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1Cell Culture TechniquesNameDate of Submission
21.The purpose of the experiments performed in week 6 was to understand variousseparation methods used in various analytes. In specific, chromatographic methods, more so theHPLC and HILIC. These two separation methods were compared to find the differences anddetermine which method is better for certain materials and not the other. In this case, it wasnoted that the HILIC makes use of hydrophilic stationary phases and a reverse phase eluent. Themost common mobile phase used in HILIC is acetonitrile alongside some small amounts of water1. The principles of reverse phase HPLC is that it uses a gradient elution instead of the isocraticelution. The biomolecules adsorb to the surface of a reverse phase matrix strongly under theaqueous environment. Additionally, the biomolecules desorb from the matrix at a narrowwindow. The beta lactam antibiotics can be separated using HPLC because they are made up ofvarious components of varied ionic strengths. These can easily be carried by the mobile phaseand eluted accordingly.2.Tetracycline is an antibiotic which can be easily separated using the RPHPLC and notHILIC. This molecule is a broad spectrum antibiotic which acts against a number of pathogenicorganisms. Tetracycline is an adaptive compound which can modify itself with relative ease viatautomerism in response to various environmental conditions2. These environments could haveeffects to the coordination behaviors of tetracycline for instance with various metal ions. This isbecause tetracycline has several potential metal binding sites and hence the ease of separation.Thus, the separation methods with tetracycline shows high limits of detection, high linear rangesand low time of analysis since it does not require an extraction phase.Fluoroquinolones:amixture of fluoroquinolones such as ciprofloxacin and sparfloxacin can be separated using theHPLC. In this case, the mobile phase is composed of acetonitrile and trifluoroacetic acid3.
33.The HILIC needs a hydrophilic type of a stationary phase in order to adsorb waterlayers and allow the analytes process of partitioning. The hydrophilic nature of the material usedto pack the column determines the thickness of the water layer and hence the rate of retention theanalyte. Additionally, the interactions between the stationary phase and the analyte includehydrogen bond formation, and electrostatic forces4. This therefore highlights the need forconsidering the chemistry of the stationary phase selected for the process. the current HILICstationary phases used are bare silica and other polar chemically bonded phases that haveionizable groups linked to the surface of silica. The ligands used in this separation process canundergo electrostatic interactions and also add extra dimensions to the separations during theseparation of ionizable materials. The HILIC stationary phases are: neutral- there have a polarsurface which does not contain any electrostatic forces, the second is the charged phase-hasstrong electrostatic forces since it has either anionic or cationic groups. Third, is the zwitterionstationary phase which has weak electrostatic interactions due to the presence of both anionic aswell as cationic groups.4. Theglycosaminoglycan need to have their components analyzed in order to determinetheir structures. Enoxaparin is an example or type of glycosaminoglycan which is used clinicallyas lovenox as an anticoagulant medication. Thus enoxaparin primarily treats deep veinthrombosis as well as pulmonary embolism. Therefore, HILIC has been widely used in theseparation of carbohydrates including glycosaminoglycan. In this method, the polar materials areseparated on the basis of the stationary phases by use of water miscible organic solvents, whilewater itself is an elutropic solvent. The enoxaparin is a low molecular weight carbohydrate inwhich the original reducing end and non-reducing end is identified by reducing the reducingportion followed by its hydrolysis by use of hydrogen peroxide. This compound is neither a