chromatography Flashcards
A tripeptide was heated with hydrochloric acid and a mixture of amino acids was
formed. This mixture was separated by column chromatography.
Outline briefly why chromatography is able to separate a mixture of compounds (3)
phase or eluent or solvent (or named solvent) is moving or mobile
M2 stationary phase or solid or alumina/silica/resin
M3 separation depends on balance between solubility or affinity
(of compounds) in each phase
The dipeptide in part (d) is hydrolysed in acid conditions and the mixture produced
is analysed by column chromatography. The column is packed with a resin which
acts as a polar stationary phase.
Suggest why lysine leaves the column after alanine
lysine has a more positive charge
it has a greater affinity or it adheres better to the stationary phase
Why do we need to
Wearing plastic gloves to hold a TLC plate
add the developing solvent to a depth of not more than 1 cm.
Allow the developing solvent to rise up the plate to the top.
Allow the plate to dry in a fume cupboard.
and are they essential or not essential
Wear plastic gloves:
Essential – to prevent contamination from the hands to the plate
Add developing solvent to a depth of not more than 1 cm3-
Essential – if the solvent is too deep it will dissolve the mixture from the plate
Allow the solvent to rise up the plate to the top:
Not essential – the Rf value can be calculated if the solvent front does not reach the top of the plate
Allow the plate to dry in a fume cupboard:
Essential – the solvent is toxic
Outline the steps needed to locate the positions of the amino acids on the TLC plate
and to determine their Rf values.
Spray with developing agent or use UV
1
Measure distances from initial pencil line to the spots (x)
1
Measure distance from initial pencil line to solvent front line (y)
1
Rf value = x / y
Explain why different amino acids have different Rf values.
Amino acids have different polarities
1
Therefore, have different retention on the stationary phase or different
solubility in the developing solvent
State in general terms what determines the distance travelled by a spot in TLC
) solubility in moving phase and retention by
stationary phase
To obtain the chromatogram, the TLC plate was held by the edges and placed
in the solvent in the beaker in the fume cupboard. The lid was then replaced
on the beaker.
Give one other practical requirement when placing the plate in the beaker.
Solvent depth must be below start line
polar compounds
The more polar the compound, the more it will adhere to the adsorbent and the smaller the distance it will travel from the baseline, and the lower its Rf value
r Less/non polar is) less attracted to (polar) plate /
stationary phase / solid
more polar- more attracted to mobile phase
A third TLC experiment was carried out using 1,2-dinitrobenzene. An identical
plate to that in Question 8.3 was used under the same conditions, but the
solvent used contained a mixture of hexane and ethyl ethanoate.
A student stated that the Rf value of 1,2-dinitrobenzene in this third experiment
would be greater than that of 1,2-dinitrobenzene in the experiment in
Question 8.6
Yes - mark on but there is NO MARK FOR YES
Solvent (more) polar or ethyl ethanoate is polar
Polar isomer more attracted to / more soluble in / stronger
affinity to the solvent (than before)
Chromatography is a technique used to show the presence of a small amount of
ETU in Zineb.
Outline how this technique is used to separate and identify ETU from a sample of
Zineb powder.
Zineb is mixed with a solvent / water
Use of column / paper / TLC
Appropriate collection of the ETU fraction
OR Appropriate method of detecting ETU
Allow ETU is an early fraction in a column or collecting a
range of samples over time, lowest retention time / travels
furthest on paper or TLC (allow 1 mark for having the longest
retention time in GLC).
1
Method of identification of ETU (by comparison with standard using
chromatography)
