On-line near infrared spectroscopy as a Process Analytical Technology (PAT) tool to control an industrial seeded API crystallization.

The final step of an active pharmaceutical ingredient (API) manufacturing synthesis process consists of a crystallization during which the API and residual solvent contents have to be quantified precisely in order to reach a predefined seeding point. A feasibility study was conducted to demonstrate the suitability of on-line NIR spectroscopy to control this step in line with new version of the European Medicines Agency (EMA) guideline [1]. A quantitative method was developed at laboratory scale using statistical design of experiments (DOE) and multivariate data analysis such as principal component analysis (PCA) and partial least squares (PLS) regression. NIR models were built to quantify the API in the range of 9-12% (w/w) and to quantify the residual methanol in the range of 0-3% (w/w). To improve the predictive ability of the models, the development procedure encompassed: outliers elimination, optimum model rank definition, spectral range and spectral pre-treatment selection. Conventional criteria such as, number of PLS factors, R(2), root mean square errors of calibration, cross-validation and prediction (RMSEC, RMSECV, RMSEP) enabled the selection of three model candidates. These models were tested in the industrial pilot plant during three technical campaigns. Results of the most suitable models were evaluated against to the chromatographic reference methods. Maximum relative bias of 2.88% was obtained about API target content. Absolute bias of 0.01 and 0.02% (w/w) respectively were achieved at methanol content levels of 0.10 and 0.13% (w/w). The repeatability was assessed as sufficient for the on-line monitoring of the 2 analytes. The present feasibility study confirmed the possibility to use on-line NIR spectroscopy as a PAT tool to monitor in real-time both the API and the residual methanol contents, in order to control the seeding of an API crystallization at industrial scale. Furthermore, the successful scale-up of the method proved its capability to be implemented in the manufacturing plant with the launch of the new API process.

Use of the kinetic plot method to analyze commercial high-temperature liquid chromatography systems. II. Practically constrained performance comparison.

Using a set of experimentally determined plate height data obtained on three commercial high-temperature HPLC supports, and evaluating their isocratic separation speed potential under the application of a set of instrumental constraints, a qualitative map of the practically achievable critical pair separation speed potential of high-temperature HPLC has been established. The obtained data show that the gain in separation speed is more strongly affected by the instrumental limitations in the high-temperature range than it is for the low temperatures. For the presently considered case of alkylbenzene separations, the potential gain in analysis time that can be obtained by going from T=30 to 120 degrees C in the presence of a typical set of instrumental limitations nevertheless remains of the order of a factor of 2-4. The study also shows that improvements on the instrumentation side (increased detector frequency, pumping flow rate, smaller extra-column volumes, ...) are indispensable to fully benefit from the high temperature advantages for all separations requiring less than 10,000 effective theoretical plates.

Use of the kinetic plot method to analyze commercial high-temperature liquid chromatography systems. I: Intrinsic performance comparison.

The present study aimed at mapping the separation speed potential of a critical pair on commercial high-temperature HPLC (HT-HPLC) supports at elevated temperatures. For this purpose, band broadening and pressure drop measurements were conducted on three different commercial HT-HPLC columns operated at various elevated temperatures but by keeping the same retention factor. The plate height data were subsequently transformed into a plot showing the minimal required analysis time needed to yield a given required effective plate number. For the considered RPLC alkylbenzene separations, it was found that the maximal gain in separation speed of the critical pair that can be obtained by varying the operating temperature from T=30 to 120 degrees C can be expected to be of the order of a factor of 3-4, if using an individually optimized column length for each considered temperature and if no secondary adsorption effects occur at the lower temperature. This gain factor, remaining more or less constant over the most relevant range of plate numbers, largely paralleled the reduction of the mobile phase viscosity accompanying the temperature increase.

Porous silicon as a stationary phase for shear-driven chromatography.

We report on the possibility to strongly increase the mass loadability and retention capacity of shear-driven chromatography (SDC) channels by growing a thin porous silicon layer on the stationary wall part. The thickness of the produced porous silicon layers was found to increase linearly with the anodisation time, and could easily be varied between 50 and 300 nm. Combining these layers with sub-microm thin flow-through channels, we believe it is the first time a sub-microm on-chip LC system with a phase ratio similar to that in packed column HPLC (i.e., Vs/Vm approximately equal to 1.5) is obtained. The chromatographic performance of the produced channels has been tested by separating binary mixtures of coumarin dyes under RP-LC conditions. The plate height measurements, yielding Hmin, approximately equal to 0.5 microm (corresponding to more than 2 x 10(6) plates/m) for a retained component with k" = 3, showed good agreement with the theoretical expectations. Due to the presence of some macroscopic defects in the prepared layers, the quality of the separations could however only be maintained over a few millimeters of the channel length. This length was however more than sufficient to separate the coumarin mixture, given the extremely small plate heights of the system.

Enhancement of DNA micro-array analysis using a shear-driven micro-channel flow system.

A very simple micro-channel flow system is used to investigate the potential gain in hybridization rate stemming from the induction of a convective flow past the surface of a DNA micro-array. Reporting on a series of preliminary experiments wherein a two-dimensional convective flow is created past the surface of a conventional micro-array slide, the analysis time could be brought down from overnight waiting (16 h) to some 10 to 30 min. The experiments open the road towards the development of novel, convection-driven hybridization systems yielding shorter analysis times, and/or lower detection limits.

Shear-flow-based chromatographic separations as an alternative to pressure-driven liquid chromatography.

It is only by developing specially designed injection and detection systems that shear-driven chromatography can become a viable alternative to HPLC. In the present paper, a dedicated zero dead-volume injection procedure is presented with which sample volumes can be injected reproducibly in the required picoliter range. In addition, a transversal detection groove system is designed which should allow to perform on-line UV-VIS absorption measurements with path lengths in the millimeter range, with an acceptable theoretical plate loss (only 20% in a 5 cm long channel) and acting as a nearly perfect wave guide.

Experimental demonstration of the possibility to perform shear-driven chromatographic separations in micro-channels.

The possibility to perform shear-driven chromatographic separations in micro-channels is demonstrated, using a novel laser-jet printed microfluidic channel system. The obtained theoretical plate numbers are in fair agreement with the theoretical calculations. Theoretical extrapolations of the separation speeds and detection limits which can be achieved when further miniaturizing the current system are presented as well.