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BTEC Applied Science Unit 2 Assignment C - Chromatography
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BTEC Applied Science Unit 2 Assignment C - Chromatography. Pictures, graphs, tables included. Referenced throughout. Awarded a DISTICTION.
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Unit 2Assignment C
Diana Duka
Practical Scientific Procedures and Techniques
Undertake chromatographic techniques to identify components in mixtures.
Chromatography
Introduction
Chromatography is a technique used to separate mixtures and isolate substances. It can be
used in drug tests to determine the components in blood plasma and during forensic
investigation for DNA fingerprinting. There are different types of chromatography because
substances have different polarities yet each has the same aim but different stationary and
mobile phases. For instance, TLC has a short stationary and mobile phase whereas in paper
chromatography the stationary and mobile phase is long. In this assignment, I will be
focusing on TLC of extracting plant pigment and amino acids, paper chromatography and I
will be researching gas chromatography. The main difference between the stationary phases
is that paper chromatography uses paper and TLC uses a glass or aluminium foil that is
thinly coated with silica or another absorbent material. There are a few factors that affect the
absorption of pigments. These factors include; polarity of molecules and hydrogen bonding.
Polarity is the electrical charge within a molecule. There are non polar molecules - in which
there are o positive or negative charge ends. Polar molecules have a negative or positive
charge at the ends. Polar molecules/ solvents used in the experiments can attract and repel
each other. The other factor affecting solubility of hydrogen bonding, which is a covalently
bonded hydrogen atom to another negatively charged atom. Hydrogen bonding is a strong
dipole-dipole force, stronger than Van der Waal forces. The more hydrogen bonds the more
soluble a molecule is. Lastly, another factor that affects the solubility is the molecular weight.
Simply put, the molecular weight is the mass of a molecule to 1 mole. This can affect the
experiment negatively as the molecular weight increases and the solubility of a substance
decreases, so there is less chance of absorption.
The experiments we conducted were extraction of plant pigments using paper
chromatography, thin layer chromatography of amino acids and extraction of plant pigments
using thin layer chromatography.
After the experiments are done, the RF value can be calculated. The RF value is the value
that the solvent moves in ratio to the distance moved by the solute, which is always less than
1. The equation to calculate the RF value is; the distance travelled by solute / distance
travelled by solvent.
Standard Rf values come in a range because components evaporate at different rates,
components have different polarity, meaning that the Rf value you get as a result of your
,experiment, it may fall into that range. It does not mean that it is incorrect or false, it simply is
the range accepted.
In this assignment I will be researching, comparing and evaluating my results.
Carotene - 0.98
Chlorophyll A - 0.59
Chlorophyll B - 0.42
Xanthophyll A - 0.28
Xanthophyll B - 0.15
Mobile phase - solvent (a liquid) that moves up the paper (water, acetone, propanone etc.)
Stationary phase - a porous solid that does not move, stays at the origin.
Chromatogram - a graph representing the substances/ components.
Soluble - substances that dissolve in a solution.
Solubility - how soluble a substance is.
Solution - a mixture of substances/ components.
Polar - molecules with uneven distribution of electrons.
Nonpolar - molecules with an even distribution of electrons.
How does paper chromatography work?
Paper chromatography is one of the many chromatographic techniques. The technique
separates mixtures in order to determine the components of the substance/ mixture. It is
mostly used to separate chlorophyll, amino acids and DNA bases. The paper acts as a solid
supporting material which has tiny water molecules in the pores. These act as a stationary
phase while ethanol, methanol, ether, propanone etc. act as the mobile phase. There are
different modes of operation; ascending, descending, radial and 2D. The most common
mode of operation is the ascending mode where a solvent reservoir is at the bottom of a
beaker, just below the solvent front on the chromatography paper. Once the solvent has
stopped moving, the solvent front is marked, this way scientists can work out the Rf value of
the mixture.
Factors affecting paper chromatography
● Solubility
● Absorption
● Solvent used
, ● Polarity of solution
Risk Assessment
Hazard Risk Precaution
● Spills (acetone, ● Irritate skin and eyes ● Wear eye protection
propanone mixture) ● Highly flammable and disposable
gloves, handle with
care.
● Wash hands if
spilled.
● Rinse eyes if solvent
goes in eyes.
● Keep away from
open flames.
● Sharp pencil ● Possible cuts to skin ● Handle with care,
extra attention
● Splashes ● Green pigment from ● When using the
spinach leaf going pestle and mortar be
into eyes/ onto gentle
clothes. ● Add a small amount
of acetone to the
spinach leaf.
How does TLC work?
Thin layer chromatography is named after the thin layer of absorbent material on the glass or
aluminium foil. This thin layer of absorbent material is usually silica, which absorbs the
substances. The glass sheet/ aluminium foil is handled with care so that the absorbent
material does not get contaminated otherwise it will alter the results or give false information.
TLC is very sensitive to environmental factors (temperature, humidity etc.) that need to be
set and kept at a constant level to complete the experiment. After the mobile phase has
stopped moving, the glass sheet/ aluminium foil is placed under ultraviolet light to view the
developed sheet. A fluorescent indicator is added to the silica that will cause it to fluoresce
under the UV light. The results will show up as dark spots. The spots are outlined with pencil
to be able to view under visible light. The results now can be compared and the Rf value can
be calculated.
Factors affecting Thin Layer Chromatography
● Polarity of substances in solution mixture
● Solubility of solution
● Absorption
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