Retention time and resolution

FIGURE 7.14 A Fractogel EMD BioSEC Superformance column (600-16) was loaded with 500 /il of BSA, ovalbumin, and cytochrome c (5/5/3 mg/ml) at I ml/min. The test covered 100 individual runs with the standard proteins as samples. The buffer system used was 20 m/VI sodium dihydrogen phosphate, 300 m/VI NaCI, pH 7.2. After each individual run the column was rinsed with I /VI NaOH (60 min with I /VI NaOH at 2 ml/min). No significant change in retention times and resolution was observed after 100 cycles. 

In robustness tests, peak measurementfanalysis parameters can also be considered. Such parameters are related to the measurement of the detector signal and they affect responses, such as peak areas, peak heights, retention time, and resolution. They allow improving the quality of these responses. These factors can be found in the data treatment software of an instrument, where often only the default settings are used by the analyst. 

Flow Rate Accuracy. One of the key performance requirements for the pump module is the ability to maintain accurate and consistent flow of the mobile phase. This is necessary to provide stable and repeatable interactions between the analytes and the stationary phase [8,9]. Poor flow rate accuracy will affect the retention time and resolution of the separation. The flow-rate accuracy of the pump can be evaluated simply by calculating the time required to collect a predetermined volume of mobile phase at different flow rate settings. For example, the flow-rate accuracy at 2 mL/min can be verified by using a calibrated stopwatch to measure the time it takes to collect 25 mL of effluent from the pump into a 25-mL volumetric flask. A calibrated flow meter can be used to determine the flow rate as well. The typical acceptance of the flow rate accuracy is listed in Table 11.1. A steady backpressure may be required, depending on the requirement of the system. 

The efficiency of a HPLC column varies with column temperature. In general, capacity factor k decreases with temperature, and hence the retention of the analysis decreases with temperature [16]. Retention drops by 1 to 3% for each increase of 1°C [17]. The ability to maintain a steady and accurate column temperature is critical to achieving the desired retention time and resolution... 

Chromatographic properties such as retention time and resolution can be improved. 

Gradient methods in HPLC depend on the dwell volume, Vd, i.e., the volume between the point of mixing eluents A and B and the column inlet, including the loop of the injector. This volume differs from instmment to instmment, so that a method can be transferred only if this volume is well defined. When there is a significant difference in dwell volume between the two systems, retention times and resolution are dramatically different. So one must be state in which range of the method is still valid. 

Addition of organic modifier strike a balance of cost, retention time, and resolution (see Figure 6.25). 

In addition to the usual advantages for chromatography, including reduction in polarity, protection of labile compounds, alteration of retention times and resolution and enhancement of sensitivity, there are further advantages to be gained from the use of derivatives in GC-MS, and these are covered in the next section. 

Retention time is also strongly influenced by the choice of stationary phase. Several factors must be taken into account when selecting a column for separating androgens. Retention time and resolution will be affected by the choice of column length, stationary film thickness and polarity, and the temperature program rate. The programmed temperature optimization of a mixture of anabolic... 

Solvents of equal strength will yield similar k values of the analytes, but in practice the observed retention times and resolutions can differ due to their selectivity properties. As an example, tert-butyl methyl ether and acetonitrile have similar strengths, but the former is basic whereas the latter is not. The same can be true for solvent mixtures. The separation of the two diastereomers shown in Fig. 1 was tried with several mixtures (on LiChrosorb SI 60) and the following results were obtained ... 

To optimize such separation systems, on the one hand the nature and concentration of the chiral selector can be varied, while on the other hand the pH and organic additives (e.g., lower alcohols) make it possible to modify retention times and resolution. For electrophoretic applications, the electric field strength and the composition of the buffer ( background electrolyte , BGE) can also support enantiomer separations. 

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