1. Chapter: Chromatography

2. Introduction to Chromatography
Definition
Chromatography is a separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium.
Components:
mobile phase: a solvent that flows through the supporting medium
stationary phase: a layer or coating on the supporting medium that interacts with the analytes
supporting medium: a solid surface on which the stationary phase is bound or coated

3. What is Chromatography?
"Chromato" "graphy" derives its name from two words as chromo= colour and graphy= writing. I.e. colour bands are formed in the procedure which are measured or analyzed. These colour bands are due to separation of individual compounds at different lengths on the column as seen in column chromatography and on paper in paper chromatography.
Chromatography is a technique for separating mixtures into their components in order to identify, analyze, purify, and/or quantify the mixture or components.
Analyze
Identify
Purify
Quantify
Separate
Mixture
Components

4. Uses for 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 the a mixture and/or the components present in the sample

5. Uses for Chromatography
Real-life examples of uses for chromatography:
Pharmaceutical Company – determine amount of each chemical found in new product
Hospital – detect blood or alcohol levels in a patient’s blood stream
Environmental Agency – determine the level of pollutants in the water supply
Manufacturing Plant – to purify a chemical needed to make a product

6. Definition of Chromatography
Detailed Definition:
Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass.
Terminology:
Differential – showing a difference, distinctive
Affinity – natural attraction or force between things
Mobile Medium – gas or liquid that carries the components (mobile phase)
Stationary Medium – the part of the apparatus that does not move with the sample (stationary phase).

7. Illustration of Chromatography
Stationary Phase
Separation
Mobile Phase
Mixture
Components
Components
Affinity to Stationary Phase
Affinity to Mobile Phase
Blue
Insoluble in Mobile Phase
Black
     
 
Red
    
Yellow 
        

9. Type of Chromatography Type of Mobile Phase
Types of Chromatography
1.) The primary division of chromatographic techniques is based on the type of mobile phase used in the system:
Type of Chromatography Type of Mobile Phase
Gas chromatography (GC) gas
Liquid chromatograph (LC) liquid
2.) Further divisions can be made based on the type of stationary phase used in the system:

10. Gas Chromatography Name of GC Method Type of Stationary Phase
Gas-solid chromatography solid support
Gas-liquid chromatography liquid-coated support

11. Types of Chromatography: based on the principle of separation
Liquid Chromatography
Name of LC Method Type of Stationary Phase
Adsorption chromatography solid support
Partition chromatography liquid-coated support
Ion-exchange chromatography support containing fixed charges
Size exclusion chromatography porous support

12. Adsorption based paper chromatography
Here the stationary layer is a solid while the mobile phase is liquid. The compounds travel on the solid surface under the influence of mobile liquid. The separation depends on the extent of physical adsorption to the solid surface.

13. Column chromatography
Principle: When a mixture of mobile phase and sample to be separated are introduced from top of the column, the individual components of mixture move with different rates. Those with lower affinity and adsorption to stationary phase move faster and eluted out first while those with greater adsorption affinity move or travel slower and get eluted out last.

14. Retention time tr
Time it takes for analyte to reach detector after sample injection
Tm = retention time for material to come through column which is not retained also called dead time or void volume
tm rate of migration is the same as the average rate of motion of the mobile phase molecules u = L/tm

15. Theory of Chromatography
1.) Typical response obtained by chromatography (i.e., a chromatogram):
chromatogram - concentration versus elution time Wh Wb
Inject
Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units

16. Note: The separation of solutes in chromatography depends on two factors:
(a) a difference in the retention of solutes (i.e., a difference in their time or volume of elution
(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation system)
A similar plot can be made in terms of elution volume instead of elution time. If volumes are used, the volume of the mobile phase that it takes to elute a peak off of the column is referred to as the retention volume (VR) and the amount of mobile phase that it takes to elute a non-retained component is referred to as the void volume (VM).
Peak width & peak position
determine separation of peaks

17. Types of Chromatography
Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)
Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)
Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)
Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

18. Principles of Paper Chromatography
Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity.
Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents.
Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.

19. Paper Chromatography Experiment
What Color is that Sharpie?

20. Overview of the Experiment
Purpose:
To introduce students to the principles and terminology of chromatography and demonstrate separation of the dyes in Sharpie Pens with paper chromatography.
Time Required:
Prep. time: 10 minutes
Experiment time: 45 minutes
Costs:
Less than $10

21. Preparing the Chromatography Strips
Cut 6 strips of filter paper
Draw a line 1 cm above the bottom edge of the strip with the pencil
Label each strip with its corresponding solution
Place a spot from each pen on your starting line

22. Red Dye 20% 50% 70% 100% 0% 1. Dyes separated – red and yellow
2. Yellow –soluble in low concentrations of isopropanol and
less soluble in high concentrations of isopropanol
3. Red – slightly soluble in low concentrations of isopropanol, and more soluble in concentrations of isopropanol >20%
20%
50%
70%
100% 0%
Concentration of Isopropanol

23. Alternative Experiments

24. Partition Chromatography
Movie
Used in GC & LC
Molecules will partition into the stationary phase based upon affinity for stationary phase & eventually partition into mobile phase again
Thin layer is coated onto inside of GC column or on small particles on LC column

25. Adsorption Chromatography
Movie
Very similar to partition chromatography
Adsorption just on surface, partition into thin layer
Not used as widely as partition used mainly in TLC & very small particles in LC

26. Ion Exchange Chromatography
Movie
Separation of either cations or anions
Separtion based on relative strength of ionic bond
Anion exchange has cations on surface
Used in LC exclusively

27. Molecular Exclusion Chromatography
Movie
Separation based on size
Small molecules get trapped in pores & take longer to get out

28. Gel Electrophoresis
Separation based on size and charge
Smaller molecules will migrate further, less tangled
Movie

29. Affinity Chromatography
Very selective
Specific binding site is used to concentrate analyte on column
Used a lot in biological applications

30. THE END