Assignment Detail:- NASC 1009 Introduction to Biosciences - University of South Australia
Chemistry Practical Experiment
Aims• Qualitative analysis• Use of chromatographic techniques• Thin-layer chromatography -TLC- and column chromatography• Separation of mixtures
Introduction
Chromatography, from the Greek words for colour and writing, is a term used to describe a variety of separation techniques- The components of a mixture to be separated are partitioned between two phases, one of which remains stationary while the other phase -mobile phase- percolates through the stationary phase, or over its surface- The mobile phase can be a liquid or a gas and the stationary phase can be a solid or a liquid - several combinations of stationary and mobile phases are thus possible-
PART A - THIN LAYER CHROMATOGRAPHY -TLC-
Procedure:Capillaries with very small diameters are used as TLC spotters- You have been provided with TLC spotting pipettes to use to spot your samples onto the TLC plate-1-- A clean spotting pipette will be needed for each sample, put them in a particular arrangement so that you do not mix up what has been spotted with what sample- If you get mixed up- get a fresh spotter or you will cause errors-
2-- Into the TLC developing jar add sufficient solvent -solvent A- to cover the bottom of the jar to about 0-5 cm- Close the lid on the jar lightly to allow a solvent atmosphere to form while preparing the TLC plate-
NOTE: when handling the TLC plates in the following steps please handle via the edges as fingerprints will interfere with the plate-
3-- Using one TLC- Take a piece of the silica gel-coated TLC sheet and lightly rule a pencil line across -approx- 1cm from the bottom of the plate, see Figure-, taking care not to score through the absorbent layer -the silica-- Then place small spots -1 mm diameter- of A, B, C and M only, on the line using the spotters -see Figure-- Where A, B and C are the dichloromethane solutions of aspirin, paracetamol and caffeine- The spots need to be small so as to diminish spreading of the spots on development- Allow the spots to dry between each application -See TLC troubleshooting page in this booklet about problems with spotting on TLC--
4-- Then show a demonstrator your TLC to ensure that you have sufficient sample -will be viewed under UV light-UV lamps are in the balance room- before developing your TLC-
5-- Stand the TLC plate in the development jar, ensuring that the sampling line is above the level of the liquid in the beaker- Cover the beaker once more with the lid and watch closely as the solvent ascends up the TLC plate via capillary action-
Take care not to disturb the jar too much during this time-
6-- When the solvent has ascended to within 1-5 cm of the top of the plate -this should take approx- 20-25 minutes-, mark the upper limit of the solvent front with a pencil, remove the plate, and allow to dry on benchtop -or in fumehood if needed--
7-- Examine the plate under a UV lamp -short wavelength, 254 nm- and mark the positions of the spots -carefully circle each spot you can see with a pencil-- Sketch your chromatogram below clearly marking the positions of the spots and the solvent front- Each spot should be carefully outlined with a pencil-
8-- CARE: UV light may be harmful to unprotected eyes- Get help from a demonstrator if you need it -to analyse the developed plate under UV light-
9-- Now measure the distance between the ruled line and the solvent front and between the ruled line and the centre of each spot- Determine the Rf value of each of the reference drugs- Calculate Rf values -see over the page for help with calculating Rf values and/or ask a demonstrator for help- and identify the drug components present in the mixture-
10-- Report your results in your report book and then proceed to the second part of the practical session-
Procedure:
1-- Tear -or cut with scissors- some spinach leaves into strips and grind them to a paste using a mortar and pestle-
2-- Add a small amount -about 5 mL- of ethanol at a time, and stir/grind until the solution becomes dark green- As ethanol evaporates more may need to be added-This is your solution of extracted pigments- Note: you may need to add more ethanol to your paste as it evaporates to make a liquid consistency-
3-- Filter the extract through a very small wad of cotton wool -or folded filter paper- in the neck of a small filter funnel into a small clean and dry test tube held in a clamp or a mini-test tube holder -see lockers-- The stationary phase for the chromatography will be fine silica powder -found in the fume hood- held in a glass column-
4-- Take a glass Pasteur pipette -this is what will be your mini glass column- and use atiny wad of cotton wool to partially restrict the narrow capillary exit-
5-- Add fine silica powder into the pipette to make the column 5-6 cm long- Optional: wear face mask if concerned about silica- Then add a small top sand layer of 2-4mm-
6-- Clamp the pipette in the vertical -using a retort stand, boss head and clamp- and place a small beaker under it- This apparatus set-up can now be moved into a spare fume hood so there is minimal risk of inhalation of solvent as you run the column-
7-- Next, using a clean plastic Pasteur pipette place 2-4 drops of the -filtered- and hopefully very green plant extract on the top of the column of silica, allow 30-60 seconds for the material to adsorb onto the top of the column and to evaporate off and dry-
8-- Add the chromatographic solvent very gently so as not to disturb the surface of the silica with your sample extract on top -10% ethanol in petroleum ether - in the fume hood- to the top of the column, to fill the rest of the pipette-
9-- Keep carefully adding the solvent to the top of the column as it passes through the sample and down the column- NOTE: Add carefully or you will disturb the silica-
10-- Take note of the coloured bands that form as you add the chromatographic solvent and watch the separation that takes place- Draw a picture-s- of it over the page-These are the purified chlorophylls and other extracts -see notes at the beginning of part B for more details--
11-- Try to identify the bands, make a note of the colours and the bands and show your demonstrator- You only need to develop the column until you see the separation-
12-- Once you have finished put any solvent waste in the container provided and your mini spinach column can go straight into the glass container in the fume hood -ask a demonstrator where this is--
13-- Pack up, tidy up and leave your bench/lockers clean and neat for the next group! Note: you may need to use detergent to clean up the mortar and pestle- Get your locker checked before leaving the lab as part of your lab participation-
Attachment:- Introduction to Biosciences-rar
Attachment:- practical report-rar
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