Chromatography qyuestopns
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State why each amino acid has a different Rf value.
each amino acid has different (relative) affinity/attraction to/solubility in stationary and mobile phases
Suggest a suitable reagent for the hydrolysis of a protein.
Conc HCl
Deduce the minimum number of amino acids present in the original mixture. https://www.physicsandmathstutor.com/pdf-pages/?pdf=https%3A%2F%2Fpmt.physicsandmathstutor.com%2Fdownload%2FChemistry%2FA-level%2FTopic-Qs%2FAQA%2FOrganic-II%2F3.16-Chromatography%2FSet-I%2FChromatography%20QP.pdf 2c
7 or seven Basically count the spots after the first chromatography
Suggest why it was necessary to use two different solvents.
Some of the amino acids did not separate/dissolve with the first/either solvent OR Some amino acids have the same Rf value or have the same affinity with the first/either solvent
State in general terms what determines the distance travelled by a spot in TLC.
(Balance between) 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
A second TLC experiment was carried out using 1,2‑dinitrobenzene and 1,4‑dinitrobenzene. An identical plate to that in part (c) was used under the same conditions with the same solvent. In this experiment, the Rf value of 1,4‑dinitrobenzene was found to be greater than that of 1,2‑dinitrobenzene. Deduce the relative polarities of the 1,2‑dinitrobenzene and 1,4‑dinitrobenzene and explain why 1,4‑dinitrobenzene has the greater Rf value.
1,2- is more polar OR 1,4- is less polar OR 1,2 is polar, 1,4- is non-polar 1 1,4- ( or Less/non polar is) less attracted to (polar) plate / stationary phase / solid OR (Less/non polar is) more attracted to / more soluble in (non-polar) solvent / mobile phase / hexane
A third TLC experiment was carried out using 1,2‑dinitrobenzene. An identical plate to that in part (c) 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 part (f) Is the student correct? Justify your answer.
Yes - mark on but there is NO MARK FOR YES Mark independently following yes Solvent (more) polar or ethyl ethanoate is polar 1 Polar isomer more attracted to / more soluble in / stronger affinity to the solvent (than before) Penalise b
A mixture of the two dipeptides J and K is analysed by gas chromatography followed by mass spectrometry (GC-MS). Explain why J and K can be separated by gas chromatography and why mass spectrometry using electrospray ionisation does not enable you to identify them. Gas chromatography explanation
Different retention times / dipeptides appear at different times. 1 Different balance between solubility in the moving phase / gas carrier and retention by the stationary phase / column OR different relative affinity for mobile and stationary phases. 1 Mass spectrometry explanation Same m/z values. 1 (Both) dipeptides / J and K have same molecular formula / Mr.
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
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
Name a suitable developing agent. State why the developing agent is needed
M1 ninhydrin M2 amino acids are colourless / to make the amino acids visible
WHy is a lid required in chormatogrpahy
M1 lid/cover (on beaker) Then any 2 from these 3 * prevents escape of vapour (from beaker) / evaporation of solvent (from beaker) * so atmosphere in beaker is saturated with solvent vapour owtte * to reduce evaporation from the plate
State why the amino acids separate on the TLC plate.
Difference in the balance between solubility in solvent/mobile phase and attraction to/retention on stationary phase