Focus time

Similarly, the focusing capability of an array is the strongest focused beam which can be steered. The simplest way to evaluate it is to test a theoretical focusing time delay law, in the near-field and in the natural direction of propagation of the array. The beam pattern characteristics depth, lateral size and length of the focal spot must be found consistent with modelling and no lobe must appear above a predetermined level. 

FIGURE 19 Effect of focusing time on clEF separation. 

The separation conditions were subsequently optimized using the same antibody molecule. Focusing times of 1, 5, 10, 15, and 20 min were studied using a separation voltage of 25 kV. The optimized focusing time for sufficient separation was determined to be 10 min (Figure 19). 

Separation voltages of 5, 10, 15, 20, 25, and 30 kV were evaluated for the same molecule using a 10 min focusing time. The results are shown in Figure 20. The data demonstrate that 25 kV is an appropriate separation voltage for this molecule. 

Capillary length Capillary inner diameter (ID) Separation voltage Separation temperature Focusing time... 

Flight-time-focused time-of-flight atom-probe... 

For high-performance analysis, it is important that the sample applied has a low salt content [1]. Salt concentrations below 10 mM is preferable concentrations above 40-50 mM should be desalted. The salt will compress the pH gradient so that it will occupy only some part of the capillary. Hereby, the resolution will decrease and the risk for protein precipitation will increase (see the section on Focusing, below). Also, focusing time will have to be increased as well as focusing/mobilization time being less reproducible. 

Too many experts and executives focusing time and effort on very narrow areas that frequently have nothing to do with the core competency of the business, essentially wasting time that could be otherwise be devoted to improving the business s competitive advantage... 

First, following the initial conditions, 4% Pharmalyte (pH 3-10), and 0.2 mg/mL sample concentration were tested for this sample. Under the conditions, as shown in the trace 1 of Figure 19.3, the sample peak height is detected at 0.1 Abs. In the next run, the sample concentration was increased to 0.6 mg/mL. The focusing time was 6 min at 600 V/cm with a 1 min prefocusing at 300 V/cm. Two pi markers were spiked into the sample for pi calibration. The results show good reproducibility in peak pattern (traces 2 and 3 in Figure 19.3). Since the separation resolution under these conditions was satisfactory, no further method development was pursued to enhance the resolution. 

A second sample was also analyzed to determine pi values of different lots in production. As shown in Figure 19.7, when focusing time was limited to 6 min, the peak pattern reproducibility was satisfactory for the application. From our experiences with cIEF method development for virus samples, cIEF may not achieve the peak pattern reproducibility that can match that of monoclonal antibodies or other protein samples. However, in most cases, the reproducibility is satisfactory for the desired applications [39]. 

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