B1: Analysis of Plasma Extracts and Drug Samples by TLC An UV lamp is used to observe the TLC plate. YOU MUST NOT PICK UP, TILT OR MOVE THE LAMP WHILE YOU ARE DOING THIS AND DO NOT LOOK DIRECTLY AT THE LAMP UNDER ANY CIRCUMSTANCES. Safety Notes: The compounds used in these experiments as "unknown" samples are drug compounds and care must be taken to avoid ingestion and skin contact. The plasma solutions must be treated as biological hazards and all work must be carried out using gloves to avoid any contact. All biological wastes and contaminated glassware must be sterilised chemically after use (see technician). Sample Acid/neutral extract, basic extract and drug powders Mobile Phases A Ethyl acetate: methanol: 0.880 ammonia solution (85:10:5) B Ethyl acetate Apparatus Silica Gel GF254 TLC plates Two TLC tanks Procedure Prepare TLC tank A (large beaker) by pouring in the Mobile Phase A to a depth of about 8 mm and TLC Tank B by pouring in Mobile Phase B. Line both tanks with solvent soaked paper to provide an atmosphere saturated with solvent vapor. Remember to label TLC tanks accordingly. Cover with a glass plate and leave to equilibrate for 15 minutes. Cut TLC plates to a suitable size to accommodate at least 4 spots then draw a pencil line about 1 cm from the bottom edge. Dissolve a small amount of each drug powder in methanol. Apply 1-2 µl spots of acid-neutral extract and dissolved powder along the pencil line of plate A. Run plate A in TLC Tank A. Apply 1-2 µl spots of basic extract and dissolved powder along the pencil line of plate B. Run plate B in TLC Tank B. Allow the spots to dry thoroughly before placing the plate in the TLC tank. Allow the solvent to migrate to within 1 cm of the top of the plate and mark the solvent front. Air-dry the plate. When examining a plate under UV light, the components show up as dark spots against a white background. Mark any spots with a pencil. Never look at the UV light directly. Match the drug sample with the extracted samples. Measure the Rf (Retention Times) for all observed spots, where \[R_{f}=\frac{\text { distance a solute migrates up plate }}{\text { distance the solvent front migrates up plate }}\] Compare the Rf values obtained from the unknown drug substances and the plasma extracts using the two solvent systems. Thinking about Making a Valid Measurement How far apart do the spots need to be for the analyst to be confident that adequate separation has been achieved? What experiments could be performed to check your prediction? Improving Your Practical Technique Work in a clean environment to avoid contamination. Only handle the TLC plate by its top corner and only lay the TLC plate down on a clean surface. Put identifying marks on the top of the TLC plate using a pencil. Consider doing duplicate samples. Prepare a relatively concentrated solution from the original samples otherwise you will not be able to see minor components on the TLC plate. Select a solvent in which the sample dissolves. Use reference materials (e.g. a pure substance) to help to identify the components in the sample. The intensity of the spots can be used as a crude guide as to the relative amount of components in the sample.