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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.
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,
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
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.
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
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cellulose fibers and forms the stationary hydrophilic phase .Cellulose
ellulose is a polymer of the
simple sugar, glucose.
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.
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].
Development
A. Ascending paper chromatography
Detection of spots
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.
For example, if a compound travels 3.0 cm and the solvent front travels 4.5 cm, the Rf is 0.67:
Solvent front
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.
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.
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
Final separation
X X
Solvent 2 Solvent 2
(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].
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.
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.
.
Result sheet
Title: …………………………………………………………………………..
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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List Rf values for all components off the TLC. (Calculation should be shown):
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
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