Chapter 4: Carry out chromatography

Chromatography is an essential technique in analytical chemistry used to separate components of complex mixtures for identification and quantification. In Kenyan analytical laboratories, such as those in pharmaceutical companies or food safety agencies, chromatography provides critical data to ensure product quality and regulatory compliance. Mastering the setup of chromatography apparatus is fundamental for accurate and reproducible results, as improper assembly can lead to contamination, poor separation, or equipment damage. This chapter focuses on the detailed preparation and configuration of chromatography equipment, emphasizing practical considerations relevant to Analytical Chemistry Technology professionals in Kenya.

4.1 Set up chromatography apparatus and equipment

Setting up chromatography apparatus means choosing the right equipment for the sample, assembling it so that the mobile phase can move over the stationary phase in a steady, repeatable way, and proving that it is ready before any analyte is separated. A badly assembled set-up cannot be rescued later: a tank that is not saturated gives a slanting solvent front, a pump that is not primed gives a drifting baseline, and a gas line that leaks gives noisy peaks. Four techniques are set up in this topic: paper chromatography, thin layer chromatography, high performance chromatography and gas chromatography. The principles of separation, the application of samples and the testing of analytes are taught in Sample preparation, Separate analytes and Test analytes, so this topic stays with the apparatus, the specifications and the order of assembly.

What every chromatography set-up needs

Whatever the technique, every set-up has the same five jobs to do. The table shows how each technique does them, so that the right equipment can be collected before work starts.

Preparing the workspace and the glassware

A set-up begins with the bench, not with the instrument. The same preparation applies to all four techniques.

• Cleanliness: Wash glassware, tanks and sample vials, rinse with distilled water and then with a little of the solvent to be used, and let them dry. A fingerprint or a trace of detergent shows up as an extra spot or peak.
• Solvent handling: Measure and pour organic solvents in a fume cupboard, keep them away from flames and hot plates, and cap the stock bottles at once. Most chromatography solvents are flammable and their vapour is harmful to breathe.
• Gloves and eye protection: Wear nitrile gloves and safety glasses. Disposable gloves are used once and discarded, never rinsed for reuse.
• Fresh solvents: Prepare mobile phases on the day of use from the grade the method asks for, and label each container with its contents, proportions, date and your initials.
• Waste: Collect used solvent in a labelled waste bottle kept apart from the aqueous waste, and keep used plates and papers in a closed solid-waste container until they are disposed of by the laboratory's waste procedure.
• Records: Write the date, solvent batch, plate or column identity and set-up checks in the laboratory notebook before the first run, so that a later fault can be traced.

4.1.1 Paper chromatography

Paper chromatography uses a strip of cellulose paper as the support and a liquid solvent as the mobile phase. The apparatus is simple and inexpensive, which is why it is widely used in teaching laboratories and for quick qualitative checks of dyes, food colours and amino acids in a food testing laboratory. Because there are so few parts, each one has to be right: the paper must hang freely, the origin must stay above the solvent, and the air in the jar must be saturated with solvent vapour.

Apparatus and equipment

Setting up the apparatus

1. Cut the paper strip so that it is about 2 cm shorter than the height of the tank, wearing gloves or holding it by the top edge only, because skin oils change how the solvent moves.
2. Draw the origin line lightly across the strip with a pencil, about 2 cm above the bottom edge. Pencil graphite does not dissolve in the solvent, whereas ink would run up the paper with the sample.
3. Mark the positions for the sample spots on the origin line with small crosses, at least 1.5 cm apart and 1.5 cm from each edge of the strip.
4. Pour the solvent into the tank to a depth of about 1 cm, so that the level will stay below the origin line when the strip is hung.
5. Cover the tank and leave it for 10 to 15 minutes so that the air inside becomes saturated with solvent vapour. A saturated tank stops the solvent evaporating from the paper surface, which would distort the solvent front.
6. Fix the top of the strip to the glass rod with a clip or staple, adjust the rod so that the bottom edge of the strip will just dip into the solvent and the strip hangs free of the walls, and check that the origin line is above the solvent level.

Checks before the run

• The strip is clean, flat and not creased or discoloured. A damaged strip is replaced.
• The tank is level on the bench, away from draughts and direct sunlight, so that the solvent front rises evenly.
• The solvent depth is below the origin line, and the lid fits.
• The spot positions are marked and the capillary tubes are clean and dry.
• The locating reagent and the drying aid are ready for the end of the run.

4.1.2 Thin layer chromatography

Thin layer chromatography uses a plate coated with a thin layer of adsorbent, usually silica gel, as the stationary phase. It is quick, needs little solvent and several samples can be run side by side on one plate, so it is common in plant extract laboratories for screening herbal products and in agrochemical residue laboratories for checking extracts before instrument analysis. The plates themselves are also used to follow extractions, as met in Carry out Extraction. The set-up differs from paper chromatography in the plate, the saturated tank and the way the spots are seen afterwards.

Apparatus and equipment

Setting up the apparatus

1. Select the plate to suit the method and check that the layer is smooth, uniform and free from chips or scratches. Keep plates in their box or in a desiccator, because the layer takes up moisture from the air.
2. If the method asks for it, activate the plate by heating it in an oven for the stated time and let it cool in a desiccator before use.
3. Cut the plate to the size needed with a plate cutter or scissors, depending on the backing, without cracking the layer. Handle it by the edges with forceps.
4. Mark the origin line lightly in pencil about 1.5 cm above the bottom edge, without breaking the layer, and mark the lanes about 1 cm apart and 1 cm from the side edges. Mark the finish line about 1 cm below the top edge.
5. Calculate the volume of mobile phase from the tank size. The solvent depth should be about 0.5 cm, which is always below the origin line. For a tank with an inside base of 12 cm by 6 cm: volume = 12 cm × 6 cm × 0.5 cm = 36 cm³ = 36 mL. Prepare about 40 mL, for example 70 parts hexane to 30 parts ethyl acetate: 40 mL × 0.70 = 28 mL hexane and 40 mL × 0.30 = 12 mL ethyl acetate.
6. Pour the mobile phase into the tank, line the back wall with filter paper and wet the paper with the solvent, close the lid and leave the tank for 15 to 30 minutes to saturate.
7. Prepare the viewing aids before the run: switch on the UV lamp or position the viewing cabinet, fill the spray bottle with the reagent, and set the oven or hot plate to the temperature the method gives.

Checks before the run

• The plate layer is intact, and the plate stands in the tank with the origin line clear above the liquid.
• The filter paper liner is wet all over, so that the tank atmosphere is saturated.
• The lid is closed and the tank is not disturbed or moved once it has been set up.
• A fresh batch of mobile phase is used for each run, because a mixture of solvents changes in proportion as the more volatile part evaporates.
• Anyone using the UV lamp wears a face shield or UV-blocking glasses, because short-wave ultraviolet light damages the eyes and skin.

4.1.3 High performance chromatography

High performance chromatography, in its liquid form (HPLC), forces a liquid mobile phase through a column packed with very small particles by means of a high-pressure pump. The fine particles give a sharp separation but resist the flow, which is why the pump must deliver a steady pressure and every fitting must hold it. HPLC is widely used in quality control laboratories of food and beverage plants for preservatives, sweeteners and colours, and in water laboratories for dissolved organic contaminants. Setting it up carefully protects the column, which is an expensive consumable.

Apparatus and equipment

Setting up the apparatus

1. Prepare the mobile phase from HPLC grade solvents and water. When a buffer is used, adjust its pH before the organic solvent is added, and measure each liquid separately before mixing, because the volume of a water and acetonitrile mixture is slightly smaller than the sum of the two. For 1 L of a 60 : 40 mixture of acetonitrile and water, measure 600 mL and 400 mL separately and then combine them.
2. Filter the mobile phase through a 0.45 µm membrane filter that suits the solvent, and degas it by sonication or by the instrument degasser. Particles block the column inlet, and bubbles cause pressure swings and a noisy baseline.
3. Fill the reservoirs, label them, make sure the inlet filters are fully covered by liquid, and place the waste line in the waste container.
4. Prime the pump: open the purge valve, run the pump at a high flow for a few minutes until no bubbles leave the purge line, and then close the valve.
5. Connect the guard column and the analytical column in the direction of the arrow marked on the column, with fingertight fittings. Over-tightening damages the fitting and the column end.
6. Start the flow at a low rate and raise it in steps to the method value, for example 1.0 mL per minute, watching the pressure on the display. A pressure that is far above its usual value points to a blockage, and a pressure that is very low points to a leak or air in the pump.
7. Switch on the detector and its lamp and allow it to warm up, then set the wavelength given in the method.
8. Equilibrate the column by pumping the mobile phase through it until the baseline and the pressure are steady. Use at least 10 column volumes, taking the column volume as the liquid held inside the packed column. For the column in the table, the empty tube holds π × r² × length = 3.1416 × (0.23 cm)² × 15 cm = 2.49 cm³. Taking the liquid inside it as about 65 % of this, the column volume is 0.65 × 2.49 = 1.62 mL, so 10 column volumes are 16.2 mL, which takes about 16 minutes at 1.0 mL per minute.

Checks before the run

• No bubbles in the solvent lines or the pump, no leaks at the fittings, and no salt crust under a connection.
• The pressure and the baseline are steady, and the detector lamp has reached its working state.
• The injection valve moves freely between its load and inject positions, and a manual injection syringe is clean and its volume markings are checked against a known volume from time to time.
• Solvent quality is acceptable: a mobile phase left standing for days can grow algae when it holds water or buffer, so it is renewed regularly.
• The buffer is washed out of the column with a water and organic mixture at the end of the day, and the column is stored in the solvent the manufacturer recommends, never left filled with buffer.

4.1.4 Gas chromatography

Gas chromatography separates compounds that can be vaporised without decomposing. The sample is injected into a hot port, carried by a gas through a long, thin column in an oven, and detected as it leaves. It is used in petroleum products laboratories for fuel composition, in environmental laboratories for volatile organic compounds, and in quality control laboratories for residual solvents. The set-up depends on gas, power, heat and electronics, and so its checks cover each of them.

Apparatus and equipment

Setting up the apparatus

1. Check the gas supply: the cylinder is chained upright, the regulator is fitted with a clean, leak-free connection, and the purifier traps are within their service life. Set the delivery pressure the instrument manual requires.
2. Fit a new septum and a clean liner in the injection port, because a worn septum leaks and a dirty liner holds back or breaks down the sample.
3. Install the column. Cut each end square with a ceramic wafer, check the cut under a magnifier, fit new ferrules and insert the injector end to the depth the manual gives. Leave the outlet end free of the detector for now. A badly cut end gives tailing peaks.
4. Turn on the carrier gas and let it flow through the column for 10 to 15 minutes before any heating. Oxygen in a hot column destroys the stationary phase.
5. Check for leaks at the injector and the column nut with an electronic leak detector. Do not spray liquid leak solution on fittings near the detector.
6. Condition a new column by heating it, in steps and with its outlet not yet joined to the detector, to a temperature not above the limit marked on the column, and holding it for the time the column maker states. Then cool the oven, fit a new ferrule, join the outlet end to the detector to the depth the manual gives and check that fitting for leaks.
7. Set the temperatures: injection port above the boiling point of the least volatile component, oven programme as the method gives, and detector temperature at or above the highest oven temperature, so that nothing condenses in it.
8. Start the detector. For a flame ionisation detector, set the hydrogen and air flows to the manual's values, for example about 30 mL per minute of hydrogen and 300 mL per minute of air, ignite the flame, and confirm ignition from a change in the baseline signal. Wait until the baseline is steady before analysing.

Checks before the run

• Hydrogen is a flammable gas. Use a hydrogen generator or a cylinder in a ventilated place, check the supply line for leaks, and never light the detector while a leak is suspected.
• The septum, liner and column nuts are free of leaks, and the carrier gas flow is steady at the method value.
• The injection syringe is rinsed several times with a clean solvent before use and its volume delivery is calibrated at intervals, so that the volume injected is reproducible.
• The baseline is flat and quiet when the oven is at its starting temperature. A rising or noisy baseline points to an impure gas, a bleeding column or a dirty detector.
• The detector jet and collector are cleaned when the signal falls or the baseline becomes noisy, and the electrical connections are checked so that no signal is lost.

Chapter Summary

This chapter explored the process of carrying out chromatography, beginning with the proper setup of chromatography apparatus and equipment essential for accurate separation. It highlighted the importance of preparing samples correctly to ensure that the analytes are suitable for the chosen chromatographic method. The separation of analytes was discussed in detail, focusing on how different components move through the stationary and mobile phases based on their chemical properties. Following separation, the chapter examined techniques to test and identify the analytes, ensuring reliable analysis results. Emphasis was placed on maintaining precision throughout the procedure to achieve clear separation and accurate detection. Overall, the chapter provided a comprehensive guide to performing chromatography from start to finish, emphasizing both practical skills and theoretical understanding.