Techniques for Separating Substances

Task 1

Demonstrate use of scientific techniques to separate substances

1. Purifying an impure solid (alum) filtration and evaporation
2. Use about 3-4 of alum which is an impure solid
3. Add 25cm3 of water which is one-quarter full of the beaker
4. Place the beaker on a gauze and tripod, over a Bunsen burner. Stir the mixture with a glass rod while heating
5. Stop heating when the mixture boiled and allow it to cool a little
6. Filter the solution which is hot into the evaporating dish
7. Leave it to cool, and if necessary cover the evaporating dish with a watch glass or filter paper overnight
8. Remove the crystals by using a pair of forceps or by decanting
9. Dry the crystals by dabbing them on filter papers (RSC 2015)

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Thin layer chromatography of halides

1. Thin layer chromatography (TLC) is a method for mixture analysis by separating compounds found in a mixture. TLC of halides has three steps, which are spotting, development and visualization
2. The sample is applied to the adsorbent layer (a solid phase), as a small solution spot, near one edge.
3. After the solvent has evaporated, the sheet that is adsorbent coated is propped more or less vertically in a container that is closed, with the edge on which the spot was applied downwards. The spot on the plate must be placed above the solvent level in the container as shown in the figure below. If it is positioned below the solvent level, the spot will be washed often thin layer plate into the developing solvent (Wisconsin University 2015).
4. The solvent at the bottom of the container, start creeping up the adsorbent layer, passes over the spot as it rises up, in the process effecting materials separation in the spot (chromatogram development)
5. The thin layer plate is taken out of the container when the solvent front has reached near the top of the adsorbent as shown in the figure below (Wisconsin University 2015).

Paper chromatography

This is a technique used for separating chemical substances that has dissolved by using their different migration rates across sheets of paper. This technique involves application of sample or test solution as a spot near one corner of the filter paper sheet. Initially the paper is impregnated with some solvent to forma stationery liquid phase. The paper’s edge close to the spot is then immersed in a separate solvent where the mixture components are soluble in varying degrees as shown in the diagram below. The paper is penetrated by the solvent through capillary action and by passing over the spot of the sample, carries along with it different sample components (Block, Strange & Zweig 2013).

The components of the sample move with the solvent flowing at velocities dependent on their solubility’s in the flowing and stationery solvents. The components separation is brought about if there exist differences in their solubility’s in the two solvents. Both solvents are evaporated before the flowing solvents reaches the papers further edge, and the locations of the components separated is identified, usually by using reagents which form coloured compounds with the substances separated. The components separated appear as individual spots on the solvent path as shown in the diagram below (Block, Strange & Zweig 2013).

Electrophoresis of amino acids

1. Obtain cellulose chromatogram strip of sheet that is 15 cm long and 5 cm wide, while wearing gloves for safety
2. Mark a plus on one end of plate using a pencil and the other end with a sign of minus like the one shown in the diagram below

1. Draw a line through the middle of the plate to divide the two ends. The ruler should be wiped with Kimwipe before touching the plate’s surface.
2. For each sample, place a tic mark on the line. The samples should be at least 1 cm from the edge and 1 cm apart
3. On one end of the plate, label what samples are on each mark of the tic
4. Each electrode compartment should be filled with ~35 mL of buffer
5. Obtain an unknown solution and two standard amino acid solutions
6. Apply small volumes of the samples by using capillary tubes to the plate. By inserting the pointed end of the capillary tube, draw sample into the tube. Samples should not be contaminated by using a capillary tube from another sample. By touching the capillary tube end to the proper mark on the line, apply the sample to the plate. Touch the plate quickly and lightly, the spots should be small on the plate. Reapply twice more the same sample to the same spot, to allow the plate to dry up between the applications
7. Using a Pasteur pipette, apply buffer to the plate once the samples have dried. Start by dropping the buffer to the plate’s one end ten to the next end, and to allow it to move in the center toward the sample spots. Continue in a manner that the buffer exactly meets at the middle line to reduce sample spots migration
8. Place the plate carefully in the apparatus in a way that the plus mark corresponds to the red jack (positive electrode) and the minus side to the black jack (negative electrode). To avoid migration of the sample, care should be taken to avoid tilting the plate in any direction.
9. Turn on the apparatus and start recording the time
10. For 45 minutes, allow the sample to run or even as along as possible because of the time constraints
11. A few minutes before removing the plate, turn on low the hot plate
12. After expiry of the time, turn off the apparatus and remove the plate carefully
13. Spray the under the hood with ninhydrin
14. Place the plate on a plate that is hot until sports appear after drying up
15. Identify the unknown solution contends by comparing to the samples of standard amino acids (Science in Motion 2015).

Centrifugation of milk

Separation of milk and cream can be done by centrifugal separation. The centrifugal method separates according to density and size I which the lighter particles of a mixture move towards the axis of rotation while the denser mixture components are moved away from the axis of rotation of the centrifuge. The centrifuge is the device which rotates the substance around a fixed axis.

1. The mixture (milk with cream) is put in two test tubes that are specially designed referred to as the centrifuge tubes
2. The tubes are then placed in two different holders
3. With the help of a handle, the holders are rotated rapidly and then stopped after sometime. The centrifuge tubes are then taken out
4. In separating cream from milk, the cram will come to the surface since it is lighter than milk and then it is skimmed off through separate outlets (Mandakini Study Institute – Patna, 2015).

Task 2

Explain How to know a sample is pure using melting point analysis? What is the result if the sample is impure?

Using melting point analysis to know whether a sample is pure, it should melt over a range of 2⁰C or less. Experiment melting points need to be reported as a range. A sample will be presumed pure if the melting point is within the degree value found in the lab handbook (CSI 2015).

A sample is impure if the results of its melting point range is lower or is wider than what is stated in the literature value because impurities affect the melting points (CSI 2015). The melting, boiling points, and chromatography techniques can be used to tell whether a substance is pure or not. Pure substances have a distinct melting point while impure substances have a range of melting points.

A substance is presumed to be pure if the melting point range is two degrees or less within the value of the book. A sample is impure if the melting point is lower or wider that the book value. This is usually because of the more impurities in the substance.

Explain How to know a sample is pure using chromatographic technique? What is the result if the sample is impure?

Using chromatographic technique, a sample is purer if it gives only one spot. However, the sample is impure if it develops two or more spots. The paper chromatography technique is a modern method of separating mixtures. The technique uses paper as the stationary surface and the liquid as the mobile phase. The idea of the technique is that the paper and the liquid have both attractions to components in the, mixture. When place on the stationary any component of the mixture that is soluble will dissolve in the liquid. Every material in the soluble will then moves along with the solvent and display its own characteristics on the paper.

A substance is aid to be pure if it leaves only one spot on the paper and it is impure if it leaves more than one spot on the paper. For the impure substance, the spots must be different. In conclusion, therefore, if a substance is pure it produces one spot/substance at the end of the process. If it impure it will produce several spots/substances at the end of the process.

1. Sample A was purer. This is because its melting point range was within the melting point value found in the literature.
2. Sample A is pure because it gave one spot, some P is impure because it is a mixture and gave two spots, and finally sample B is a no soluble substance in solvent

References

Block, R. J., Le Strange, R., & Zweig, G. (2013). Paper Chromatography A Laboratory Manual. Burlington, Elsevier Science. http://public.eblib.com/choice/PublicFullRecord.aspx?p=1837501.

CSI, (2015). Melting Point Tips and Guidelines. [online] Csi.edu. Available at: http://www.csi.edu/ip/physci/faculty/rex/mptips.htm [Accessed 2 Jun. 2015].

Mandakini Study Institute – Patna, (2015). Mandakini Study Institute – Patna: CHAPTER 4 – CHEMISTRY. [online] Kuntal.org. Available at: http://kuntal.org/msipstudentslogin/pages/view/1053/chapter-4-chemistry [Accessed 2 Jun. 2015].

Royal Society of Chemistry, (2015). TeacherExpt:Purification of an impure solid – Learn Chemistry Wiki. [online] Rsc.org. Available at: http://www.rsc.org/learn-chemistry/wiki/index.php?title=TeacherExpt:Purification_of_an_impure_solid&oldid=7385 [Accessed 2 Jun. 2015].

Science in Motion, (2015). Electrophoresis of amino acids. [online] Google.com. Available at: http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=9&cad=rja&uact=8&ved=0CEEQFjAI&url=http%3A%2F%2Fwww.science-in-motion.org%2Fchem%2Flabs%2Felectrophoresis%2Fprocedural%2520labs%2FElectrophoresis%2520of%2520Amino%2520Acids.doc&ei=QaltVZTlAojVoATBqIDgAg&usg=AFQjCNGpzsIVDoK8X3Rbw3CV18gT0WR9sg&sig2=anwir2eVfX9yKIklqAwAFg&bvm=bv.94911696,d.cGU [Accessed 2 Jun. 2015].

Wisconsin University. (2015). Thin Layer Chromatography. ChemViews. Available at: http://www.chem.wisc.edu/courses/342/Fall2004/TLC.pdf [Accessed 2 Jun. 2015].

Herman Bailey

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