The term electrophoresis is defined as :
The movement or flow of charged particles under the influence or action of external electric field. In electrophoresis narrow peaks of graphs are formed that are easy to study. Instrumentation is very simple, Working principle of electrophoresis technique is very simple. Cost per sample in electrophoresis is less.
At start this technique was used by biochemists, biologists and clinical chemists. For many years electrophoresis has been the powerhouse method for separating proteins (enzymes, hormones, antibodies) and nucleic acids (DNA, RNA).
Types of Electrophoresis:
There are following types of electrophoresis.
- Gel Electrophoresis
- Capillary electrophoresis
In gel electrophoresis material is moved in gel plate by applying voltage on the basis of size or charges.
The main instrumentation of Gel electrophoresis is shown here in diagram.
In diagram you can see there is a tray on which agarose gel is placed . On gel sample wells are also shown in which sample material is placed. On one side positive electrode is attached and on the other negative electrode is attached. A potential difference is created across the gel. Gel is present in a solution that is buffer solution. Buffer solution helps in flow of molecules in gel.
Any of the specie is neutral then it can’t be analyzed. Because analyte solution should be in the form of cations and anions. For example if the DNA or protein strands are separated these strands are separated on the basis of their size or charges. The mixture is filled in sample wells. By applying electricity a potential difference is created.
Negatively charged species move toward positive electrode and pass through gel particles having more negative charge moves rapidly towards the positive electrode. On the other hand if a specie is small sized then it will move fast towards respective electrode. As a result of this movement separation patterns are formed. This technique is known as gel electrophoresis.
Now in above mentioned diagram gel plates are shown . When wells are loaded with DNA sample strands move towards electrode. Small sized strands moves rapidly , large sized strands will not move so in this way separation is done. One of the strand is taken as a standard strand just like that of high liquid pressure chromatography with the help of standards peaks the peak of analyte is analyzed. In electrophoresis standard strand is used whose nature is determined.
A buffer filled silica capillary having internal diameter of 10 to 100μm and 40 to 100cm long in length is used in capillary electrophoresis. A potential of 5 to 3 KV is applied across the electrodes.
Capillary electrophoresis is latest form of gel electrophoresis. In case of gel molecules consume time to move from one side to other. So that’s why gel plate is replaced by a capillary tube in which gel is filled.
In comparison to gel electrophoresis it has less operational cost, small size instrument, less time consuming and better analysis is done with this .
The main instrumentation of capillary electrophoresis is shown in given below diagram.
In this two buffer reservoirs are used in which positive and negative electrodes are dipped. On one side there is anode and on the other side cathode. For example if species are in the form of cations these were added in the positive electrode reservoir.
By adding sample overall length of the reservoir is increases in order that the solution will reached to capillary after mixing. By applying potential difference species are separated on the basis of charges and sizes. Detector give signals and by any of the spectroscopic instrument, analytical technique results will given out.
Why capillary electrophoresis is better than gel electrophoresis?
- Because in gel electrophoresis particles consume time in flow from one side to other. capillary electrophoresis consume less time.
- Instrumentation of capillary electrophoresis is small as compared to gel electrophoresis.
- There is less operational cost as that of gel electrophoresis by the capillary electrophoresis.