tailieunhanh - HPLC A Praactical User'S Guide Part 4

Tham khảo tài liệu 'hplc a praactical user's guide part 4', kỹ thuật - công nghệ, cơ khí - chế tạo máy phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | PARTITION 51 Table Relationship of efficiency to flow rate Efficiency Changes with Particle Size Packing diameter mm Plates meter Flow rate mL min 10 30 000 5 50 000 3 100 000 times 2-fold while increasing efficiency only by due to increased diffusion. Finally we have variables affecting efficiency that can be controlled at the time of the run. These are pump flow rate extracolumn volumes in the instrument used and the method of calculation. Flow rate is the major efficiency variable that I use during methods development. Generally halving the flow rate will increase separation around 40 . I do much of my scouting at min knowing that I can improve separation by dropping to min. Plotting of efficiency versus flow rate shows that each diameter of packing has its own optimum flow rate. Efficiency decreases at higher flow rates. In the microparticulate packings large packing diameters show a more rapid loss of efficiency with increasing flow rate than do smaller packings. Decreasing extracolumn volumes is critical to HPLC success. The most important volumes are those immediately adjacent to the column zero-deadvolume end-fittings inlet and outlet tubing diameters and detector cell volumes. From the time the sample enters the injector until it exits the detector nothing must add increased mixing space. Tubing from injector to column must be in for 5-mm and 10-mm packings with tubing lengths no more than 4-6 in for the 5-mm. Use tubing about 3 in long or less for 3-mm packing. Zero-dead-volume endcaps and connectors must be prepared correctly so that tubing ends butt firmly against the fitting. We covered the preparation of compression fittings in Chapter 3 but if you find efficiency drops after you change a fitting check the dead-volume fit. For detector cells the rule of thumb is 8-12 mL anything larger acts increasingly as a mixer for your already separated bands. Tubing volumes outside the critical injector-detector