Modifying conventional high-performance liquid chromatography systems to achieve fast separations with Fused-Core columns: a case study.

The theoretical increase in performance from the use of high efficiency columns with conventional HPLC equipment is generally not observed due to the design limitations of such equipment, particularly with respect to extra-column dispersion (ECD). This study examines the impact of ECD from a Waters Alliance 2695 system on the performance of 2.7 µm HALO C(18) Fused-Core superficially porous particle columns of various dimensions. The Alliance system was re-configured in different ways to reduce extra-column volume (ECV) and the ECD determined in each case as a function of flow rate up to a maximum of 2 mL/min. The results obtained showed a progressive decrease in ECD as the ECV was reduced, irrespective of the flow rate employed. However, this decrease in ECD was less than theoretically expected for the lower ECV configurations. The inability to reduce the actual extra-column dispersion further was attributed to additional dispersion associated with the design/volume of the auto-injector. This was confirmed by making sample injections with a low dispersion manual injection valve, instead of auto-injection, for the two lowest ECV configurations studied. In each case, the measured and predicted ECD values were in good agreement. The auto-injector module is an integral part of the Alliance 2695 instrument and cannot be easily modified. However, even with autosampler injection, for a 3mm ID × 100 mm Fused-Core column approximately 70% of the maximum plate count (∼84% of the resolution or more) could still be obtained in isocratic separations for solutes with k ≥ ∼4.5 when using the lowest ECV configuration. This study also highlights some of the problems inherent in trying to measure accurately the true extra-column dispersion of a chromatographic system and compares the results obtained to those theoretically predicted. Using this same lowest volume instrument configuration, two real-world pharmaceutical methods were scaled to separations that are ∼3-3.5-fold faster, while still maintaining comparable data quality (resolution and signal-to-noise ratios).

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time I. Peak capacity limitations.

The separation of samples that contain more than 15 to 20 analytes (n > 15-20) is typically difficult and usually requires gradient elution. We have examined the reversed-phase liquid chromatographic separation of 24 samples with 8 < or = n < or = 48 as a function of temperature T and gradient time tG. The required peak capacity was determined for each sample, after selecting T and tG for optimum selectivity and maximum sample resolution. Comparison of these results with estimates of the maximum possible peak capacity in reversed-phase gradient elution was used to quantify the maximum value of n for some required sample resolution (when T and tG have been optimized). These results were also compared with literature studies of similar isocratic separations as a function of ternary-solvent mobile phase composition, where the proportions of methanol (MeOH), tetrahydrofuran (THF) and water were varied simultaneously. This in turn provides information on the relative effectiveness of these two different method development procedures (optimization of T and tG vs. % MeOH and % THF) for changing selectivity and achieving maximum resolution.

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time II. Two-run assay procedures.

By optimizing column temperature T and gradient time tG, complex samples can often be separated by means of reversed-phase high-performance liquid chromatography (RP-LC). Conclusions reached in Part I suggest that the complete separation of such samples will be difficult, however, when more than 15-20 components are present in the sample. An alternative approach is to carry out two separations with different conditions (T, tG) in each run. The combination of results from these two runs then allows the total analysis of the sample, providing that every sample component is adequately resolved in one run or the other. Examples of this approach, carried out by means of computer simulation, are shown here for several samples of varying complexity. Also considered is the ability of a single separation where T and tG are optimized to enable the separation and analysis of one or more individual sample components from complex mixtures (e.g., drugs in animal plasma), including the resolution of isomeric compounds from each other.

Simultaneous variation of temperature and gradient steepness for reversed-phase high-performance liquid chromatography method development. I. Application to 14 different samples using computer simulation.

The optimized reversed-phase HPLC separation of 14 different samples is reported, based on simultaneous changes in temperature and gradient steepness. Four experimental runs are required for each sample, following which preferred conditions can be predicted using computer simulation software (DryLab). The overall accuracy and effectiveness of this method development approach is discussed, with particular attention to the use of resolution maps provided by the software. These maps are useful for maximizing resolution for the total sample, for optimizing the separation of a smaller number of selected sample compounds, and as an initial step in the separation of more demanding samples.

Combined use of temperature and solvent strength in reversed-phase gradient elution. I. Predicting separation as a function of temperature and gradient conditions.

It has been shown previously that computer simulation based on two initial experiments can predict separation in reversed-phase gradient elution as a function of gradient conditions (gradient steepness, gradient range and gradient shape) and column conditions (column length, flow-rate and particle size). The present study extends this capability for changes in temperature. Four initial experiments (two different gradient times, two different temperatures) provide input data that allow predictions of separation as a function of temperature as well as gradient and column conditions. A semi-empirical relationship, tR = a + bT, is able to relate gradient retention time tR to column temperature T (other conditions constant). The accuracy of this approach has been evaluated for 102 solutes and a variety of experimental conditions, including the use of five different HPLC instruments (four different models).