How Electrophoresis Advances Research and Diagnostics
Electrophoresis is known as the movement of the dispersed or colloidal particles that are suspended in a given fluid because of their interactions with the electric field that is uniform partially. The electrophoresis process of the positively charged particles called cations is often referred to as cataphoresis. On the other hand, the electrophoresis of the negatively charged particles called anions is referred to as anaphoresis. It is caused due to the presence of an interface that is charged and lies between the surface of the particle and the surrounding fluid. It makes for the basis for different analytical techniques that are used in Chemistry to separate the molecules according to their charges, size, or binding affinity.
The electrophoresis process is also used in laboratories for separating the macromolecules depending on their size. This technique includes the application of a negative charge so that the proteins would move towards the positive charge. Electrophoresis is also widely used in the analysis of DNA, RNA and different proteins. We will discuss the electrophoresis uses and the application of electrophoresis in chemistry in this article.
(image will be uploaded soon)
Application of Electrophoresis in DNA Analysis and DNA Fragmentation
Amongst the most important applications of the process of electrophoresis is in the analysis of DNA analysis the studying of DNA fragments. DNA is known for the negative charge consistency that it holds and it is affected due to the presence of the electrical current. When pressure is exerted on it by the electric field, DNA starts to break down into small and large fragments. This happens because different kinds of DNA fragments get affected by the current on different scales. A test medium like an agarose gel or an acrylamide gel is used for freezing the separated fragments of a particular DNA strand. The electric field is then removed and the study of these fragments is carried out.
Application of Electrophoresis in Protein Detection
A type of electrophoresis known as immunoelectrophoresis is common and used for analysing several kinds of proteins’ existence and how they behave chemically in different environments. When irregular molecules of proteins are formed they get triggered because of various medical conditions like multiple sclerosis, kidney failures and even different kinds of cancers. The irregular proteins are detected by the method of electrophoresis on the urine or blood samples and frequently checking its results for any kind of deviations from standard forms and amounts of the protein. Immunoelectrophoresis is also used for the detection of specific proteins which are called immunoglobulins.
Application of Electrophoresis for Testing Antibodies
Electrophoresis plays a variety of essential functions when it comes to the testing of antibiotics.
The most common applications of the process of electrophoresis in this field are the testing of antibiotics to check their purity. Electrophoresis is used in a solution that consists of the antibiotic to be tested in the form of a paper strip. This strip is impregnated with a capillary or the antibiotic which is filled with the drug.
Electrophoresis is also used for determining the strength of the antibiotic having a vital importance in regards to administering the exact dosages. Also, the antibiotic research field has a common ground along with the genetic testing field. Hence, electrophoresis also helps in discovering the genes which tend to indicate the resistance to a specific kind of antibiotics.
Factors affecting Electrophoresis
Below are the factors that affect the process of electrophoresis.
Electric Field
The electric field is one of the major factors affecting the process of electrophoresis. The movement of ions in electrophoresis depends on the current, resistance, and voltage in the electric field.
During electrophoresis, the voltage of the electric field affects the travel time of the molecules being separated. With higher voltage, the DNA will be able to travel through the gel much faster. However, excessively high voltages can melt the gel or even cause distortion of DNA bands.
The distance travelled by the ions in electrophoresis is directly proportional to the time and current. Since the current is conducted between electrodes, the increase in voltage leads to an increase in the number of charges towards each electrode.
Resistance of electrophoresis depends on the thickness of the gel, size, buffer conductivity, temperature, and the amount of buffer. It usually decreases with an increase in the temperature. The amount of resistance will determine whether the circuit is a bad conductor or a good one.
Sample
The size, shape, and charge of the sample, which is being separated during electrophoresis, affect its migration rate. With an increase in the charge, the rate of migration will also increase. Moreover, the rate of migration is also affected by the change in the size of the molecule and the shape of the sample. It is inversely proportional to the size and shape of the sample.
Buffer
The migration rate of the compound is affected by the buffer, which stabilises the pH levels of the supporting medium. Some of the most commonly used buffers for the electrophoresis process are EDTA, formate, Tris, citrate, barbitone acetate, and pyridine. The migration of the sample used can be affected if the buffer binds to the molecules, which is why it should never bind to them.
The proportion of the current carried by this buffer will increase while the current carried by the sample being separated will decrease. At low ionic strength, the proportion of the current carried by the buffer will decrease and the one carried by the sample will increase.
Heat generation is oftentimes a problem that occurs during electrophoresis. The heat generated during electrophoresis changes the density and viscosity of the electrophoretic media. It can also damage the equipment by melting the plastics, cracking the glass plates, warping, etc.
In case the conductivity of the supporting medium is high, the process of electrophoresis becomes more difficult. It is because solutions with high conductivity lead to a lower field strength per current and the heat load on the system increases.
Supporting Medium
The supporting medium used in electrophoresis can affect the migration rate of the sample. Usually, the inert medium is preferred during the process of electrophoresis. This medium, however, can cause molecular sieving, absorption, or electro-osmosis that can affect electrophoresis. Absorption can cause tailing of the sample, which causes a disruption in the movement of the sample. It also reduces the rate and resolution of the separation during electrophoresis.
FAQs on Applications of Electrophoresis in Chemistry
1. What is the fundamental principle of electrophoresis?
The fundamental principle of electrophoresis is the movement of charged molecules through a solution or matrix (like a gel) under the influence of an applied electric field. Positively charged ions (cations) migrate towards the negative electrode (cathode), while negatively charged ions (anions) move towards the positive electrode (anode). The rate of migration depends on the molecule's net charge, size, and shape, allowing for effective separation.
2. What are the major applications of electrophoresis in chemistry and biology?
Electrophoresis is a versatile technique with several crucial applications, including:
DNA and RNA Analysis: Separating DNA or RNA fragments by size, which is essential for DNA fingerprinting, genetic mapping, and PCR product analysis.
Protein Separation: Analyzing proteins from a sample to determine their size, quantity, and purity, or to detect specific proteins in medical diagnostics.
Pharmaceutical Quality Control: Testing the purity of antibiotics and vaccines by separating the active components from any impurities.
Forensic Science: Used extensively in creating DNA profiles from crime scene samples to identify suspects.
3. How does agarose gel electrophoresis specifically help in separating DNA fragments?
Agarose gel electrophoresis is ideal for separating DNA fragments for two main reasons. First, the agarose gel forms a porous matrix that acts as a molecular sieve. Smaller DNA fragments navigate this mesh more easily and travel farther than larger fragments. Second, the phosphate backbone gives DNA a uniform negative charge-to-mass ratio. This means that in an electric field, DNA fragments migrate solely based on their size, not their charge, allowing for reliable and predictable separation.
4. Why is electrophoresis considered an essential tool in forensic science for DNA fingerprinting?
Electrophoresis is essential in DNA fingerprinting because it allows forensic scientists to visualise the unique genetic blueprint of an individual. The technique separates specific DNA fragments called Variable Number Tandem Repeats (VNTRs), which vary in length between individuals. When separated by gel electrophoresis, these fragments create a distinct banding pattern, similar to a barcode. By comparing the pattern from a crime scene sample to that of a suspect, a definitive match can be made, making it a powerful tool for criminal investigation and paternity testing.
5. How does the charge-to-mass ratio of a molecule affect its movement during electrophoresis?
The charge-to-mass ratio is a critical factor determining a molecule's mobility in electrophoresis. A molecule with a higher net charge will experience a stronger electrostatic force, causing it to move faster towards the oppositely charged electrode. Conversely, a larger molecule (higher mass) will face more frictional drag from the medium, slowing its movement. Therefore, molecules with a high charge-to-mass ratio migrate the fastest, while those with a low ratio move the slowest, enabling separation based on these distinct physical properties.
6. What are the main types of electrophoresis techniques used in laboratories?
Several types of electrophoresis are used, each suited for different applications:
Gel Electrophoresis: Uses a gel matrix (like agarose or polyacrylamide) as the support medium. It is the most common method for separating proteins and nucleic acids.
Capillary Electrophoresis (CE): Performed in a narrow capillary tube, offering high speed, excellent resolution, and automation. It is used for separating a wide range of small and large molecules.
Paper Electrophoresis: An older technique using a strip of filter paper as the support medium, primarily used for separating smaller molecules like amino acids and small proteins.
7. What are some common challenges or limitations encountered when performing electrophoresis?
Despite its utility, electrophoresis has certain limitations. A primary challenge is heat generation from the electric current, which can denature sensitive molecules like proteins or affect the gel's consistency, leading to distorted separation bands. Other issues include preparing gels of a consistent concentration, avoiding contamination of samples, and the potential for overlapping bands if molecules have very similar sizes and charges, which can make interpretation difficult. Proper technique and controlled conditions are crucial to overcome these challenges.
