1. Separation Science has grown to become one of the most useful scientific tools. Used extensively in diagnostic and clinical science, Separation Science is relied upon by scientists, physicians, law enforcement officials and the general public to provide quantified information about our health, our food, our environment, our products we use and in almost every aspect of our modern society. Different samples and sample matrices present distinct challenges to the separation scientist and therefore there are different techniques to separate and quantify and then qualify these samples. Smaller and smaller sample availability presents further challenges to analysis. Phase Partitioning and Extraction Chromatography Electrophoresis These are the main techniques today used by separation scientists. Each technique has its own place and value. 7/23/2013HONESTY IS THE BEST POLICY 2

2. A technique for separating the components of a mixture of charged molecules (proteins, DNAs, or RNAs) in an electric field within a gel or other support. The history of electrophoresis dat es back to the work of Arne Tiselius in the 1930. Capilary electrophoresis Gel electrophoresis There are two major types of electrophoresis. 7/23/2013HONESTY IS THE BEST POLICY 3 3. 7/23/2013HONESTY IS THE BEST POLICY 4 4. First introduced by Hjerton in 1967. Jorgenson and Lucaks demostrated its high resolution power in 1980. It has helped in sequencing 14.9 billion base-pairs in Human-Genome Project in just 9 months. 7/23/2013HONESTY IS THE BEST POLICY 5 5. 7/23/2013HONESTY IS THE BEST POLICY 6 6. 7/23/2013HONESTY IS THE BEST POLICY 7 7. 7/23/2013HONESTY IS THE BEST POLICY 8 8. 7/23/2013HONESTY IS THE BEST POLICY 9 9. Hydrostatic (or pressure) injection Electrokinetic injection By gravity (most common) 7/23/2013HONESTY IS THE BEST POLICY 10 10. The force exerted on a charged particle depends on electric field,E(volts/cm), and charge q on the particle. This electrophoretic force causes the particle to move and is given as: Felec = q.E The particle however shall experience resistance on account of the viscosity of the solvent. This resistance,which is itself a force is proportional to velocity,that is: Fresistnace = f.v Where f is the frictioanl coefficient and v is the velocity of the particle under observation. f depends on viscosity of solvent , and size and shape of the particle. The bigger and asymmetrical the particle,the slower is its motion through the solvent. 7/23/2013HONESTY IS THE BEST POLICY 11 11. When electric field is applied to charged particles, they begin to migrate. The electrophoretic and frictional forces oppose each other and the particles velocity increases until the forces are balanced. Fresistance = Felec f.v = q.E v/E = q/f This property v/E is called electrophoretic mobility and it is characteristic property of each particle. The electrophoretic mobility is proportional to the ionic charge of a sample and inversely proportional to any frictional forces present in the buffer. When two species in a sample have different charges or experience different frictional forces, they will separate from one another as they migrate through a buffer solution. 7/23/2013HONESTY IS THE BEST POLICY 12 12. Electroosmotic flow is observed when an electric field is applied to a solution in a capillary that has fixed charges on its interior wall. Charge is accumulated on the inner surface of a capillary when a buffer solution is placed inside the capillary. In a fused-silica capillary, silanol (Si-OH) groups attached to the interior wall of the capillary are ionized to negatively charged silanoate (Si-O-) groups at pH values greater than three. Attracted to the negatively charged silanoate groups, the positively charged cations of the buffer solution will form two inner layers of cations (called the diffused double layer or the electrical double layer or Debye layer) on the capillary wall. The first layer is referred to as the fixed layer because it is held tightly to the silanoate groups. The outer layer, called the mobile layer, is farther from the silanoate groups. 7/23/2013HONESTY IS THE BEST POLICY 13 13. The mobile cation layer is pulled in the direction of the negatively charged cathode when an electric field is applied. Since these cations are solvated, the bulk buffer solution migrates with the mobile layer, causing the electroosmotic flow of the buffer solution. The rate of EOF is dependent on the field strength and the charge density of the capillary wall. The wall's charge density is proportional to the pH of the buffer solution. The electric double layer is the basis for a category of phenomena known as electrokinetic effects. Electro-osmosis is one of the electrokinetic effects. 7/23/2013HONESTY IS THE BEST POLICY 14 14. Since the electroosmotic flow of the buffer solution is generally greater than that of the electrophoretic flow of the analytes, all analytes are carried along with the buffer solution toward the cathode. Even small, triply charged anions can be redirected to the cathode by the relatively powerful EOF of the buffer solution. Negatively charged analytes are retained longer in the capillary due to their conflicting electrophoretic mobilities. The small multiply charged cations migrate quickly and small multiply charged anions are retained strongly. 7/23/2013HONESTY IS THE BEST POLICY 15 15. 7/23/2013HONESTY IS THE BEST POLICY 16 16. N=L/H The term theoretical plates N refers to an explanation of the resolving power of a column where the column was viewed as a stack of closely related but discrete layers (or plates). The more plates in the column the greater the resolving power of the column but as the column has a definite length, more plates equals thinner plates. In other words N and H are inversely proportional. As N increases (for a given column) H decreases. where L is the length of the column packing (usually in cm - this is a constant for a given column). H is the height of a theoretical plates. 7/23/2013HONESTY IS THE BEST POLICY 17 17. 7/23/2013HONESTY IS THE BEST POLICY 18 18. Finally,The efficiency of capillary electrophoresis separations is typically much higher than the efficiency of other separation techniques like HPLC. Capillary electrophoresis separations can have several hundred thousand theoretical plates. On account of these justifications, the CE is becoming principle workhorse in forensic and clinical labs. 7/23/2013HONESTY IS THE BEST POLICY 19 MARKET SEGMENT ESTIMATED SHARE(%) PHARMACEUTICAL INDUSTRY 35 BIOSCEINCE 35 CHEMICAL INDUSTRY 20 FOOD/BEVERAGES 5 OTHERS 5 19. On the flip-side, the CE has got certain serious disadvantages. In single-capillary instrments, samples are analyzed sequentially (one at a time). The rate at which samples can be processed is called throughput. 7/23/2013HONESTY IS THE BEST POLICY 20 20. 7/23/2013HONESTY IS THE BEST POLICY 21 21. Gunshot residues are produced when a firearm is discharged and can be deposited on the hands of the perpetrator. In targets and weapons, these residues comprise unburned powder, particles from the primer and case of the cartridge. The mode of separation most widely used for this type of analysis is MEKC. The separation and identification of organic gunshot and explosive constituents by MEKC was first applied by Northrop. The separation was rapid and efficient providing the separation of 26 organic gunshot and explosive constituents in less than 10 min. 7/23/2013HONESTY IS THE BEST POLICY 22 22. GUNSHOT RESIDUES PAKISTAN ARMY AGAINST MILITANTS IN FATA 7/23/2013HONESTY IS THE BEST POLICY 23 23. 7/23/2013HONESTY IS THE BEST POLICY 2 24. Verification and authentication of printed documents just from the inks that have been used has long been a major problem for forensic scientists, customs officials and historians. The ability to differentiate between inks is of great interest in forensic science because it allows evaluation of the authenticity of a suspicious document. 7/23/2013HONESTY IS THE BEST POLICY 25 25. Modern inks contain dozens of chemical components (e.g., acid or basic dyes, organic or inorganic colour pigments, surfactants, antio xidants, viscosity adjusters, resins, glycol and glycerol, waxes, oils, and Pigments). Forensic-ink chemists face a very complex, challenging analytical problem in carrying out this type of analysis, as sophisticated writing instruments and printing technologies have emerged. Since ink-analysis links its roots to handwriting-analysis, an analysis often found very useful in forensics and can be well understood in a Hollywood movie Sherlock Holmes. 7/23/2013HONESTY IS THE BEST POLICY 26 26. There are two major categories of pens: ballpoint and non- ballpoint. However, the former account for more than 80% of the cases requiring ink analysis. Initially, many techniques such as UV-VIS, IR and HPLC were harnessed to differentiate ink samples. For the forensic scientist, perhaps the most important characteristic of CE is the extraordinarily small sample requirement (nanoliters), which is often advantageous in minimizing the destruction of the document being tested. In 1991, Fanalli and Schudel reported the first CE method for analyzing inks. 7/23/2013HONESTY IS THE BEST POLICY 27 27. DNA Typing 7/23/2013HONESTY IS THE BEST POLICY 28 28. It is a very important technique and is becoming central in separation science. But at the same time, it has been found to carry certain serious issues with it. So it can be referred to as a necessary evil. 7/23/2013HONESTY IS THE BEST POLICY 29 29. 7/23/2013HONESTY IS THE BEST POLICY 30 30. 31