How to simplify HPLC using low-pressure valves. Rheodyne's Technical Note 3 shows eight ways of using lowpressure switching valves to simplify method development and routine analysis in high-pressure liquid chromatography. These economical 2-position and 6-position Teflon valves can be inserted before the pump or after the column. Among uses described in detail are switching between reservoirs to select the correct mobile phase for a particular routine analysis. Switching between several different solvents during method development when seeking maximum selectivity. Switching to a flushing solvent. Switching effluent back to the reservoir to conserve solvent And switching effluent to a fraction collector.
Send for Tech Note #3 All techniques are fully described in this well-illustrated 6-page technical note. Contact Rheodyne, Inc., RO. Box 996, Cotati. Calif. 94928. U.S.A. Phone (707) 664-9050.
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Figure 3. Comparison of absorbance (a) and fluorescence (b) for the detection of aflatoxins B,, B2, G-, and G2 fui LC analysis. How this is approached depends primarily upon the nature of the compound of interest. For example, if the compound of interest were strongly fluorescent, one would try to employ a fluorescence detector for the analysis. The cleanup would be directed toward reducing the fluorescent background. It is possible to have many coextractives present in the final sample solution but if they do not fluoresce, they will not interfere in the analysis. However, it must also be kept in mind that when too much coextracted material is present it can interfere in the chromatography by distorting peaks or altering retention values. Even though chromatograms may appear relatively clean because of selective detection, the repeated injection of "dirty" samples can lead to contamination and to decreased column life. To keep the chromatographic system functioning well as long as possible, it is good practice to inject as little sample as necessary to obtain a reliable result. This is especially important for trace analysis and of lesser importance for formulations analysis. Pump and Injector Requirements The minimum requirements for pumps are that they deliver essentially pulseless flow over the range 0.5-2.0 mL/min at high detector sensitivity. The noise, of course, depends upon the type of detector but generally a pump that produces less than 1% noise above the detector noise at 0.01 absorbance units full scale (AUFS) on a UV detector is satisfactory. Most popular pumps in use today, including
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the dual and single piston reciprocating pumps, easily meet this requirement. The most useful injection ports are those incorporating a syringe-loop configuration. These permit injection of different volumes up to the volume of the loop. Also, highly reproducible injections can be made when the full loop volume is used. They are easy to use and are preferred to stopped-flow injection or injection via a septum. Although all syringe-loop injectors are rated for operation at 3000 psi or more (which is more than adequate for most LC applications), those capable of operating at 6000 psi permit the use of higher flow-rates for rapid mobile phase changes or column conditioning or cleaning. Detectors The prime requirement for LC detectors that are to be used for trace analysis is high sensitivity. This is very important when one is considering the quantities of sample extract that must be injected to produce a reliable peak for the component of interest. If detector sensitivity is poor, then more sample equivalent must be injected, and if done on a regular basis this will lead to shortened column life. Sensitivity becomes increasingly important if extremely low levels (e.g., parts per billion), are to be determined. For most applications, detectors sensitive to 1-50 ng of substance are required. This of course eliminates the refractive index detector for application to trace analysis. Selectivity, another important factor in detection, can be considered to
