1. LU 3: Separation Techniques

2. Classification of separation techniques
Basis of Separation Separation Technique
size filtration; dialysis; size exclusion chromatography
mass and density centrifugation
complex formation masking
change in physical state distillation; recrystallization
change in chemical state precipitation; ion exchange; electro-deposition; volatilization
partitioning between phase extraction; chromatography

3. What is Chromatography?
Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.
Greek word chroma – “color” and graphein – “to write”.
Separate
Analyze
Identify
Purify
Quantify
Components
Mixture

4. Chromatography
Separations method - differences in partitioning behavior (migration rates) between a flowing mobile phase and a stationary phase to separate the components in a mixture.
Column (or other support: e.g: TLC) holds the stationary phase and the mobile phase carries the sample through it.

5. Terms in Separation Techniques
Stationary phase: a phase that is fixed in place either in a column or on a planar surface.
Mobile phase :a phase that moves over or through the stationary phase, carrying the analyte with it.
Eluent: a solvent used to carry the components of a mixture through stationary phase.

6. Uses of Chromatography
Chromatography is used by scientists to:
Analyze – examine a mixture, its components, and their relations to one another
Identify – determine the identity of a mixture or components based on known components
Purify – separate components in order to isolate one of interest for further study
Quantify – determine the amount of a mixture and/or the components present in the sample

7. Chromatography Basics
How it works:
sample is loaded onto the polar stationary phase.
Polar compounds will adsorb onto the stationary
phase to a greater extent than non-polar
compounds.
The mobile phase (eluting phase) helps “push” or
elute the compounds either down a column (for
CC) or up a plate (for TLC).
The main concept to consider in chromatography is polarity.

8. Polarity & Intermolecular Attractive Forces
More polar compounds will be more attracted to silica gel than non-polar compounds due to intermolecular attractive forces
- a dipole-dipole interaction.
The more non-polar compounds - will travel more easily and more quickly through the stationary phase.
The mobile phase helps carry the compounds through the stationary phase.
Separation of compounds in a mixture is possible because compounds have different polarities. Non-polar compounds will elute first and polar compounds will elute last.
Silica gel, [SiO2]n

9. Components that are weakly retained by stationary phase will move through the system more rapidly

10. Paper Chromatography / Thin Layer Chromatography (TLC)
Method for testing the purity of compounds and identifying substances.
Useful technique because it is relatively quick and requires small quantities of material.
Substances - distributed between a stationary phase and a mobile phase.
The stationary phase: a piece of high quality filter paper/porous paper.
The mobile phase: developing solution that travels up the stationary phase, carrying the samples with it.

11. Paper Chromatography / Thin Layer Chromatography (TLC)
Solid-liquid technique.
Two phases are a solid (stationary phase) and a liquid (mobile phase).
Solids most commonly used in chromatography are silica gel (SiO2) and alumina (Al2O3). Both of these adsorbents are polar.
Silica is also acidic. Alumina is available in neutral, basic, or acidic forms.

13. Spotting a TLC plate with sample
Running the TLC plate in solvent

14. Theory of paper chromatography
A small sample of a mixture is placed on porous paper which is in contact with the solvent.
The solvent moves through the paper due to capillary action and dissolves the mixture spot.
Components of the mixture with a stronger attraction to the paper  move more slowly.

15. Thin Layer Chromatography (TLC)
Visualization There are various techniques to visualize the compounds. 1. Sulfuric acid/heat: destructive, leaves charred blots behind. 2. Iodine: semi-destructive, iodine absorbs onto the spots, not permanent. 3. UV light: non-destructive, long wavelength (background green, spots dark), short wavelength (plate dark, compounds glow).

16. Uses of TLC
To determine how many components there are in a mixture (is it really pure?)
To determine the best solvent conditions for separation on a column.
To monitor the composition of fractions collected from column chromatography.
To detect the existence of toxin (e.g. TTX) from the biological samples.

17. Thin Layer Chromatography (TLC)
Advantages
Sensitive, fast, simple and inexpensive analytical technique.
It is a micro technique; as little as 10-9g of material can be detected, although the sample size is from 1 to 100 x 10-6 g.

18. Retention Factor (Rf)
Rf = distance spot traveled from origin line/distance
of solvent front

20. Thin Layer Chromatography (TLC)
The Rf depends on the following parameters:
-solvent system
-absorbent (grain size, thickness)
-amount of material spotted
Ideally Rf value: 0.2 ≤Rf ≤0.8.
Adjust the Rf with solvent polarity. Rf increase with increasing polarity.

21. Thin-Layer Chromatography: A Two-Component Mixture
More polar!
Less polar! s o l v e n t f r i g
mixture c m p B A
Increasing Development Time

22. Calculate the Rf value

24. Which samples plotted on the TLC plate below composed of more than one substance?

25. Answer :
Samples 1 and 3 were composed of more than one substance, as because during the TLC experiment two or more spots separated from the original sample.
Sample 2 may have been composed of more than one substance, but if it was, the substances did not separate during the experiment.