Questions on chromatography: TLC, column and gas

Question 1

Describe how thin-layer chromatography is used to identify the amino acids in a mixture, using standards of known amino acids.

Accepted Answer

Indicative content

a pencil baseline is drawn about 1 cm from the bottom of a silica plate, handled only by its edges
small spots of the mixture and of each standard are placed on the baseline with capillary tubes and allowed to dry
the plate stands in a covered tank of solvent whose level is below the baseline, until the solvent nears the top
the solvent front is marked in pencil as soon as the plate is removed, and the plate is dried
the plate is sprayed with ninhydrin in a fume cupboard and warmed, so the amino acids show as purple spots
the Rf of each spot is calculated and compared with the standards on the same plate; a match identifies the amino acid

Levels

Level 3 (5–6 marks) all stages are present in order: the pencil baseline, small spots of the mixture and each standard, the covered tank with the solvent below the baseline, marking the solvent front, developing with ninhydrin in a fume cupboard, and the Rf comparison with standards on the same plate
Level 2 (3–4 marks) the plate is spotted, run and compared with the standards; the solvent level below the baseline, the marked solvent front, the ninhydrin step or the Rf values are not there
Level 1 (1–2 marks) the mixture is spotted and run, often with the baseline drawn in ink or the solvent above the spots, and the colourless amino acids are never made visible

Question 2

Calculate the Rf value of each spot given by a mixture of two amino acids, at 2.9 cm and 6.1 cm from the baseline with the solvent front 7.6 cm from it, and identify each amino acid from the Rf values in the table, measured on the same kind of plate in the same solvent.

Accepted Answer

M1 Rf = distance moved by the spot ÷ distance moved by the solvent front = 2.9 ÷ 7.6 A1 Rf = 0.38, with no unit, so the lower spot is alanine M1 Rf of the upper spot = 6.1 ÷ 7.6 A1 Rf = 0.80, to two significant figures, so the upper spot is phenylalanine; reject an answer with a unit

Question 3

Calculate the percentage of each component in a mixture from the gas chromatogram peak areas in the table, assuming the detector responds equally to each.

Accepted Answer

M1 total area = 3.6 + 8.1 + 1.3 + 2.0 = 15.0 cm² M1 percentage of each = its area ÷ 15.0 × 100; reject the use of peak heights A1 24% and 54% for the first two components A1 8.7% and 13% for the last two, to two significant figures

Question 4

Explain the limitations of gas chromatography alone for identifying the components of a mixture, and how GC–MS overcomes them.

Accepted Answer

B1 GC identifies a component only by matching its retention time to a standard run under the same conditions B1 different compounds with similar properties can have the same retention time B1 a compound for which no standard was run, or a new one, cannot be identified at all B1 in GC–MS each separated component passes into a mass spectrometer, and its mass spectrum is matched against a database of known spectra

Question 5

Compare thin-layer chromatography with gas chromatography as methods of analysing a mixture.

Accepted Answer

B1 in TLC the mobile phase is a liquid solvent; in GC it is an unreactive carrier gas B1 in TLC the stationary phase is a solid such as silica; in GC it is usually a liquid on a solid support in a column B1 TLC identifies components by Rf value; GC by retention time, each compared with standards B1 GC gives the relative amounts of the components from peak areas, which TLC does not, but GC works only for compounds that vaporise without decomposing

Question 6

Evaluate a student's identification of a spot with Rf 0.45, on a plate run on Monday, as leucine, because a leucine standard gave Rf 0.45 on a plate run on Friday.

Accepted Answer

B1 the equal Rf values are consistent with the spot being leucine B1 Rf depends on the solvent, the plate and the temperature, which may have differed between the two days B1 other amino acids can have the same Rf as leucine in a given solvent B1 a sound identification runs the standard on the same plate as the unknown, and confirms it in a second solvent or by another technique

Question 7

Explain why HPLC rather than gas chromatography is used to measure the sugars in a fruit juice, and why the mobile phase in HPLC is pumped through the column at high pressure.

Accepted Answer

B1 sugars do not vaporise without decomposing, so they cannot be carried through a GC column as a gas B1 HPLC uses a liquid mobile phase, so the sugars stay in solution and need not be vaporised B1 the stationary phase is very small particles packed tightly, so the solvent would barely flow under gravity B1 the small particles are used because their large surface area gives a much sharper separation

Question 8

Suggest which solvent in the table should be used to separate compound X from compound Y on a silica column, giving reasons.

Accepted Answer

B1 solvent B, because it gives the largest difference between the Rf values of X and Y, so the bands separate B1 in solvent A both compounds are held so strongly that they barely move down the column B1 in solvent C both move almost with the solvent and come out together; an Rf near 0.3 for the wanted compound suits a column

Question 9

Explain how thin-layer chromatography on silica separates a polar compound from a non-polar compound.

Accepted Answer

B1 silica is polar, with O–H groups on its surface B1 the polar compound is adsorbed more strongly onto the silica, so it spends more time on the stationary phase and moves a shorter distance B1 the non-polar compound is held weakly and spends more time dissolved in the mobile phase, so it moves further and has the higher Rf

Question 10

Suggest how a chemist could find out whether two compounds that give spots with the same Rf value on a silica plate in one solvent are the same compound.

Accepted Answer

B1 run the two on the same plate in a different solvent, or on a different stationary phase B1 different compounds will usually give different Rf values in at least one of the new conditions B1 if they still match in every system, and give a single spot when spotted together, they are very likely the same compound

Question 11

Describe how a mixture is separated in gas chromatography.

Accepted Answer

B1 the sample is injected and vaporised, and an unreactive carrier gas such as helium carries it through a long coiled column in an oven B1 the column holds a liquid stationary phase with a high boiling point, coated on a solid support B1 components more soluble in the liquid, or less volatile, take longer to reach the detector and so have longer retention times

Question 12

Outline how column chromatography is used to obtain a pure sample of one component of a mixture.

Accepted Answer

B1 a column is packed with a slurry of silica or alumina in the solvent, and the mixture is added at the top B1 eluent is run through continuously and the components move down as separate bands at different rates B1 each band is collected as a separate fraction at the tap and the solvent is evaporated to leave the component

Question 13

Explain why, in gas chromatography, propanone (boiling point 56 °C) has a shorter retention time than butan-1-ol (boiling point 118 °C), and what raising the oven temperature does to both.

Accepted Answer

B1 propanone is more volatile, so it spends more of its time as vapour in the carrier gas and is carried through faster B1 butan-1-ol forms stronger intermolecular forces, including hydrogen bonds, so it dissolves more in the liquid stationary phase and is held back B1 a higher oven temperature shortens both retention times, as both spend more time in the gas phase

Question 14

Calculate the concentration of caffeine in a diluted energy drink that gives an HPLC caffeine peak of area 24.3 units, using the calibration results in the table, measured under the same conditions.

Accepted Answer

M1 area is proportional to concentration, with gradient 15.2 ÷ 0.10 = 152 units per g dm⁻³ M1 concentration = 24.3 ÷ 152; allow a value read from a plotted line A1 0.160 g dm⁻³, to three significant figures

Question 15

State what is meant by the stationary phase and the mobile phase in thin-layer chromatography.

Accepted Answer

B1 the stationary phase is the solid layer that does not move, silica or alumina on the plate B1 the mobile phase is the solvent that moves up the plate, carrying the components with it

Question 16

Explain why the baseline on a TLC plate is drawn in pencil and placed above the level of the solvent.

Accepted Answer

B1 pencil is graphite, which does not dissolve in the solvent or move up the plate, whereas ink would B1 above the solvent level the spots are not dissolved off the plate into the solvent in the tank

Question 17

Explain why a lid is placed on the tank while a TLC plate is being developed.

Accepted Answer

B1 the lid keeps the air in the tank saturated with solvent vapour B1 so the solvent does not evaporate from the plate as it rises and the front moves evenly

Question 18

Suggest two ways of making the colourless spots of amino acids visible on a TLC plate.

Accepted Answer

B1 spray with ninhydrin and warm, which turns amino acids purple B1 use a plate containing a fluorescent dye and view it under ultraviolet light, where the spots appear dark; or expose the plate to iodine vapour

Question 19

Identify the components of a solvent mixture from the retention times in the table, where the standards were run under the same conditions as the mixture.

Accepted Answer

B1 the peak at 1.8 min matches propanone and the peak at 4.6 min matches ethyl ethanoate B1 no peak appears at 2.9 min, so ethanol is not detected in the mixture

Question 20

Explain why percentages calculated from gas chromatogram peak areas may not be the true percentages, and how external calibration avoids the problem.

Accepted Answer

B1 the detector does not respond equally to every compound, so equal amounts of two compounds can give different peak areas B1 calibration with standards of the same compound at known concentrations relates that compound's own peak area to its concentration

Amino acid	Rf
Serine	0.27
Alanine	0.38
Valine	0.60
Phenylalanine	0.80

Retention time / min	Peak area / cm²
1.5	3.6
2.2	8.1
3.0	1.3
4.4	2.0

Solvent	Rf of X on a silica plate	Rf of Y on a silica plate
A	0.05	0.08
B	0.30	0.55
C	0.85	0.90

Caffeine / g dm⁻³	Peak area / units
0.10	15.2
0.20	30.4
0.30	45.6
0.40	60.8

Sample	Retention times / min
Mixture	1.8, 4.6
Propanone standard	1.8
Ethanol standard	2.9
Ethyl ethanoate standard	4.6