TLC lab protocol

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School

McGill University *

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Course

210

Subject

Chemistry

Date

Dec 6, 2023

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pdf

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Uploaded by SuperHumanArtLark19

Name: Student number: 1 TLC labs Work in groups of 4 Teaching Assistants will assign you a group # to label your samples Groups: • a: Spirulina (Cyanobacteria) • b: Dulse (Red Algae) • c: Spinach Leaves We are extracting water-insoluble photosynthetic pigments today. You will be using a pipette today – see the pipette guide at the end of this protocol for proper use. You will first use acetone (an organic solvent ) to extract pigments from plant tissue. After centrifugation (and filtration in the case of spirulina), to remove cell sediments and other solids, you will use thin layer chromatography (TLC) to separate the mixture of pigments. These pigments will show up at different distances along the TLC strip. Here is an explanation of the process: TLC uses a thin plastic plate coated with silica gel. You will apply samples of your plant extract near the bottom of the strip of TLC with a thin glass tube called a capillary tube . The TLC strip is then placed vertically in a suitable solvent. As the solvent is wicked up, it passes through the sample and carries the compounds upwards. Different compounds dissolved in the solvent adsorb to the silica gel to different degrees; the more polar a molecule, the more strongly it is adsorbed. (Note: adsorb means to adhere to the surface of another compound without forming a chemical bond and is different from absorbing.) Relatively polar compounds stay at the bottom of the TLC strip, while less polar ones are carried by the solvent nearer the top. Each student group will make separate chromatograms from the acetone extracts of either cyanobacteria (spirulina), red algae (dulse), or the leaves of a land plant (spinach). At the end of the experiment, you will compare the various TLC strips and answer some questions in the lab quiz.

Name: Student number: 2 Extraction and filtering: The first few steps depend on which group you are in – follow the instructions specific to your plant tissue (see the table): • Weigh out the tissue in small “weigh boats” on a scale; subsequently, transfer the tissue to a mortar and pestle to grind: o Spirulina : weigh out 0.5 g powder o Dulse : 1.5 g powder o Spinach : 2 leaves ( no need to weigh) • In the case of spinach, you need to add 2 g of sand • For dulse and spinach samples, you have to grind strenuously, but spirulina powder is very fine already • Add acetone (organic solvent) to extract the pigments (follow table for volumes); grind more for the case of dulse and spinach • Use the 1000 μL micropipette to transfer the liquid extract to a 1.5 ml tube ; transfer roughly 1 ml to 1.5 ml of liquid extract; try to avoid debris (solid bits) • Label your tube with your group # e.g., A1 (group #1 doing spirulina extractions). • Centrifuge the samples at top speed for 1 min (make sure the tubes in the centrifuge are balanced — check with TA before running). Your extract will be the supernatant (liquid) and the cell debris will form a pellet at the bottom of the tube (pellet). • Set the tubes aside on a rack – do not stir and avoid dislodging the pellet. Spirulina Dulse Spinach 0.5 g 1.5 g Take 2 baby spinach leaves and transfer to mortar-pestle Add 2 g of sand Grind Grind thoroughly Grind thoroughly Add 2 ml of acetone Add 2 ml of acetone Add 5 ml of acetone Grind some more! Grind some more! Filter your sample through a Buchner flask with filter paper Transfer liquid into 1.5 ml tube Transfer liquid into 1.5 ml tube Transfer to a new 1.5 ml tube Centrifuge (top speed) for 1 min Centrifuge (top speed) for 1 min Centrifuge (top speed) for 1 min

Name: Student number: 3 Application & separation: Near the top edge of each TLC strip, label with your group #; use a pencil very gently; otherwise, the silica gel will crack off. • Using a capillary tube, apply a row of spots along a line about 1.5 cm from the bottom of the TLC strip (see Figure 1). Control the size of spots by firmly holding your finger over the tube's top, then gently touching the strip. You want to gently aspirate (draw liquid) into the capillary tube so there is not too much volume in the tube. • Only a small amount of extract is wicked out. Allow spots to dry out completely (10 seconds) and apply another row (3 spots) on top of the first. Apply 15 spots (or 5 x 3 spots across the line of application). • If you do not apply to the same line – or apply too much liquid – the bands of separated pigments will be blurred. • Add 300 μl TLC solvent (toluene : acetone; 3:2 ratio) to the bottom of the TLC tube (enough to fill the bottom curve of the tube ~0.5 cm). • Avoid letting the solvent touch/run down the sides of the tube. • If you are not ready to run the TLC, use a rubber stopper to close the tube (avoid solvent evaporating). • Lower a TLC strip gently into the tube (using forceps and holding the strip from the top) – leave the tube in the rack while adding the TLC strip, and do not jostle it afterwards. • Remove your strip from the tube when the solvent front (wet TLC) comes within 2-3 cm of the top; you can observe the separation of pigments while it is in the tube and draw it out on the next page, indicating colours and pigment predictions. • Bring your TLC strip to the TA; discuss and draw a depiction on the board. Disposal and clean-up: • When all chromatograms are done, pour all the solvent from tubes into a designated chemical waste jar • Pour contents of flasks and test tubes into chemical waste and wash thoroughly with water (be gentle – be careful not to break glass) • Dispose of 1.5 ml tubes in the BioHazard bin • Throw out TLC strips in the garbage once TAs have viewed them • Throw out sand/slurry from mortar-pestle in the garbage; use a paper towel to wipe it out and then wash it in the sink • Collect capillary tubes in a beaker and hand them over to the TA. These are very fragile and expensive. Treat with care.

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Name: Student number: 4 TLC LAB ASSIGNMENT 1. Do all the samples have carotenoids? (yes / no) 1 pt. 2. Which samples do not have chlorophyll b? (2 pt) 3. Which set of pigments are the most polar? (1 pt) 4. Mark the separation of pigments below: (3 pts for correct band identification) Figure 1

Name: Student number: 5 Eppendorf tube (1.5 ml)