1. Chromatography General

2. Chromatographic Process

3. Chromatographic Systems

4. Chromatographic Techniques
TLC/PC
PC-Paper Chrom
HPLC
GC/SFC

5. 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)

6. Different sorptions explained
Sorption problems
ADsorption
ABsorption
Different sorptions explained

7. Chromatograhy – Mechanism of Separation
Partition
Ion exchange
Adsorption

8. Chromatography – Mechanism of Separation
Affinity
Size Exclusion

9. Chromatogram – Basic Parameter
tR = retention time
tm = dead time H
W1/2
1/2H
unretained

10. 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

12. Chromatography – Peak Broadening

14. 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

15. Chromatography – Packing Effect on Broadening

16. Chromatography - Equilibrium
Amobile Astationary

17. Molecular diffusion (B) – in mobile phase
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

18. 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)

19. 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

20. Chromatography – van Deemter Plot
Plate height (cm) Cu
Mass transfer A
Multipath effect
B/u
Diffusion (Longitudinal)
Mobile phase velocity

21. Chromatography - Resolution
DtR
tR1 tR2
R = 2(tR1 – tR2)/Wb1 – Wb2
Response
100%
Baseline resolution
for Gaussian
shape peaks = 1.5
Wb1
Wb2

22. 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

23. Chromatography - Resolution

24. Chromatography Qualitative Analysis Quantitative 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

25. Chromatogram – Basic Parameter
tR = retention time
tm = dead time H
W1/2
1/2H
unretained

26. IS α - Cholestane 1 RRT1 = RT1/RTIS RRT2 = RT2/RTIS RRT3 = RT3/RTIS 34 1
RRT1 = RT1/RTIS
RRT2 = RT2/RTIS
RRT3 = RT3/RTIS 3
Cholesterol
Accurate to e few
digits (2) at fourth
Decimal Point
IS α - Cholestane 2 5

27. END

28. Chromatography - Methodology

29. Peaks Broadening