PAPER CHROMATOGRAPHY

- Chromatography is a family of analytical chemistry techniques for the separation of

mixtures.

- It was the Russian botanist Mikhail Tsvet (Mikhail Semyonovich Tsvet) who invented the
first chromatography technique in 1901.
- The separation of molecules depends on differences of 1- size 2- shape 3- mass 4- charges
5- solubility and 6- adsorption.

Types of Chromatography:
1- Adsorption chromatography.
2- Partition chromatography e.g. paper chromatography
3- Gel-filtration chromatography.

Uses of chromatography:

- Government laboratories used to check

 for approved dyes in food

 that vegetables contained tiny amounts of pesticides and herbicides

Advantages of using chromatography:

1. Require very minute amount for identification.

2. Can be used to identify substances that cannot be easily melted or distilled.

1 Page Organized by Lecturers: Sharifa Al-Ghamdi& Huda Al-Shaibi

All types of chromatography involve interaction between:
1- The mixture to be separated.
2- The stationary phase.
3- The mobile phase.

Principle of Paper Chromatography:

- Method of separating and identifying both colored and colorless mixtures.

- Mixtures can be solids, liquids or gases but their components must be able to dissolve in
the same solvent to different extents.

- Generally involves 2 phases:

 stationary phase solid support e.g. water on paper

 mobile phase solvent or a gas

- Test mixture is applied onto the chromatography paper and a solvent is then allowed to
pass over the paper. As the solvent does so, the components of the mixture travel along with
it.

- The stationary phase retards the passage of the components of the sample. When
components pass through the system at different rates they become separated in time.

The solvent used depends on the substance to be separated

The components will travel at different rates over paper depending on:

 their solubility in the solvent

 how well the dyes adsorb on the chromatography paper

Generally, the more soluble the component is in the solvent and the less it adsorb onto the
chromatography paper, the faster it would move with the solvent on the paper and hence
the spot appears further up the paper

2 Page Organized by Lecturers: Sharifa Al-Ghamdi& Huda Al-Shaibi

Result of chromatography is known as the >>>> Chromatogram

Types of Paper Chromatography:

- There are three types of paper chromatography:

I- Ascending Paper Chromatography:

- Solvent running up the paper by capillary action.

II- Descending Paper Chromatography:

- Solvent running down the paper by both capillary action and gravity.

Advantage of the descending method over the ascending method:

- Good for long pieces of paper thus better separation.

- Aided by gravity thus faster.

III- Two-Dimensional Paper Chromatography:

- The mixture is separated in the first solvent which should be volatile then after drying the
paper is turned through 90 and separation is carried out in the second solvent. After
location, a map is obtained and compounds can be identified by comparing their position
with a map of known compounds developed under the same conditions.

Stationary Phase:

- In paper chromatography, cellulose in the form of paper sheets makes an identical

support medium. >>>> WHY?

- Because it has the ability to adsorb water molecules between cellulose fibers and forms a
stationary hydrophilic phase.

3 Page Organized by Lecturers: Sharifa Al-Ghamdi& Huda Al-Shaibi

- Paper: Watman No. 1 of high quality is the paper most frequently used for analytical
purposes.

Mobile Phase:

- In paper chromatography, mobile phase is a mixture of solvents.

- The choice of solvent depends on the mixture investigated:

1- If the compounds move close to solvent (A) front >>>>> these compounds are highly
soluble in solvent A

2- If the compounds are crowded around the origin >>>>> these compounds are not
sufficiently soluble in solvent B.

Therefore, a suitable solvent for separation would be an appropriate mixture of both

solvent A & B. As a result R f values of the components of the mixture are spread across
the length of the paper.

Retention Factor(R f ):

- The retention is measured as the retention factor Rf, the run length of the compound

divided by the run length of the solvent front:

- Unknown substances could be identified by the Rf values

Rf = dist. Moved by the substance

dist. Moved by the solvent

- The Rf of a compound often differs considerably between experiments and

laboratories due to variations of the solvent, the stationary phase, temperature, and the

4 Page Organized by Lecturers: Sharifa Al-Ghamdi& Huda Al-Shaibi

setup. It is therefore important to compare the retention of the test compound to that of

one or more standard compounds under absolutely identical conditions.

Detection of Spots:

- After development, the spots corresponding to different compounds may be located by

their color
- However, most compounds are colorless and are visualized by:
1- Spraying the paper with specific reagents.
2- Dipping the paper in a solution of the reagent in a volatile solvent.
3- Fluorescent substances can be visualized by ultraviolet (UV) light.

SEPARATION OF AMINO ACIDS BY

PAPER CHROMATOGRAPHY

- Separation and identification of amino acids are operations that must be performed
frequently by biochemists. The 20 amino acids present in proteins have similar structures.

5 Page Organized by Lecturers: Sharifa Al-Ghamdi& Huda Al-Shaibi

However, each amino acid is unique in polarity and ionic characteristics. In this
experiment, we will use paper chromatography to separate and identify the components of
an unknown amino acid mixture.
- The solvent mixture contains several components, one of which is usually water and
another of which is a more non-polar solvent. As the solvent mixture moves up the paper
by capillary action, the water in the mixture binds to the hydrophilic paper (cellulose) and
creates a liquid stationary phase of many small water droplets. The non-polar solvent
continues to move up the paper forming a liquid mobile phase. Since amino acids have
different R-groups, they also have different degrees of solubility in water vs. the non-polar
solvent. An amino acid with a polar R-group will be more soluble in water than in the non-
polar solvent, so it will dissolve more in the stationary water phase and will move up the
paper only slightly. An amino acid with a hydrophobic R-group will be more soluble in the
mobile non-polar solvent than in water, so it will continue to move up the paper. Different
amino acids will move different distances up the paper depending upon their relative
solubilities in the two solvents, allowing for separation of amino acid mixtures.

- The movement of amino acids can be defined by a quantity known as Rf value, which
measures the movement of an amino acid compared to the movement of the solvent. At the
start of the chromatography, the amino acid is spotted at what is called the origin. The
chromatography is then performed, and the procedure is stopped before the solvent runs
all the way up the paper. The level to which the solvent has risen is called the solvent front.
The Rf value of an amino acid is the ratio of the distance traveled by the amino acid from
the origin to the distance traveled by the solvent from the origin.
- Since Rf value for an amino acid is constant for a given chromatography system, an
unknown amino acid can be identified by comparing its Rf value to those of known amino
acids.

Application:
Materials:
1- Filter paper: Watman No.1.
2- Solvent system: Butanol: glacial acetic acid: water.

6 Page Organized by Lecturers: Sharifa Al-Ghamdi& Huda Al-Shaibi

3- Ninhydrine reagent.
4- Standard amino acids and mixture of unknown.
Procedure:
1- Draw a light pencil line 1-2cm from the bottom of the paper.
2- Place a single drop of compound at intervals 2cm.
3- Dry with hair dryer.
4- Dip the paper in the jar with one of the edges of the paper to which the sample of the
spot is adjacent into the solvent.
5- Allow to run.
6- Remove the paper.
7- Determine the solvent front.
8- Dry.
9- Spray the paper with ninhydrin.
10- Dry the paper.

After some time

References:

www.wikipedia.org

7 Page Organized by Lecturers: Sharifa Al-Ghamdi& Huda Al-Shaibi

 RESULTS & LAB REPORT

- Calculate the R f and then identify the unknown amino acids in the mixture.

- Present your results in a good and full lab report.

8 Page Organized by Lecturers: Sharifa Al-Ghamdi& Huda Al-Shaibi

 `1

9 Page Organized by Lecturers: Sharifa Al-Ghamdi& Huda Al-Shaibi