Introduction to Modern Liquid Chromatography, Third Edition part 9. High-performance liquid chromatography (HPLC) is today the leading technique for chemical analysis and related applications, with an ability to separate, analyze, and/or purify virtually any sample. Snyder and Kirkland's Introduction to Modern Liquid Chromatography has long represented the premier reference to HPLC. This Third Edition, with John Dolan as added coauthor, addresses important improvements in columns and equipment, as well as major advances in our understanding of HPLC separation, our ability to solve problems that were troublesome in the past, and the application of HPLC for new kinds of samples. . | 36 BASIC CONCEPTS AND THE CONTROL OF SEPARATION ti 80 B 70 B min 50 B min min Figure Origin and measurement of peak width a Measurement of peak width b peak 3 of Figure as a function of B shown with the same time scale for each peak. Because an ideal chromatographic peak has the shape of a Gaussian curve Section peak widthis sometimes slcscnbcd in teams of the standard deviation a of the Gaussiancurve where w 4a and therefore Equation can be expressed in other forms for example N 25 tR W5a 2 where W5CT is the so-called 5 a peak width. Equation can be rearranged to give w 4 - G . or replacing by Eq. W 4N- 5io l k PeakWidthand the Column Plate NumberN 37 Because values of N are approximately constant for the different peaks in a chromatogram Equation tells us that peak width W will increase in proportion to retention time. A continual increase in peak width from the beginning to the end of the chromatogram is therefore observed for example see the chromatogram of Figure . The area for a given solute peak normally remains approximately constant when retention time is varied by a change in B temperature or the column so peak height hp times peak width W will also be constant. For this situation h constant constant P Tr W That is as tR increases peak height decreases. An example is shown in Figure for peak 3 of Figure as a function of B. A reciprocal change is seen in peak height and width as B is varied as predicted by Equation . Dependence of N on Separation Conditions We will begin by summarizing some practical conclusions about how the column plate number N varies with the column the sample and other separation conditions. In following Section we will examine the theory on which these conclusions are based. N can also be described by N L H where H L N is the column plate height. H is a measure of column efficiency per unit .
