Method
Each group is given 2 unknown compounds to identify
Thin Layer Chromatography
- Prepare solution with 10ml ethanol in 90ml hexane and place in a sealed chromatography tank at least 20 minutes before analysis
- Prepare two fine needles using Bunsen burner.
- Take a few crystals of the unknown compound and dissolve it in 1ml of chloroform then spot the solution onto the chromatoplate using the 2 needles prepared.
- Place the chromtoplate in the sealed tank.
- Remove the plate once the solvent has travelled near to the top and use UV light to visualise the spots and determine the Rf value.
Melting Point Determination
- Seal one end of a melting point tube in the Bunsen burner and avoid curvature of the tube.
- Place a few crystals of the unknown compound into the tube and tap it down to the sealed end.
- Use the melting point equipment to determine the melting point, settings it initially at 55°C and increasing it by 2°C at a time.
Ultra violet Spectroscopy
- Prepare 10ml dilute solution of Metroclopromide in acetonitrile.
- set the UV spectrophotometer to record absorption from 200nm-400nm wavelengths
- run a background spectrum of the solvent using a quartz cuvette
- run a sample spectrum for the Metroclopromide sample using the matched UV cuvette
- Record the wavelength of maximum absorption and the absorption value which this occurs.
Results
Calculation of Rf for Unknown B Calculation of Rf for Unknown G
Rf =3.9/9.2 Rf =3.2/9.2
Rf= 0.42 Rf= 0.35
Fig 1: A table showing the melting points and Rf values for the unknown compounds B and G
Fig 2: Absorbance against wavelength graph for Metroclopromide diluted in acetonitrile
Wavelength for maximum absorption = 302nm
Absorption value = 1.86
50% acetonitrile and 50% metroclopromide
Discussion
Identification
The melting point of unknown B is 65-67°C and therefore falls into group 1. Once the Rf value was calculated at 0.42 it is possible to identify it as the compound chloro-m-cresol.
The melting point of unknown G is 125-127°C and therefore falls into group 4. Once the Rf value was calculated at 0.35 it is possible to identify it as the compound methyl-4-hydroxybenzoate.
The unknown compounds were determined mainly from the melting point because this is a more accurate method, whereas the Rf value can deviate largely from the correct value. To verify the identified compound is correct it is possible to mix the unknown sample with a sample of the compound it’s thought to be and then determine the melting point of the sample, if identification of the unknown was incorrect then the melting point could be lower or a lot higher than is stated in textbooks.
Why do substances melt?
The melting point of a solid is the temperature at which it changes from its solid state to a liquid.
There are attractive intermolecular forces between all molecules within a solid; these forces pull the material together whilst the kinetic energy of each particle pushes them apart. For melting to occur the kinetic energy has to dominate over intermolecular forces. As we increase the temperature of a solid the particles within it gain more kinetic energy and move faster and eventually possess enough energy to break the attractive forces between each other and pull away to form a liquid.[1]
Types of attractive forces include (in decreasing strength), ionic bonding, hydrogen bonding, dipole-dipole interactions and
van der waals.
Ionic bonds are the strongest bonds and therefore require a lot of heat energy to break them; hydrogen bonds are present in both compound B and G due to the free –OH and O on methyl-4-hydroxybenzoate. There are more sites for hydrogen bonding in methyl-4-hydroxybenzoate and so the melting point is a lot higher because there are more bonds to break.
Fig3: Structure of chloro-m-cresol Fig4: Structure of methyl-4-hydroxybenzoate
What type of separation mechanism operates in thin layer chromatography?
A spot of the sample is applied to the plate and then dipped into a solvent, usually ethanol or water. The solvent moves up the plate by a capillary action. When the solvent meets the sample it dissolves it and the sample is carried up by the solvent. The different compounds will travel at different rates depending on their solubility in the solvent. If a polar solvent is used then the polar parts of the sample will travel up the plate faster than the non polar parts of the sample.[2]
The compounds will also differ in the strength of their adsorption to the adsorbent on the plate. This produces an ascending chromatographic separation.
UV spectroscopy is used to determine the wavelength at which absorbance is a maximum, this wavelength is characteristic to each compound and provides information on the electronic structure of the compound.[3]
Conclusion
Unknown compound B is chloro-m-cresol and unknown compound G is methyl-4-hydroxybenzoate. Chloro-m-cresol is used as a preservative for adhesives, glues, polymer dispersions and emulsions whilst methyl-4-hydroxybenzoate is used for preservatives in food, cosmetics, sunscreens and shampoos.
These analytical methods used are very important in drug characterization to help identify unknown compounds. Thin layer chromatography and melting point determination are both cheap, simple and quick to carry out and therefore are the most useful to use, although the most accurate method is melting point determination if carried out appropriately[4].
Other identification methods that can be used are nuclear magnetic resonance and UV spectroscopy.
References
1:Rob Lewis and Wynne Evans (2001) Chemistry 2nd Edition: Palgrave Foundations: p154
2:http://en.wikipedia.org/wiki/Thin-Layer_Chromatography
3:http://www.chm.davidson.edu/ChemistryApplets/spectrophotometry/AbsorbanceSpectrum.html
4: http://orgchem.colorado.edu/hndbksupport/meltingpt/meltingpt.html
