Student Number: xxx
The aim of this experiment was to determine the concentration of aspartame in a sample of diet Coke® using HPLC as investigation tool.
High Performance Liquid Chromatography (HPLC) is one of the most widely used analytical techniques of nonvolatile organic compounds. Chromatographic processes can be described generally as mass-transfer between stationary and mobile phase. In HPLC, a liquid mobile phase is used to separate individual components of complex mixtures. Stationary phase can be either liquid or solid (like that in experiment).
The standard design of HPLC involves a core of four to five functional elements. These elements are: a filtered solvent reservoir of mobile phase and degassing system, the pump, an injector, a column (optionally housed in an oven) and detector/data system. Test mixtures are dissolved in solvent and micro injections are forced to flow under a high pressure through the column. In the column, the mixture separates into its components and these are detected and recorded on chromatogram. The resolution of various components is determined by extent of interaction between the solute components and the stationary phase. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.1
Diagram 1: Typical HPLC Design
Individual Modules of HPLC Instrument
The modular HPLC unit in the experiment was essentially composed of an isocratic pump, an auto sample injector, a thermostatically controlled column, a diode array or variable wavelength detector and a computer with control and data analysis.
There are 4 choices of pumps available: Isocratic, Gradient, Binary and Quaternary. The isocratic pump delivers constant mobile phase compositions, while the gradient delivers variable mobile phase compositions. A binary gradient delivers two solvents whereas a quaternary; four. For the more simple separations, as in the case of the experiment, the isocratic pump is opted for. Typically mobile phases are delivered in 10ml volumes per minute at immense pressures ca. 50Kpa.
The column is central to the success of HPLC and the correct choice must be made to fit purposes. Columns come in 4 specifications: analytical (internal diameter (ID) 1.0-4.6mm; 15-250mm), preparative (ID>4.6mm; 50-250mm), capillary (ID 0.1-1.0mm; various lengths) and nano (ID < 1.0mm). The column tubing is constructed from stainless steel (the most popular), glass or an inert PEEK polymer. The packing materials in the columns are composed of porous particles in dimensions of 5μm, 3.5μm and 1.8μm. Owing to the minute sizes of the particles a high pressure is needed to force the analyte- mobile phase through the stationary phase. These particles usually have a chemically bonded phase (such as C18) on their surface that interacts with the analyte components. Retention times are driven by the choice of column packing and mobile phase.
Diagram 2: Examples of Columns
The auto sample portion of the module unit can sample up to 120 plus vials. Operator just loads the carousel with samples and sampler will automatically measure appropriate μml volume, inject it, flush the injector and resume with the next sample.
At the detection end there is again a choice of systems to use. These range from the spectroscopic, to refractive index, to fluoresce detection. The type used in experiment was spectroscopic.
This uses a beam of...