Here, we will focus on the use of different analytical techniques, particularly in terms of analyzing data to provide structural information about compounds. Here, your knowledge of core chemistry will help you to do this.
Some methods of detection and analysis
There are five main areas of analysis that you need to be familiar with:
1. Electrophoresis and DNA Fingerprinting
2. Proton-NMR Spectroscopy
3. X-ray Crystallography
5. Mass Spectrometry
In each of the case you need to be able to explain how the technique exactly works and what Information it provides. You may be expected to interpret data from one or more of these techniques to solve a problem.
In this part, we will discuss Electrophoresis and DNA fingerprinting.
We know that amino acids form zwitterions in aqueous solution and that the exact nature of a zwitterion depends on the pH of the solution.
This effect can be seen when an electric potential is applied to a mixture of the amino acids glycine, lysine and glutamic acid at pH 7 using the set-up showing in the following Figure:
Apparatus for electrophoresis
If the power supply is connected and the apparatus left for a period of time the amino acid mixture is separated as shown in the following Figure:
Results of the electrophoresis of a mixture of three amino acids
We can explain how different amino acids are affected by looking at the structures of glycine, lysine and glutamic acid at pH 7:
Some Points to be noted at pH 7:
- Glycine carries one of each type of charge so it is attracted equally to both electrodes and hence does not move
- Glutamic acid carries an extra negative charge and moves towards the positive electrode
- Lysine carries an extra positive charge and hence moves towards the negative electrode
The same process can be used to separate protein fragments (peptides):
The velocity of the amino acids is related directly to the voltage applied across the plate. Other factors can affect the relative velocities of samples.
- The smaller molecules move faster than larger molecules carrying the same charge
- A molecule with large side chains moves more slowly than a straight-chain molecule with the same charge and Mr
- The pH of the buffer influences the extent of ionization, and hence movement and direction
This has become a ‘high-profile’ analytical technique due to its use in forensic science to screen crime suspects. It uses the same technique as electrophoresis but rather than relying on filter paper as the medium, a gel is used here.
In recent years, the technique has been refined so that only a tiny amount of DNA is needed. This can be extracted from the material such as cheek cells, blood, hair, semen or skin. The DNA is firstly treated with enzymes to break it into fragments that can be analyzed by electrophoresis. It is possible to start the analysis with as little as 0.2 nanograms (2 × 10-10 grams) of DNA.
If the amount of DNA is very small, a technique called the polymerase chain reaction (PCR) can be used to produce more copies of the DNA.
The figure shows the apparatus for carrying out gel electrophoresis. Samples of the DNA fragments are put into small ‘wells’ in the gel near the cathode. Since the phosphate groups on the DNA fragments are negatively charged and are attracted to the anode while smaller fragments moving faster than larger ones.
The results can be stained to help them show up or if the DNA fragments are treated with radioactive phosphorus where a photographic print can be obtained. A typical example is shown below,
A simple example to show the results of gel electrophoresis
In this simplified example, it is relatively straightforward to pick out the DNA of both the victim and suspect 2 from the DNA samples collected at the crime scene.
Related- X-ray Crystallography
Although the use of DNA in crime detection gets a great deal of publicity, it has also a number of other important uses:
- Establishing the father of a child in a paternity case and making links within a family from samples obtained from both living and deceased relatives
- Establishing links between archaeological samples of biological origin, such as animal skins
- In medical applications where the presence of a particular polypeptide or protein can be an early indication of a problem without symptoms, for instance in newborn babies
Things to remember:
- Amino acids may be positively charged, negatively charged or neutral. This means they can be separated on buffered filter paper when an electric potential is applied
- The speed and direction of movement in electrophoresis depend on the size of the amino acid or peptide, the size of any side chains present and the charge carried at the pH of the buffer used
- DNA is broken into fragments using enzymes called restriction enzymes
- The amount of DNA can be increased using the polymerase chain reaction (PCR)
- The phosphate groups in DNA fragments are negatively charged enabling them to move when an electric potential is applied
So, DNA fingerprinting has a wide range of applications including providing forensic evidence, establishing genetic links between individuals, archaeology involving specimen of biological origin and early diagnosis of genetic diseases.