Introduction

Chromatography, Color writing. "Chroma" is a Greek roots prefix for color and "graphy" is
a Greek roots suffix for writing. It is used to analyze, identify, purify & quantify the compounds.
[1]
Chromatography is the physical separation of a mixture into its individual components .

 Analyze
 Identify
 Purify
 Quantify

Mixture Components

The components to be separated are distributed between two phases, a stationary and mobile
phase. A mixture which contains the solutes is separated into pure components by passing it over
the stationary phase (an insoluble material) to which the substances stick to varying degrees. The
mobile phase, solvent (liquid or gas) is carrying the solutes over the stationary phase.

Separation based on the different interactions of the compounds with the two phases. Substances
that stick tightly to the stationary phase move very slowly, while those that stick loosely or do
not stick at all move rapidly.

Chromatography can be an analytical method, in which the investigator learns the number and
nature of the components in a very small amount of a mixture, but does not actually isolate them.
A common analytical method is silica-gel thin-layer chromatography,TLC. Or it can be a
preparative method, in which the investigator uses a large quantity of the mixture to obtain
useable amounts of each component. A common preparative method involving the same phases
is silica-gel column chromatography.

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All chromatographic systems needs:
 A stationary phase (a solid, or a liquid supported on a solid).
 A mobile phase (a liquid or a gas)
 Sample molecules (mixture for separation)

Applications of chromatography
In any chemical or bio-processing industry, the need to separate and purify a product from a
complex mixture is a necessary and important step in the production line. This separation of
mixtures is useful to us in various ways. For examples,

 Pharmaceutical industry uses chromatography to isolate penicillin and other antibiotics.

 Proteins can even be separated into amino acids.
 Chromatography is also used in crime scene investigation for DNA and RNA sequencing.
 In many scientific studies to identify unknown organic and inorganic compounds.
 Government laboratories used to check dyes in food and vegetables contained tiny
amounts of pesticides and herbicides.

Types of Chromatography

There are many forms of chromatography, but all forms work on the same principle:
1. Partition Chromatography which includes a liquid film coated in an inert suitable
support.
2. Adsorption Chromatography which includes finely divided solid functioning as an
adsorbing surface & they are divided finely to increase their surface area.
3. Ion Exchange Chromatography (which is reversible step) which includes ionic groups
(ionic means holding different charges) which are attached to an inert material; this
method is used in purifying water for example & the competition will be between the
sample (water considered mobile phase also) & the stationary phase directly.
4. Gel Chromatography (also called molecular sieving/Gel filtration/Gel
permeation/Molecular exclusion) which depends on a suspension of a polymer having a
suitable pore size (like agar) & is an important method for some analysis kinds such as
separating hormones, enzymes & biological fluids; AGAR itself is a polymer with pores,
so small particles will enter into the pores & might leave only in case it found a larger
pore to enter in it

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Partition
artition chromatography

Partition chromatography is the he distribution of solutes between two immiscible phases. The
solute will distribute itself
self between the two phases according to its solubility in each phase, this
is called partitioning. It is mainly used for separation of molecules of small molecular weight.
In partition chromatography one solvent usually water molecules bound to supporting phase
which is in the form of a paper [1]. Partitioning occurs between the bound water which is the
stationary phase and the solvent which is the mobile phase.

Paper chromatography

Paper
aper chromatography is an analytical technique for separating and identifying both
colored (e.g. pigments) and colorless (e.g. amino acids) mixtures.

Figure 1: Paper chromatography [2].

In paper chromatography, the stationary phase is a very uniform absorbent paper. Cellulose (non
polar) in the form of paper sheets makes an ideal support medium where water is adsorbed to the
[1]
cellulose fibers and forms the stationary hydrophilic phase .Cellulose
ellulose is a polymer of the
simple sugar, glucose.

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awi, W. M. ~3~
The key point about cellulose is that the polymer chains have -OH
OH groups sticking out all around
them..The cellulose fibers attract water vapour from the atmosphere
atmosphere as well as any water that
was present when the paper was made. You can therefore think of paper as being cellulose fibres
with a very thin layer of water molecules bound to the surface.

Non-polar molecules in the mixture that you are trying to separate will have little attraction for
the water molecules attached to the cellulose, and so will spend most of their time dissolved in
the moving solvent. Molecules like this will therefore travel a long way up the pa
paper carried by
the solvent. They will have relatively high Rf values.

On the other hand, polar molecules will have a high attraction for the water molecules and much
less for the non-polar
polar solvent. They will therefore tend to dissolve in the thin layer of water
around the cellulose fibres much more than in the moving solvent.

Because they spend more time dissolved in the stationary phase and less time in the mobile
phase, they aren't going to travel very fast up the paper.

If you want to identify the spots in the mixture, you obviously can't do it with comparison
substances on the same chromatogram as we looked at earlier with the pens or amino acids
examples. You would end up with a meaningless mess of spots. You can, thoug
though, work out the
Rf values for each of the spots in both solvents, and then compare these with values that you
have measured for known compounds under exactly the same conditions.

Choice of paper

Whatman chromatography papers are the most widely used papers for
chromatography worldwide. This acceptance and usage reflect the purity,
high quality, and consistency of Whatman papers. These qualities are relied
upon by chromatographers and are essential to successful, reproducible
chromatography. Whatman chromatography
hromatography paper media are made from
specially selected cotton cellulose. They are rigorously quality controlled for
characteristics important to the chromatographer and to ensure uniformity within the grade.

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awi, W. M. ~4~
• Whatman No. 1 MM is the paper most frequently
frequently used for analytical purpose.
• Whatman No. 3 MM is a thick paper is best employed for separating large quantities of
material but the resolution is inferior to Whatman No. 1.
• Whatman Nos. 4 and 5 are convenient for a rapid separation, although the spots are less well
defined. The paper may be impregnated with a buffer solution before use or chemically
modified by acetylation [1].

Choice of Solvent

It is depend on the mixture investigated. If the compounds move close to the solvent front in
solvent A then they are too soluble, while if they are crowded around the origin in solvent B then
they are not sufficiently soluble. Therefore, a suitable solvent
solvent would be an appropriate mixture of
both solvent A & solvent B, so that the Rf values of the components of the mixture are spread
across the length of the paper.
The pH may also be important in a particular separation because many solvents ccontain acetic
acid or ammonia to create a strongly acidic or basic environment [1].

Spotting the paper

The sample (10-20

20 ul) is spotted onto the paper by the capillary glass
in contact with the paper so that the spot is no larger than 1 mm in
diameter, then raise the capillary and do it this procedure in all samples
[1]
.

Development
A. Ascending paper chromatography

Solvent running up the paper or TLC by capillary action. It is most

employed and has the advantage that separation can be carried out in two
[1]
dimensions . Ascending is simple and inexpensive compared with
descending and usually gives more uniform migration with less diffusion of
the sample "spots".
Solvent

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B. Descending paper chromatography Solvent

In descending paper chromatography, the chromatogram is held

vertically, and the spot of dye is drooped on the top of the chromatogram.
Solvent drips off the bottom of the paper by gravity. It is convenient for
compound which has similar Rf values. Descending chromatography is
faster because gravity helps the solvent flow but it’s difficult to set the
apparatus.

Detection of spots

Most biological compounds are colorless and are visualized by:

1. Spraying the paper by specific reagents, for example, Ninhydrin (Triketohydrindane hydrate).
2. Dipping in a solution of the reagent in a volatile solvent, for example, Iodine vapors.
3. Fluorescence compounds can be visualized with ultraviolet light.
4. Radioactive spots can be located with a detector, or the chromatogram can be pressed against
X-ray film for minutes or hours to expose the film.

Retention factor (Rf)

Different compounds should move different distances on the plate or on the paper; however the
exact distance a particular compound moves depends on how far the solvent is allowed to rise up
the plate. The further the solvent moves, the further the spots travel. Some compounds in a
mixture travel almost as far as the solvent does; some stay much closer to the base line.To take
this variation into account, the ratio of the two distances is calculated and reported. This ratio, is
called the retention factor. Rf allows identification of individual compound in a mixture when
compared to one or more standard compounds under absolutely identical conditions to that of the
test compound [1]. When comparing two different compounds run under identical conditions, the
compound with the larger Rf is less polar because it interacts less strongly with the polar
adsorbent on the paper.

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Specifically, the retention fraction is defined as the fractional distance the spot moves compared
to the distance travelled by the solvent front. The Rf is constant for a particular compound as
long as you keep everything else constant - the solvent system, the stationary phase, temperature,
amount of material spotted, pH- for example. It corresponds to a physical property of the
compound. Spots with the same Rf values within experimental error and the same appearance are
likely to be the same compound.

For each compound it can be worked out using the formula:

Rf = Distance from baseline to spot, x

Distance from baseline to solvent, y

For example, if a compound travels 3.0 cm and the solvent front travels 4.5 cm, the Rf is 0.67:

Solvent front

New position of compound

3.0 cm 4.5 cm Rf = 3.0 = 0.67
4.5

Origin

Adsorption chromatography

Adsorption chromatography is oldest form of chromatography and was first used in 1903 by
the Russian botanist Mikhail Tswett. It utilizes a mobile liquid or gaseous phase that is adsorbed
[1]
onto the surface of a stationary solid phase . The equilibrium between the mobile and
stationary phase account for the separation of different solutes.

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Thin Layer Chromatography
hromatography (TLC)

Thin layer chromatography (TLC) is among the most useful

tools for assaying the purity of organic compounds. Many
compouns with varying functional groups may be used as the
stationary phase and several types of interactions can aid in
developing the desired separati
eparation (i.e. Van der Waals forces,
electrostatic interactions, hydrogen bonding,
bonding etc.).

The stationary phase consisting of very a thin layer of adsorbent

material, usually the very polar silica gel (oxides of silicon)
silicon) on a glass or plastic plate
plate, aluminum
[3]
oxide, or cellulose immobilised onto a flat, inert carrier sheet . Aluminum Oxide for
Chromatography is a white to off white, fine-grained
fine grained powder, highly porous form of aluminum
oxide. Its surface is more polar than that of silica gel and reflecting the amphitrical nature of
alumina,, has both acidic and basic characteristics.
The mobile phase is a solvent (or mixture of solvents) that is less polar than silica gel. Typical
solvents are hydrocarbons like hexane (C6H14, very non-polar), acetone (CH3COCH3, moderately
polar), and ethanol (C2H5OH, very polar).
pol The process is similar to paper chromatography but in
the TLC have advantages of faster runs, better separations, and can choice between different
stationary phases.

Separation of the experimental solution based on the polarity Solvent front

of the components of the compound.
Component A
More polar
Non-polar compounds will be less strongly attracted to the
Component B
Plate and will spend more time in the moving phase. Less polar
This compound will move faster and will appear closer to the top of Origin X
the plate.

Polar compounds will be more strongly attracted to the plate and will spend less time in the
moving phase and appear lower on the plate.

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 The separation of amino acids by two-dimensional
(Paper chromatography & Thin Layer chromatography)

Principle
Two-dimensional paper chromatography (Two-way paper chromatography) involves using
two solvents and rotating the paper 90° in between. This useful for separating complex mixtures
of similar compounds for example amino acids.
Ninhydrin reacts with all α - amino acids to give a purple color. Other compounds also react if
present, and these include primary and secondary aliphatic amines and some non- aromatic
heterocyclic nitrogen compounds. The imino acids, proline and hydroxyl proline, react to give a
yellow color [1].

Turn 90°
Solvent front

Result
Apply sample X X
X
Solvent 1 Solvent 1

After turn 90° the separation

carried out in the second solvent

Final separation

X X
Solvent 2 Solvent 2

Figure 2: Separation of mixture by two- dimensional paper chromatography.

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Apparatus Ascending paper chromatography

 Chromatography glass jar with lid

Support rod
 Chromatography paper or filter paper: Whatman No.1,
Glass jar
(10 cm x 10 cm)
Clip
 Capillary tubes or pasteur pipettes
Solvent front
 Hairdryer or drying oven at 105°C
Paper

Chemicals & other material

Sample X
Solvent 1
 For paper, n-butanol: glacial acetic acid: D.W. [4:1:5 v/v] Solvent for development

(FLAMMABLE, TOXIC)
 For TLC, ethanol: water [7:3 v/v]
Figure 3: Paper chromatography.
Solvent 2
 For paper, phenol: D.W. [4:1 v/v]. Care, phenol can give nasty burns on the skin.
Add 125 ml of water to a 500 gm bottle of phenol and allow standing overnight. Just before
use, add a few drops of 0.88 ammonia to the solvent and mix well.
 For TLC, n-butanol: acetic acid: D.W. [8:2:2 v/v]). The solvent should be made up fresh on
the day.
 Ninhydrin reagent (Dissolve 0.2 gm in 100 ml of aceton just before use).
 Mixture of amino acids. Prepare small volumes of 10 gm/liter solutions in 1 molar HCl
(IRRITANT), a drop of acid is needed to bring the compound into solution. Alanine, asparatic
acid, cysteine HCl, cystein, glutamic acid, glycine, histidine HCl, hydroxyproline, leucine,
isolucine, lysine HCl, methionine, ornithine HCl, phenylalanine, proline, serine, threonine,
tryptophan, tyrosine, valine] [1].

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Procedure
1. Examine the amino acids map provided in figure 4, and then select five amino acids whose
Rf values differ widely.
2. Obtain paper or TLC plate (10X 10) and place it onto a paper towel. Be careful to touch the
paper or TLC sheet only on the edges, without touching your fingers to the surface.
3. Using a pencil, draw a faint line (on the powdery side of the TLC), about 1 cm from the
bottom and about 1 cm from top of both paper and TLC (Solvent front). The line should be
parallel to the bottom edge. Do not allow the pencil to dig into the coating on the plate at all.
Then faintly draw one small hash mark, about 2 cm towards the left corner.
4. Spot 10-20 ul of a single spot of amino acids mixture onto the paper or the plate (up to a total
of 3 times). To do this, dip one end of the capillary tube into the top of the solution so that
only the clear portion of the liquid enters the capillary tube. Then lightly and quickly touch
the capillary tube to the surface of the paper or the TLC plate. Try to keep the spot as small
possible, preferably around 1mm, Dry the spot in a current of air.
5. Use forceps to place the paper or TLC into the jar. Make sure that when the TLC plate is
placed in the solvent bath, the solvent does not immerse the spot of the sample mixture. The
solvent is below the spot, but then moves up across the spot, carrying the components of the
sample mixture up the plate at different rates. If the spot is immersed in the solvent at the
beginning, it will simply dissolve in the solvent and not rise up the plate.
6. Position the paper or the plate so that it is not touching the sides of the jar, and make sure it
does not curve or buckle in the jar. Covering the jar makes the atmosphere in the jar saturated
with solvent vapor. Saturating the atmosphere in the jar with vapor stops the solvent from
evaporating as it rises up the paper or the TLC plate.
7. As the solvent slowly travels up the paper sheet or TLC plate, the different components of
the mixture travel at different rates. As the solvent migrate up the paper or the TLC plate, do
not disturb the jar.
8. Watch the paper sheet or the TLC plate carefully. Once the solvent migrates almost to the top
of the plate use forceps to remove the plate and let the solvent evaporate from it in the hood.

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9. Arrange the frame so that the second edge to which the spot was adjacent now dips in the
second solvent. Cover the jar with the lid. Once the solvent migrates almost to 1the top of the
plate use forceps to remove the plate and immediately mark the solvent front with a pencil,
before the solvent evaporates.
10. Dry the paper or the TLC plate by hairdryer. Rapidly spray the paper or the TLC plate
through the Ninhydrin reagent and allow the acetone to evaporate. Develop the colors by
heating at 105° C for 2-3 minute or using a hairdryer. The outcome of chromatography
experiment is a chromatogram..
11. Carefully circle the spots with a pencil and mark the centre of each spot. Place your name on
the front or back of your TLC plate in the result sheet.

Note:

 Each student will clean their benches and dispose of the waste chemicals.

 Each student will submit a lab report the following week to report their findings.

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 Amino acid Solvent Solvent
1 2
Alanine 0.22 0.54
Arginine 0.11 0.59
Aspartic acid 0.13 0.15
Glutamic acid 0.16 0.25
Glycine 0.17 0.40
Histidine 0.07 0.69
Isoleucine 0.55 0.86
Leucine 0.60 0.86
Lysine 0.10 0.48
Methionine 0.40 0.80
Phenylalanine 0.58 0.89
Proline 0.30 0.91
Serine 0.10 0.36
Threonine 0.22 0.50
Tryptophan 0.47 0.83
Tyrosine 0.32 0.64
Valine 0.47 0.77

Figure 1:
er chromatography Separation of a mixture of amino acids in two dimensions on
-water 4:1:5 v/v Whatman No. 1 paper chromatography.
.

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awi, W. M. ~ 13 ~
References:
1. Plummer D.T., An introduction to Practical biochemistry, Tata McGraw – Hill.
2. http://www.columbia.edu/cu/biology/courses/c2005/hand04.html
3. http://en.wikipedia.org/wiki/Thin_layer_chromatography

To learn more information click on these links:

4. Chromatography-online
5. Chromatography menu
6. Chromtutorial
7. Biochemlab

Result sheet
Title: …………………………………………………………………………..

Test sample: ……………………………………

Group partner: ……………………… ……………………….. ……………………….

Answer the following:

Fasten your TLC plates here or sketch it, and properly annotate them including all Rf values.
Note the color and calculate the Rf value of each spot.

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How many spots are separated? …………………………………………
Are all of the Amino acid composition of the mixture separated? If not, which of them is not
separate. Why?

………………………………………………………………………………………………………
………………………………………………………………………………………………………
………………………………………………………………………………………………………

Distance moved by amino acid spot (X)

Spot Distance Spot color

Number (cm)
1
2
3
4
5

Distance in centimeters solvent moved (Y) ………cm.

List Rf values for all components off the TLC. (Calculation should be shown):

Rf = Distance moved by compound X

Distance moved by a solvent y

Table 1: Rf values of the separated components

Number of the spot 1 2 3 4 5

Rf value of spot

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Table 2: Rf values measured for standard compounds under exactly the same conditions

Amino Acid Histidine Proline Alanine Leucine Aspartic cysteine Argenine Tyrosine
Polarity of the Basic Neutral Neutral Neutral Acidic Neutral Basic Neutral
amino acid Polar Non-polar Non-polar Non-polar Polar Slightly Polar polar
Polar
Rf value of 0.139 0.232 0.286 0.542 0.192 0.333 0.180
known

Compare Rf values in Table 1 with that have been measured for standard compounds, Table 2,
then identify the amino acid composition of the mixture.

Spot number 1 2 3 4 5
Amino acid composition
of the mixture

What is the location reagent? …………………………………………………

Why we use two solvents?

………………………………………………………………………………………………………
………………………………………………………………………………………………………

Why is proline and hydroxyproline give yellow color with ninhydrin?

………………………………………………………………………………………………………

What spot is polar and what is non-polare? Why?

………………………………………………………………………………………………………
………………………………………………………………………………………………………
………………………………………………………………………………………………………

Give brief description of your observations?

………………………………………………………………………………………………………
………………………………………………………………………………………………………
………………………………………………………………………………………………………

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………………………………………………………………………………………………………
………………………………………………………………………………………………………

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