Columns length change

In this case, the gradient volume is halved. The second way is to keep the gradient volume constant time is adapted at the same rate as the column length changes and, at the same time, the flow rate is changed such that the same quantity of eluent is always flowing through the column ... 

Often, the retention time is used but, as discussed above in Section 2.3, this absolute parameter changes with column length and flow rate and this precludes the use of reference data obtained in other laboratories. To make use of these reference data, the capacity factor (k ), which removes such variability, must be employed. 

We can reduce column length to maintain constant efficiency and save analysis time because compounds will elute earlier. For example, if we consider a 4.6 x 50 mm column with 5 fim particles and change to modem sub-2-micron (e.g., 1.8 fim) particles, we should achieve the same efficiency with a column only 18 mm long because N - L/dp. With the same flow rate, the compounds should elute 2.8 times earlier (5 /.un/1.8 /an). 

As mentioned in Section 11.8.4, the parameters that are most important for a qualitative analysis using most GC detectors are retention time, tR adjusted retention time, t R and selectivity, a. Their definitions were graphically presented in Figures 11.16 and 11.17. Under a given set of conditions (the nature of the stationary phase, the column temperature, the carrier flow rate, the column length and diameter, and the instrument dead volume), the retention time is a particular value for each component. It changes... 

Qualitative and quantitative analyses with HPLC are very similar to those with GC (Sections 12.7 and 12.8). In the absence of diode array, mass spectrometric, and FTIR detectors that give additional identification information, qualitative analysis depends solely on retention time data, tR and C (Remember that tR is the time from when the solvent front is evident to the peak) Under a given set of HPLC conditions, namely, the mobile and stationary phase compositions, mobile phase flow rate, column length, temperature (when the optional column oven is used), and instrument dead volume, the retention time is a particular value for each component. It changes only when one of the above parameters changes. Refer to Section 12.7 for further discussion of qualitative analysis. 

Furthermore, it is possible to increase the efficiency by a factor 3 when changing particle size from 5 to 1.7 pm while maintaining an equivalent column length, and decrease analysis time by a factor 9 while maintaining equivalent efficiencies [21],... 

The band profiles are obtained as the change of concentration against time at the column outlet. To better understand the effect of mobile phase dispersion, column length, mobile phase velocity, and other parameters, we introduce some normalization. We can rewrite Equation 10.8, using... 

It is seen from figure 2 that changing the particle diameter from I to 20 micron results in an efficiency change from about 3500 theoretical plates to nearly 1.5 million theoretical plates and furthermore, this very high efficiency is achieved at an inlet pressure of only 3000 p.s.i.. It is also seen that the maximum available efficiency increases as the particle diameter increases. This is because, as already discussed, if the pressure is limited, in order to increase the column length to accommodate more theoretical plates the permeability of the column must be increased to allow the optimum mobile phase velocity to be realized. It is possible to increase the inlet pressure to some extent, but ultimately the pressure will be limited and the effect of particle diameter will be the same but at higher efficiency levels. 

In a similar way to particle diameter and column length, there are also some practical limits that must be Imposed on the analysis time. It must be again emphasized that these limits are arbitrary in nature, and may be changed by the user if so desired. A minimum analysis time of one minute Is recommended to allow time for sample manipulation and fraction collection and at the other extreme a maximum analysis time of one hour is considered acceptable. It is seen that a practical window exists between a separation ratio of 1.01 and 1.3. Furthermore, at a separation ratio of 1.3, extremely low inlet pressures must be employed to ensure the analysis time is not less than 60 seconds. Thus, the limitation of analysis time to a minimum of 60 seconds, means that for very simple mixtures, columns with excess efficiencies must be used. As has already been suggested, such columns can be overloaded and the excess analysis time also traded in for increased sample load. 

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