Created with MindGenius Business 2005® Instrumentation (1) Instrumentation (1) Origins Preparative column chromatography (Glass columns) Internal diameter: cm Length: cm Particle size: mm (large) Flow rate: tenths of ml/min, under gravity N<100/m Developments Automation Many different types (see later) Engineered columns – can takes extreme pressure and temperature Reduction in column size (Typical Liquid Chromatography: Internal diameter: 4 -10mm, Length: cm, Particle size: 5 -10µm) N>10,000/m

Created with MindGenius Business 2005® Instrumentation (2) Instrumentation (2)

Created with MindGenius Business 2005® Instrumentation (3) Instrumentation (3) 1) Gas and Solvent Reservoirs: Contain mobile phase There may be more than one reservoir Isocratic elution uses a constant mobile phase composition (only one reservoir necessary) Gradient elution uses more than one, two pumps and a mixing system deliver a mobile phase that varies with time Mobile phase may need pretreatment: e.g. Filtering: Prior to placing in the solvent reservoirs, or using an in-line filter Sparging (degassing): by sonication or by bubbling an inert gas (e.g. Helium) through the solvent Guard Column (positioned after injection port): Provides filtration and preconditioning to protect the column 2) Pumping systems (not Gas chromatography) Generate high pressures (up to 6000psi) Generate variable flow rates ( ml/min) Accurate and reproducible rates, independent of column back-pressure Be “pulse-free” Corrosion resistant

Created with MindGenius Business 2005® Instrumentation (4) Instrumentation (4) 3) Sample addition facility (injection port) Sampling Loop (see Skoog for diagram) Syringe injection, through a septum (<1500psi) Stop-flow, direct injection onto top of column packing Pump (larger volumes) 4) Column (& oven)- See later for stationary phases by type Usually stainless steel (may be glass) Length: cm, up to several m for GC i.d (internal diameter) mm-cm Particle size of packing 5 or 10μm >10,000 plates/m Most common packing is silica based, may also have alumina, zirconium, polymeric, ion-exchange resin Short columns are quicker to use but have lower N Small particle size gives higher N but needs higher pressure to maintain flow A column oven or a water jacket may be used to provide a stable temperature or to allow chromatography at different temperatures

Created with MindGenius Business 2005® Instrumentation (5) Instrumentation (5) 5) Detectors (More detail later) Generally the most expensive part of the instrument Choice depends on analyte properties, and required sensitivity (and money available) Performance is measured in terms of Mass Limit of Detection (LOD) which is the mass that gives a signal 5 times the standard deviation of the noise, using 10ml of a sample of Mr =200 6) Output Chart recorder: contains a drive-mounted pen that moves according to the current supplied by the detector output. May include a second pen, driven by an integrator which “draws” the cumulative signal over time, giving the area under a peak Integral processor: uses instrument hardware and drop-down menus to allow modifications of conditions, and or inspection/reprocessing of chromatograms PC - based: output and controls are driven by specialist software written for use on a PC Can allow the spectrum to be reprocessed and reprinted

Created with MindGenius Business 2005® Instrumentation (6) Instrumentation (6) Automatic Integration and Reprocessing – treat with Caution Automatic processing parameters must be set so that the instrument can “recognise” peaks correctly This includes setting how the instrument recognises: Baseline Peak Start and Finish (time) Non retained peak (to ignore) Detectivity (size of baseline fluctuation to ignore) This can be altered after a run to improve peak recognition – but will NOT improve poor chromatographic performance