1. Laboratory class on next Wednesday
Please, everyone bring a laboratory coat
On Mon or Tue, instructions for a laboratory class will be uploaded on the Department’s portal, please, download them

2. Chromatography

3. Characteristics of elution
tR – retention time
Retention time is the time for a sample to travel through a chromatographic column
t0 – hold-up time
Hold-up time is the time necessary for the mobile phase to travel through a chromatographic column
k’ – retention factor
Retention time (tR) can be used for identification purposes but this characteristic depends on the flow rate. The retention factor (k’) is the parameter that does not depend on the flow rate. Therefore it is more preferable for qualitative analysis

4. Figures of merit for separation
Selectivity (a)
Resolution (Rs)
Selectivity (a) shows potential of a given stationary phase to the separation of a pair of compounds. It can give a false concept of a real resolution as the latter depend on the widths of peaks. The resolution (Rs) value takes into account the dispersion of peaks, since is more useful for the selection of media for chromatographic separation.
Selectivity does not depend on the widths of peaks whereas Resolution does.

5. Theoretical plate concept and the broadening of chromatographic peaks
There are several reasons causing the broadening of chromatographic peaks. Those are (1) slow kinetics of mass transfer, (2) diffusion in the pores of the adsorbent particles, and (3) axial dispersion in the interparticle volume.
The lower the rate of mass transfer processes, the broader the chromatographic peak.
N = 400
N = 100
A parameter characterizing the broadening of chromatographic peaks is the number of theoretical plates N. It relates to the apparent dispersion Da of a peak as N = uL/2Da (u is the linear flow velocity, L the length of a column).
The larger N, the narrower the peak
N = 50
Retention time depends only on the affinity of an analyte to the stationary phase. However the width of a chromatographic peak is a function of kinetics of mass transfer processes in a chromatographic column. This dependence is reflected in a concept of the theoretical plate. According to the plate theory, a chromatographic column is divided by a number of small sections (plates). The length of a plate is enough for adsorption equilibrium to be established under dynamic conditions, that is while a flow of an analyte passes through the column.

6. N Resolution and the broadening of chromatographic peak
Dependence on N N
N = Rs = 0.6
N = Rs = 1.0
N = Rs = 3.2

7. Resolution and the Loading
Loading is the amount of a sample applied to chromatographic separation
Loading = 1

8. Resolution and the Loading

9. Resolution and the Loading

10. Types of chromatography with respect to loading Maximal Loading
Separation
Column diameter
~ mg – mg
~ 100 mg
~ g
Analytical
Semi-preparative
Preparative
2 - 5 mm
10 – 20 mm
20 – 50 mm
Amount of an adsorbent needed for the separation of a certain amount of a sample can be evaluated according to an approximate rule 1:20. We need 20 g of an adsorbent per 1 g of a sample.

11. Comparison Analytical vs. Preparative chromatography
Analytical requirements
Precision
Accuracy
Sensitivity
Reproducibility
Robustness
Diluted samples, expensive solvents and adsorbents
Preparative requirements
Recovery
Product purity
Productivity
Costs
Concentrated samples, cheap solvents and adsorbents

12. Chromatography
Planar
Column
TLC
(Thin layer
chromatography)
Paper

13. Thin layer chromatography
Phase systems for TLC
Adsorbent
Eluent
Typical analytes
Polar adsorbents
Silica (SiO2)x
Alumina (Al2O3)
Non-polar adsorbents
Polymers
ODS-grafted silica
non-aqueous solvents
aqueous solvents
non- or medium-polar organics
polar and ionogenic organics

14. Silica gel Structure of silica
Silica gel is a granular porous form of silica
Due to the surface hydroxyls it has a strong affinity towards polar organics that are able to hydrogen bonding and strong dipole-dipole interactions.
Such compounds will longer retain on the silica whereas non-polar components will be eluted first

15. Silica gel Nontreated silica Acid washed analysis of acidic components
Base washed analysis of basic components

16. ODS modified Silica gel
ODS – octadecylsilane
hydrophobic surface
Hydrophobic (non-polar) compounds are stronger retained whereas hydrophilic compounds are eluted first

17. TLC Plate Support 200 mm for analytical chromatography
1000 mm for preparative chromatography
The adsorbent layer is prepared from an adsorbent, a binder, and, optionally, a fluorescent indicator
Adsorbent layer
Support
(Metallic or glass)