EXPERIMENT 4

CHROMATOGRAPHY
MT633 Inorganic and Organic Chemistry
Laboratory

Department of Biochemistry-Faculty of Pharmacy Presentation by: Gladys I. Bathan, MSc and Alifa Camille N. Santos, MSc
OBJECTIVES
1. Separate the colored components of plant leaves using column chromatography.

1. Determine the purity of the components using thin layer chromatography

1. Measure the Rf values of the colored components in TLC.

1. Understand how structure and functional groups affects chromatographic separations.

CHROMATOGRAPHY

-from the Greek words “kroma” and “graphein” which mean color and to draw a
graph or to write (MS Twett, 1906).

- widely used for:

- qualitative analysis (analytical)

- recovery of desired components in a sample mixture (preparative)

BASIC PRINCIPLE

-components are separated based on their affinity for two phases:

Stationary Phase is a layer or coating on the supporting medium (column or

planar surface) which interacts with the analytes.
- may be solid or liquid

Mobile Phase is the part of the chromatographic system which carries the solute
across the stationary phase.
- may be liquid or gas
TYPES OF CHROMATOGRAPHY
Based on mechanism of separation
1. Partition
• Particles are separated between liquid mobile and stationary phases
e.g. paper chromatography

2. Adsorption
• Particles are adsorbed in the solid support
• Particles are separated between liquid mobile phase and solid stationary
phase
e.g. column chromatography, thin layer chromatography
TYPES OF CHROMATOGRAPHY
Based on the direction of mobile phase movement
1. Ascending
- capillary action of solvent
-Movement of the solvent carrying the component of interest is from bottom to top
(i.e. TLC)
2. Descending

- gravitational pull to the solvent

- Movement of solvent carrying the component of interest if from top to bottom (i.e.
CC)
TYPES OF CHROMATOGRAPHY
Based on the nature of mobile and stationary phase

1. Normal Phase Chromatography

Stationary phase: POLAR
Mobile phase: NON-POLAR

2. Reverse Phase Chromatography

Stationary phase: NON-POLAR
Mobile phase: POLAR
TYPES OF CHROMATOGRAPHY
Based on the type of support

1. Planar

-The stationary phase is on a flat support/panel (i.e. TLC and Paper

Chromatography)

2. Column

- The stationary phase is placed in a cylindrical glass support or column (i.e. HPLC
and GC)
TYPES OF CHROMATOGRAPHY
Based on the concentration of solvent

1. Isocratic
- Addition of solvent mixture of fixed composition

2. Gradient

- There is a continuous change in the composition of

the mobile phase over a period of time (e.g. polarity,
pH or ionic strength).
COLUMN CHROMATOGRAPHY
- stationary phase is held in a narrow tube through which
the mobile phase is forced under pressure or under the
effect of gravity

Stationary phase
-solid silica in the columnar support
-polar

Mobile phase
-different solvents (liquid)
-nonpolar
COLUMN CHROMATOGRAPHY
Separation of molecules will depend on their affinities to the stationary and mobile phases.
Least polar molecules - eluted first
Most polar molecules - eluted last

Polar components (b) adsorb more strongly to the polar silica and elute after the less polar components (a), which
move more quickly with the non-polar (relative to silica) solvent.
COLUMN CHROMATOGRAPHY
Elute – to remove/extract/wash the molecule from the
stationary phase

Eluent/Eluant- the mobile phase used to elute the molecules

Eluate – molecules eluted together with the eluent, collected

at the end of the column
COLUMN CHROMATOGRAPHY
Based on the nature of stationary and mobile phases
NORMAL PHASE
Based on the direction of movement of solvent
DESCENDING
Based on concentration of solvent
ISOCRATIC/GRADIENT
Based on type of support
COLUMN
Based on mechanism of separation
ADSORPTION
COLUMN CHROMATOGRAPHY SETUP
Column
wet packing of silica
dry packing of silica
Iron stand
Iron clamp
Solvents
Receiver of eluates/fractions
COLUMN CHROMATOGRAPHY STEPS

- packing
- washing
- loading
- development
- collection and evaluation
RETENTION TIME (Rt)

-a measure of the time taken for a solute to pass

through a chromatography column

-↓Rt= more non-polar

-↑Rt = more polar

PAPER CHROMATOGRAPHY
Stationary phase
-water in the solid cellulose (paper) support
-polar
Mobile phase
-different solvents (liquid)
-nonpolar
PAPER CHROMATOGRAPHY
• Separation is based on the affinity of solvent
molecules between mobile and front
stationary phases

Less polar molecules-travel farthest direction of

mobile phase
More polar molecules- travel least

solvent,height is line of
lower than the line origin
of origin
PAPER CHROMATOGRAPHY
Based on the nature of stationary and mobile phases
NORMAL PHASE
Based on the direction of movement of solvent
ASCENDING
Based on concentration of solvent
ISOCRATIC
Based on type of support
PLANAR
Based on mechanism of separation
PARTITION
THIN LAYER CHROMATOGRAPHY (TLC)
- components of the sample separate according to how much they adsorb on the stationary phase and how
much they dissolve in the mobile phase.
Stationary phase
-solid silica in the solid glass or
aluminum support
-polar
Mobile phase
-different solvents (liquid)
-nonpolar
THIN LAYER CHROMATOGRAPHY (TLC)
• Separation is based on the affinity of
molecules between mobile and stationary solvent
phases front

Less polar molecules-travel farthest, direction of

↑Rf mobile phase
More polar molecules- travel least,
↓Rf
solvent,height is lower
line of
than the line of origin origin
THIN LAYER CHROMATOGRAPHY
(TLC)
THIN LAYER CHROMATOGRAPHY (TLC)
Based on the nature of stationary and mobile phases
NORMAL PHASE
Based on the direction of movement of solvent
ASCENDING
Based on concentration of solvent
ISOCRATIC
Based on type of support
PLANAR
Based on mechanism of separation
ADSORPTION
STEPS IN THIN LAYER CHROMATOGRAPHY
1. Sample/Standard Application
-small spots of standard/sample are applied to avoid overlapping and tailing
during development
2. Development
-separation of molecules between mobile and stationary phases
-equilibration (saturation with mobile phase) of chamber is done to hasten
development
3. Visualization
-use of visualizing agents to view the spots
4. Evaluation
-computation of Rf values
5. Documentation
RETENTION FACTOR (Rf)

-used to aid in the identification of a substance by

comparison to standards.

-Two substances that have the same Rf value may be

identical

-↓Rf = more polar and ↑Rf = more

non-polar
 Criteria Column Chromatography Thin-Layer Chromatography
Application Preparative Analytical

Inert Support for Stationary Glass cylinder Metal Sheet: Pre-coated TLC
Phase plate on one side

Application of Sample Using a dropper Using a capillary tube

Development Step gradient elution Isocratic

Linear gradient solution

Principle of Separation Adsorption Adsorption

Direction of Solvent Front Descending Ascending

Evaluation Retention time Retention factor

Adsorption of Depends on Structure
-The adsorption strength of compounds increases with increasing polarity of
functional groups, as shown below:
Olefins
(weakly adsorbed)
(nonpolar) Ethers
Esters
Lactones
Aldehydes
Amines
Phenols
(strongly adsorbed)
(more polar) Acids
Adsorption of Depends on Structure
-In general, the molecular polarity of organic compounds is affected by the following
factors:

-Number of carbon atoms

-↑ # of C atoms = more non-polar

-Presence of electronegative atoms

-↑ e- difference between elements = more polar covalent bond

 Moringa oleifera
(Malunggay)

Pigments isolated:

- Xanthophylls
- Chlorophyll B arranged in
- Chlorophyll A decreasing
polarity
- Pheophytin
- Carotene
Pheophytin
 Capsicum frutescens
(Siling labuyo)

Pigments isolated:

- Capsorbin (orange) arranged in

- Capsanthin (red) decreasing
polarity
- Carotene (red orange)
SUPPLEMENTARY VIDEOS
• Paper Chromatography

https://www.youtube.com/watch?v=iPpy4khqtks

• Thin-layer Chromatography

https://www.youtube.com/watch?v=lj5OWzhZSac
https://www.youtube.com/watch?v=rMGQavOMAmc

• Column Chromatography

https://www.youtube.com/watch?v=UmWMlKJAdSk
Online learning activity
1. Make an article about the health benefits of one (1) of the colored components from leaves or vegetables
(Spinach leaves, Bell pepper, Carrots, Malunggay, etc.). Note: No repetition of compound among groups.
2. The article must have the structures, and its reactions. This is to be used in your discussion how this compound
plays an important role in the body to keep and improves health, and prevention of defects/diseases.
3. Take an original photo of a dish cooked with theses leaves/vegetables. Caption the photo.
4. Give a good title to your article.
5. Write your names in alphabetical order, and my name last as your editor.
6. Minimum of 300 to Maximum of 1000 words.
7. Cite references.
8. Find a magazine, journal, or newspaper where you think this manuscript can be published. For example, Purple
Gazette, Manila Bulletin, Varsitarian, etc. Give the link.
End