Thin-layer, column and gas chromatography

The diagram shows a gas chromatogram for a sample containing four isomers with

The carrier gas is nitrogen and the stationary phase is polar.

Which of the four isomers in this sample is the most abundant?

Endomorphin - 2 is a peptide with the amino acid sequence shown.

Tyr–Pro–Phe–Phe–NH₂

Each amino acid is represented by a three-letter abbreviation.

Tyr = tyrosine Pro = proline Phe = phenylalanine

Figure 2 shows part of the structure of endomorphin - 2, showing the Tyr–Pro–Phe– part of the molecule.

The –NH₂ at the end of the amino acid sequence of endomorphin-2 shows that the terminal functional group is an amide, not an acid.

Complete the structure of endomorphin-2 in Figure 2.

Use the structure in Figure 2 to draw the skeletal formula of proline, Pro.

A student hydrolyses a sample of endomorphin - 2 to break it down into its constituent amino acids.

The student analyses the resulting mixture by thin-layer chromatography, TLC.

State a reagent and the conditions needed for the hydrolysis.

Figure 3 shows the apparatus used for the TLC.

There is a piece of the apparatus missing from Figure 3. This omission will result in an inaccurate chromatogram.

Identify the missing piece of the apparatus.

State and explain why this piece of the apparatus is needed.

State why the amino acids separate on the TLC plate.

When the solvent has risen up the TLC plate, the student removes the plate from the beaker and sprays it with a developing agent.

Figure 4 shows the result.

Name a suitable developing agent.

State why the developing agent is needed.

Determine the R_f value for Tyr.

The protein fibroin can be broken down into amino acids using an enzyme.

A student uses thin-layer chromatography (TLC) to identify these amino acids.

The student identifies two of the amino acids as alanine and serine.

Use Figure 3 to calculate the R_f value of the unknown amino acid. Show your working.

Use your R_f value and Table 1 to identify the unknown amino acid.

The amino acids cannot be seen as they move during the experiment.

State how the amino acids can be made visible at the end of the experiment.

State why each amino acid has a different R_f value.

This question is about thin-layer chromatography (TLC).

• A protein was hydrolysed to form a mixture of amino acids.

• A spot of this mixture was added to a TLC plate and the plate placed vertically in a small volume of solvent 1.

• When the solvent front reached nearly to the top of the plate, the plate was removed and allowed to dry.

• The plate was turned anticlockwise through 90° and placed vertically in a small volume of solvent 2.

• When the solvent front reached nearly to the top of the plate, the plate was again removed and allowed to dry.

• Figure 2 shows the final TLC plate.

Suggest a suitable reagent for the hydrolysis of a protein.

Suggest how the positions of the amino acids on the TLC plate were located.

Deduce the minimum number of amino acids present in the original mixture.

Suggest why it was necessary to use two different solvents.

Cyclohexene (boiling point = 83 °C) can be prepared by the dehydration of cyclohexanol (boiling point = 161 °C) using concentrated phosphoric acid.

A student prepared cyclohexene by placing 10 cm³ of cyclohexanol (density = 0.96 g cm^–3) into a round-bottomed flask. 3 cm³ of concentrated phosphoric acid were then carefully added to the flask. The student added a few anti-bumping granules and set up the apparatus shown in Figure 3.

• The student heated the mixture and collected the liquid that distilled at temperatures below 100 °C

• The distillate was poured into a separating funnel and washed by shaking with sodium carbonate solution.

• Periodically, the separating funnel was inverted and the tap opened.

• The aqueous layer was discarded and the final organic product was dried using anhydrous calcium chloride.

• After the product was dried, the drying agent was removed by filtration under reduced pressure.

The student collected 5.97 g of cyclohexene in the experiment.

Calculate the percentage yield of cyclohexene.

Describe a test-tube reaction, on the product, to show that the cyclohexanol had been dehydrated. State what you would observe.

Suggest why sodium carbonate solution was used to wash the distillate.

Explain why it is important to open the tap of the separating funnel periodically.

Give a property of anhydrous calcium chloride, other than its ability to absorb water, that makes it suitable as a drying agent in this preparation.

Describe the apparatus used to remove the drying agent by filtration under reduced pressure. Your description of the apparatus can be either a labelled diagram or a description in words.

A sample of cyclohexene has been contaminated with cyclohexanol. The cyclohexene can be separated from the cyclohexanol by column chromatography. Silica gel is used as the stationary phase and hexane as the mobile phase.

Explain why cyclohexene has a shorter retention time than cyclohexanol.

Explain how an infrared spectrum would confirm that the cyclohexene obtained from the chromatography column did not contain any cyclohexanol.

Nitrobenzene reacts when heated with a mixture of concentrated nitric acid and concentrated sulfuric acid to form a mixture of three isomeric dinitrobenzenes.

Write an equation for the reaction of concentrated nitric acid with concentrated sulfuric acid to form the species that reacts with nitrobenzene.

Name and outline a mechanism for the reaction of this species with nitrobenzene to form 1,3-dinitrobenzene.

Name of mechanism

Mechanism

The dinitrobenzenes shown were investigated by thin layer chromatography

In an experiment, carried out in a fume cupboard, a concentrated solution of pure 1,4-dinitrobenzene was spotted on a TLC plate coated with a solid that contains polar bonds. Hexane was used as the solvent in a beaker with a lid.

The start line, drawn in pencil, the final position of the spot and the final solvent front are shown on the chromatogram in Figure 3

Use the chromatogram in Figure 3 to deduce the R_f value of 1,4-dinitrobenzene in this experiment.

Tick (✓) one box.

State in general terms what determines the distance travelled by a spot in TLC.

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.

A second TLC experiment was carried out using 1,2-dinitrobenzene and 1,4-dinitrobenzene. An identical plate to that in Question 8.3 was used under the same conditions with the same solvent. In this experiment, the R_f 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 R_f value.

Relative polarities

Explanation

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 R_f 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

Is the student correct? Justify your answer.