The most precise measurements must be made on the peak width and so a comparator with a graticule that is calibrated in units of at least 0.1 mm should be used. Comparators give accurate results, but care must be taken over the measurement procedure. A diagram depicting the measurement of peak width is shown in figure 33.
Figure 33. Measurement of Peak Width
The measurements should be taken from the inside of one line to the outside of the adjacent line, to reduce errors resulting from the finite width of the ink line drawn of the chart. The measurement is repeated using the alternate edges of the line and an average taken of the two readings. Three replicate runs should be made and values obtained should not differ by more than 3%.
Peak Area Measurements
The peak area is the integration of the mass per unit volume (concentration) of eluted solute eluted with respect to time. At constant flow rate and concentration sensitive detector, the integration will also be with respect to volume flow of mobile phase through the column. Thus, peak area is proportional to the mass of solute. Measurement of peak area accommodates peak asymmetry and even peak tailing without compromising the simple relationship between peak area and mass. Thus, peak area measurements give more accurate results under non-ideal chromatographic conditions. Unfortunately, whether a chart recorder is used or computer data acquisition, both systems are based on time as the variable and not volume of mobile phase. Thus, peak area measurements demand good flow control so that time measurements are linearly related to volume flow of mobile phase. The flame ionization detector responds to mass of solute entering it per unit time and, thus, peak area with respect to time is truly related to solute mass and is independent of column flow rate. Unfortunately, there is no equivalent detector in LC or, at least, not one that is commercially available.
Peak areas can be measured manually. The peak area is obtained as the product of the peak height and the peak width at 0.6065 of the peak height (2s). However, the peak shape should be Poisson, Gaussian or close to Gaussian. Another method involves the use of a planimeter but this is very tedious and not very accurate. The most accurate manual method of measuring peak area is to cut the peak out and weigh it. This last procedure can accommodate any type of peak malformation and still provide an accurate value for skewed or malformed peaks. Cutting the peaks out and weighing them is the most accurate manual method for measuring peak areas.
