Electrophoresis Lecture Notes - King Salman International University - 2024

Field of Pharmacy Sciences Bachelor of Pharmacy-PharmD (Clinical Pharmacy) Program Advanced Instrumental Analysis (PMC510) Lecture 6: Electrophoresis Dr. Amir Shaaban Farag Date : Nov./19 /2024 The cours...

Field of Pharmacy Sciences Bachelor of Pharmacy-PharmD (Clinical Pharmacy) Program Advanced Instrumental Analysis (PMC510) Lecture 6: Electrophoresis Dr. Amir Shaaban Farag Date : Nov./19 /2024 The course is designed to introduce the student to modern methods of instrumental analysis. In modern analytical chemistry, the focus of the course is on trace analysis, and therefore methods for the identification, separation and quantitation of trace substances will be described. *Approximately 66% of all products and services delivered in the US rely on chemical analysis of one sort or another. *Approximately 250,000,000 chemical determinations are performed in the US each day. NIST, 1991, from Managing the Modern Laboratory, 1(1), 1995, 1-9.  The term electrophoresis means any experimental technique that is based on movement of charged particles (ions, molecules, macromolecules) in electric field in liquid medium. Any electrically charged particle dissolved in aqueous solution, when placed to a constant electric field, will start to migrate towards the electrode bearing the opposite charge; the speed of the particle movement will be directly proportional to the applied voltage and particle charge, but inversely proportional to the particle size. Any molecules that differ in size and/or charge can be separated from each other in this way. o The electrophoretic analysis can in principle be applied to any particles that are charged under given experimental condition, such as small cations or anions, organic acids, amino acids, peptides, saccharides, lipids, proteins, nucleotides, nucleic acids, etc… o However, the by far commonest subjects of electrophoretic separation are proteins and nucleic acids. o Electrophoresis of macromolecules is normally carried out by applying a thin layer of a sample to a solution stabilized by a porous matrix. Under the influence of an applied voltage, different species of molecules in the sample move through the matrix at different velocities  Ivanovich Arne Strakhov Tiselius (1807) (1931)  As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species and as a separation technique.  Types of electrophoresis: A) Moving boundary (Capillary electrophoresis) B) Zone electrophoresis (Gel electrophoresis)  Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve ". Gel electrophoresis can provide information about the molecular weights and charges of proteins, the subunit structures of proteins, and the purity of a particular protein preparation. It is relatively simple to use, and it is highly reproducible. The most common use of gel electrophoresis is the qualitative analysis of complex mixtures of proteins and nucleic acids (DNA & RNA). Gel is a colloid in a solid form (99% is water). Different types of gels which can be used are; Agar and Agarose gel, Starch, Polyacrylamide gel.  A linear polysaccharide, used in conc as 1% and 3%, the gelling property are attributed to both inter- and intramolecular hydrogen bonding, mainly used in separation of DNA & RNA. Pore size is controlled by the % of agarose used; large pore size are formed with lower conc and vice versa. Advantages Disadvantages Easy to prepare and small Resolution is less compared to concentration of agar is required. polyacrylamide gels Resolution is superior to that of filter Different sources and batches of agar paper. tend to give different results and purification is often necessary Large quantities of proteins can be separated and recovered. Sharp zones are obtained due to less adsorption  Frequently referred to as ‘‘PAGE’’, Cross-linked polyacrylamide gel are formed from the polymerization of the monomer in presence of small amount of N,N'-methylene- bisacrylamide. Made in conc. between 3-30% acrylamide, low % has large pore size and vice versa, mainly used in separation of proteins. Most widely used method for analysing protein mixture qualitatively. Useful for monitoring protein purification – as separation of protein is based on the size of the particle. Can also be used for determining the relative molecular mass of a protein. o Gel powder is mixed with an electrophoresis buffer (conducts the electric current) and heated to a high temperature until all of the agarose powder has melted. o The molten gel is then poured into a gel casting tray (casting unit) and a “comb” is placed at one end to make wells for the sample to be pipetted into. o Once the gel has cooled and solidified (it will now be opaque rather than clear) the comb is removed. o The gel is then placed into an electrophoresis tank and electrophoresis buffer is poured into the tank until the surface of the gel is covered. o A dye is added to the sample of DNA prior to electrophoresis to increase the viscosity of the sample which will prevent it from floating out of the wells and so that the migration of the sample through the gel can be seen. o A DNA marker (also known as a size standard or a DNA ladder) is loaded into the first well of the gel. The fragments in the marker are of a known length so can be used to help approximate the size of the fragments in the samples. o The prepared DNA samples are then pipetted into the remaining wells of the gel. o When this is done the cover is placed on the electrophoresis tank making sure that the orientation of the gel and positive and negative electrodes is correct (we want the DNA to migrate across the gel to the positive end). o The electrical current is then turned on so that the negatively charged DNA moves through the gel towards the positive side of the gel. o Shorter lengths of DNA move faster than longer lengths so move further in the time the current is run. o The distance the DNA has migrated in the gel can be judged visually by monitoring the migration of the loading buffer dye. o Once the DNA has migrated far enough across the gel, the electrical current is switched off and the gel is removed from the electrophoresis tank. o To visualize the DNA, the gel is stained with a fluorescent dye that binds to the DNA and is placed on an ultraviolet transilluminator which will show up the stained DNA as bright bands. o Alternatively, the dye can be mixed with the gel before it is poured. o If the gel has run correctly the banding pattern of the DNA marker/size standard will be visible. o It is then possible to judge the size of the DNA in your sample by imagining a horizontal line running across from the bands of the DNA marker. You can then estimate the size of the DNA in the sample by matching them against the closest band in the marker.  Jorgenson and Lukacs published papers showing the ability of capillary electrophoresis to perform separations that seemed unachievable. Employing a capillary in electrophoresis had solved some common problems in traditional electrophoresis. For example, the thin dimensions of the capillaries greatly increased the surface to volume ratio, which eliminated overheating by high voltages. The increased efficiency and the amazing separating capabilities of capillary electrophoresis spurred a growing interest among the scientific society to execute further developments in the technique. o A typical capillary electrophoresis system consists of a high-voltage power supply, a sample introduction system ‘‘a capillary tube’’, a detector and an output device. o Some instruments include a temperature control device to ensure reproducible results. This is because the separation of the sample depends on the electrophoretic mobility and the viscosity of the solutions decreases as the column temperature rises. o Each side of the high voltage power supply is connected to an electrode. These electrodes help to induce an electric field to initiate the migration of the sample from the anode to the cathode through the capillary tube. o The capillary is made of fused silica and is sometimes coated with polyimide. Before the sample is introduced to the column, the capillary must be flushed with the desired buffer solution. There is usually a small window near the cathodic end of the capillary which allows UV-VIS light to pass through the analyte and measure the absorbance. A photomultiplier tube is also connected at the cathodic end of the capillary, which enables the construction of a mass spectrum, providing information about the mass to charge ratio of the ionic species. 1.CE has a flat flow, compared to the pumped parabolic flow of the HPLC. The flat flow results in narrower peaks and better resolution. 2.CE has a greater peak capacity when compared to HPLC—CE uses millions of theoretical plates. 3.HPLC is more thoroughly developed and has many mobile and stationary phases that can be implemented. 4.HPLC has more complex instrumentation, while CE is simpler for the operator. 5.HPLC has such a wide variety of column lengths and packing, whereas CE is limited to thin capillaries.