HPLC methods efficient transfer

As shown in Figure 3.8, an increased injection volume can have a negative effect on the column efficiency. Therefore, the injection volume should be appropriately scaled down when an HPLC method is transferred to a UHPLC method or vice versa to achieve the same sensitivity and avoid overloading or detector saturation and extracolumn band-broadening. The scaling factor is mainly based on column dimension, as shown in Eq. (3.26). However, lower injection volumes than calculated can often be used on UHPLC to achieve the same sensitivity due to enhanced peak heights from use of the high-resolution columns and low carryover from the injector ... 

The major advance in the way in which column eluate is deposited on the belt was the introduction of spray deposition devices to replace the original method which was simply to drop liquid onto the belt via a capillary tube connected directly to the outlet of the HPLC column. These devices, based on the gas-assisted nebulizer [5], have high deposition efficiencies, transfer of sample can approach 100% with mobile phases containing up to 90% water, and give constant sample deposition with little band broadening. 

Flow Rate Efficiency Flow rates may be slightly lower than in other HPLC methods to maximize resolution and improve mass transfer kinetics. 

Reproducibility during column preparation is a significant problem in CEC. Preparation methods involving pumped slurries were all found to produce generally highly efficient (> 200,000 plates/m for 3-pm ODS particles), but within a batch of columns packed by the same method, the relative standard deviation (RSD) of EOF, and migration time and retention factor of a standard were [23], respectively, 7-14%, 5-22%, and 9-30%. These values are particularly relevant to considerations of the transfer of HPLC methods to CEC. 

Short column with STM particles (< 400 bar) Potential use with conventional HPLC Significant reduction in analysis time Easy method transfer Higher efficiency Relatively good variety of column chemistry Two-fold increase in speed for SIM... 

Method transfer issues aside, HPLC offers less separation efficiency than observed in other separation techniques such as capillary electrophoresis (CE), GC, and SFC. In fact, it is typically difficult to separate more than 15-20 compounds in a single HPLC run, necessitating the use of two or more runs for complex samples. As a result, other techniques continue to receive considerable interest. It is, however, safe to say that there are no emerging techniques that will significantly reduce the utilization of HPLC in the short term. 

Please measure the dead volume of your isocratic equipment. Before suspecting the column as the cause of a bad resolution, you should rule out the dead volume. The effect of a dead volume that is too large is an unnecessarily low efficiency and broad and/or tailing peaks. My advice on the subject is to use a generally accepted maximum value for the dead volume for all isocratic HPLC equipment in the laboratory. Such a general rule will help in obtaining comparable results from the different equipment and you will obtain similar chromatograms in method transfers. 

Table 2.2 Example for the attempt to improve resolution through efficiency increase when transferring from HPLC to UHPLC. All numbers are theoretically modeled on the basis of the artificial method 1 ( method 2 is not feasible in practice).

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