HPLC and Gas Chromatography Practical Considerations - Science Assignment Help

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Practical Considerations in Paper and Thin Layer Chromatography (TLC) - These are both forms of PLANAR chromatography (see lecture 3), usually involving a PARTITION mechanism in which the stationary phase is polar (e.g. water) and the mobile phase is non-polar (e.g. butanol). This type of retention is termed a conventional or FORWARD PHASE system (i.e. a polar stationary phase with a non-polar mobile phase). During chromatography it is most important that the atmosphere around the paper or plate be saturated with solvent vapour. This is achieved by sealing the chromatography chamber to prevent any evaporation of the mobile phase solvents. Evaporation of the more volatile mobile phase will lead to distortion of the chromatogram. Advantages of TLC over paper chromatography

• TLC is QUICKER (the solvent equilibrates and moves faster).

• TLC IS MORE SENSITIVE - strongly acidic or oxidative reagents can be used in the detection procedures (these reagents cannot be used with paper).

• TLC IS MORE ADAPTABLE to a wide variety of samples through the availability and use of various support matrices on the plates (e.g. silica gel, alumina, calcium sulphate, etc.). 2-Dimensional Chromatography This is a variant, or extension, of TLC used to resolve complex mixtures of constituents. Mixtures can be resolved very well by 2-stage (2 dimensional) chromatography in which 2 solvent systems are used with the plate being turned through 90° in between each stage. Such separation will produce a "map", or a pattern of spots that is very distinctive of a particular applied mixture and can be used for identification (e.g. for a certain blend of drugs). 91326 Analytical Biochemistry Supplementary Notes to Lecture 4 Spring 2021 Page 2 N. Nassif

Practical Considerations in Liquid Chromatography (LC) Liquid chromatography (LC) refers to the use of a column containing a stationary phase through which a liquid mobile phase is passed. In liquid chromatography, it is most important to minimise peak broadening to obtain better resolution (i.e. narrow sharp peaks in the chromatogram). During LC, peaks broaden due to DIFFUSION (increases with time), HYDRODYNAMIC EFFECTS OR EDDYING (increases with faster flow), SLOW EQUILIBRATION OR MASS TRANSFER between phases (increases with faster flow). In PARTITION and (especially) SIZE-EXCLUSION systems, peak broadening is minimised by applying the sample to the column in the smallest possible volume. In ADSORPTION, ION-EXCHANGE and AFFINITY systems (and sometimes partition) the sample may be "concentrated" at the top of the column by applying it in a strong retention solvent. Then the eluting power of the mobile phase may be increased gradually throughout the run. Altering the eluting power of a mobile phase in this way is referred to as GRADIENT elution.

High Performance Liquid Chromatography (HPLC) HPLC overcomes most peak-broadening effects in LC associated with fast flow by using finely divided stationary phase particles (produces less eddying, faster mass transfer), and relatively pulse-free pumping systems. It transforms LC into a much FASTER technique with very wide applications for almost all types of biochemical and environmental analytes. The pump might have to deliver high pressure with some column types (hence the original abbreviation HPLC). The ability to use a wide variety of stationary phases allows separations based on any of the retention mechanisms (least suitable for size-exclusion using conventional gels, but still applicable using materials that resist compression and clogging). HPLC is a separation technique that involves the injection of a small volume of liquid sample into a tube packed with tiny particles (3 to 5 micron (μm) in diameter, called the stationary phase), where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. In principle, LC and HPLC work the same way except that the speed, efficiency, sensitivity and ease of operation of HPLC is vastly superior.

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