Electrophoresis and chromatography are both forms of molecular sieving. They are incredibly useful and widely used tools that allow researchers to separate complex subjects into identifiable parts.
What is the difference between chromatography and electrophoresis?
It is likely that you will either do a thin-layer/paper chromatography experiment or an electrophoresis experiment during your A Level, rather than both, so it can be useful to know the difference between the two as well as the similarities. Both of these techniques use substances that act as sieves to separate out mixtures, and in fact, electrophoresis is really just a particular form of chromatography. There are many other forms of chromatography used in research, including gas chromatography and affinity chromatography. In all of these forms, chromatography takes advantage of the difference between the mobile and stationary phases. The mobile phase is the substance that is able to move across a stationary phase, allowing our sample mixture to move with it. The stationary, or adsorbent, phase is the substance that takes up the particles of subject mixture passing through. The extent to which parts of the sample move allows for its constituent pieces to be identified, and variation between the separation patterns of sample mixtures allows us to determine differences between samples.
Both thin-layer and paper chromatography use a solvent mobile phase which is drawn up a stationary phase by capillary action. The biggest difference between these methods are the different stationary phases. The stationary phase in thin-layer chromatography (TLC) is often a silica gel or cellulose on an inert substrate. The type of mobile phase used and the ease with which the sample can bind to the stationary phase will determine the pattern created. For example, a silica gel plate is very polar, meaning that strongly polar molecules in your sample will move less far before binding to the stationary phase than less polar ones. It is common to use a silica gel plate and a nonpolar solvent to separate out the pigments in cholophyll, as the solvent allows you to separate the pigments based on how polar they are.
There are advantages to using TLC over paper, depending on the sample that you are using. It may be possible to run a TLC more quickly than paper chromatography and with more samples on the same paper as the spreading zones are often smaller. This may also make it easier to perform two-way chromatography. Smaller spreading zones also improves the sensitivity of detection if you are trying to determine what your spots are made from, and TLC plates are often more heat resistant to allow you to develop spots on your plate more easily, and may be more resistant to the use of strong solvents in the mobile phase.
Gel electrophoresis is usually used for the analysis of DNA. There are numerous ways that DNA samples can be prepared before they are run on an electrophoresis gel, although as there may only be one or just a few copies of the gene or DNA fragment of interest present, the sample usually needs to be amplified by a process called the polymerase chain reaction (PCR). PCR can generate thousands or even millions of copies of your sample using thermal cycling of the DNA with a DNA polymerase enzyme. This enzyme makes copies of the DNA, then copies of the copies, so the number of DNA samples increases exponentially in a relatively short period of time, making them much easier to visualise.
Once the DNA samples are ready, they can be placed in small wells in an agarose gel, in a tank such as the one pictured above. The tank is filled with buffer solution to allow an electric current to pass through, pulling the electrically charged DNA particles through the agarose gel. The size of the DNA sequence will determine how quickly it moves through the gel, meaning sections of DNA can be separated by size. This enables us to see, for example, the presence of a gene that has been inserted into a genetically modified organism that is not present in the wild-type.
How do researchers use these techniques?
What is CRISPR?
CRISPR (Clustered regularly interspaced short palindromic repeats) are short sections of repeated DNA that can be found in prokaryotic organisms like bacteria. Their discovery was considered unusual because prokaryotes have comparatively small, compact genomes, without much of the non-coding DNA (sometimes and erroneously called junk DNA) found in eukaryotes. It was discovered that the sequences of DNA between these repeats were, in fact, exact matches to virus DNA, meaning CRISPR is actually a form of acquired defense system for prokaryotes against viruses. The bacteria is able to recognise the virus DNA using CRISPR associated proteins (Cas) and if it comes into contact with that DNA sequence, the Cas protein immediately cuts and destroys it.
CRISPR is very useful for a bacteria to be able to recognise and fight against viruses, but scientists quickly realised that the system could be used as a way of highly targeted gene editing. This is a huge advantage over traditional genetic modification methods, as previous editing methods were much less accurate. The gene that you were trying to insert or remove from a genome was much less likely to find exactly the right place in the genome, making it more expensive and time consuming to successfully accomplish. CRISPR also appears to work across prokaryotic and eukaryotic organisms, so its potential applications are enormous.
There are important medical applications for CRISPR. There are emerging CRISPR treatments for diseases such as cancer, where genes that promote uncontrolled cell division can be swapped for those that instead trigger cell death - and this idea has already been shown to work in mice. Any disease with a genetic component could potentially be treated by CRISPR, although the editing of the germ line would affect future generations who would have no way of consenting to such a procedure - which goes against one of the fundamental principles of medical ethics.
In the Laboratory Confessions podcast researchers talk about their laboratory experiences in the context of A Level practical assessments. In this episode we look at separation of biological compounds using thin-layer or paper chromatography and the use of appropriate instrumentation to record quantitative measurements such as a colorimeter or a potometer.
What do your measurements mean?
In a gel, you would run all of your experimental samples alongside a molecular-weight size marker, usually called a DNA ladder. The ladder, marked 'ELP' in the image here, allows you to determine the approximate molecular weight of your samples. It is worth noting that the molecular weight is inversely proportional to the distance travelled along the gel, so the ladder marks out a logarithmic scale. It may be that you are only interested in the presence or absence of a band on your electrophoresis gel, so the addition of the DNA ladder is not vital to your experiment. Nevertheless it is usual practice to include a ladder when running a gel in the laboratory.
To analyse the movement of your sample in thin-layer or paper chromatography, the key thing that is of interest is the distance that the different parts of the sample move in relation to the overall movement of the solvent. It is vital to quickly mark the solvent front in pencil once a chromatography plate is complete so you can accurately measure these distances. The distance the spot has moved divided by the distance the solvent front moved is called the retention factor, or Rf. The Rf values that you calculate can be compared to standard values (usually given as a range, although you may be asked to calculate your own - they will vary depending on the solvent and plate used) which will tell you what was contained in your original sample.
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