization method
Chromatogram – Basic Parameter
tR = retention time
tm = dead time
W1/2
1/2H
unretained
H
4
RRT1 = RT1/RTIS
RRT2 = RT2/RTIS
RRT3 = RT3/RTIS
3
IS α - Cholestane
Cholesterol
1
Accurate to e few
digits (2) at fourth
Decimal Point
2
5
END
Chromatography - Methodology
Peaks Broadening