Separation properties of the MIL-125(Ti) Metal-Organic Framework in high-performance liquid chromatography revealing cis/trans selectivity.

Monodisperse MIL-125(Ti) Metal-Organic Framework crystals were synthesized and studied as stationary phase in high performance liquid chromatography (HPLC). Different pure compounds and model mixtures (including stereoisomer mixtures) were injected from which chromatographic parameters, including selectivities and resolution factors, were determined to evaluate the adsorption properties and separation performance of MIL-125(Ti) in liquid phase. The MIL-125(Ti) framework displayed a trans selectivity for cis/trans difunctionalized cyclohexane molecules with high selectivity and resolution for 1,3-dimethylcyclohexane and 4-ethylcyclohexanol. The slurry-packed column was further characterized via van Deemter analysis. Fitting of the van Deemter equation through the experimental points allowed to define the contributions of the different processes to plate height with a significant proportion of the A-term, reflecting the importance of a good crystal packing. Although high in comparison to commercial HPLC stationary phases, a very good plate height of around 50µm was found.

Predictive elution window stretching and shifting as a generic search strategy for automated method development for liquid chromatography.

We report on the possibilities of a new method development (MD) algorithm that searches the chromatographic parameter space by systematically shifting and stretching the elution window over different parts of the time-axis. In this way, the search automatically focuses on the most promising areas of the solution space. Since only the retention properties of the first and last eluting compounds of the sample need to be (approximately) known, the algorithm can be directly applied to samples with unknown composition, and the proposed solutions are not sensitive to any modeling errors. The search efficiency of the algorithm has been evaluated on an extensive set of random-generated in silico samples covering a broad range of different retention properties. Compared to a pure grid-based search, the algorithm could reduce the number of missed components by 50% and more. The algorithm has also been applied to solve three different real-world separation problems from the pharmaceutical industry. All problems could be successfully solved in a very short time (order of 12 h of instrument time).

Accurate determination of extra-column band broadening using peak summation.

The present contribution addresses the problem of the accurate determination of the extra-column band-broadening dispersion by applying the method of moments (MOM) on pulse response experiments. The MOM provides the only mathematically rigorous way to determine the variance of a pulse response, but suffers from the fact that the obtained variance values usually depend very strongly and unpredictably on the selected integration boundaries. In the present study, it is investigated whether the MOM cannot be made more accurate by repeating the pulse response experiment a number of times, then add all measured peaks (after retention time alignment) and subsequently perform the integration on this summed peak. Testing this approach for a number of different integration boundary detection methods consistently more accurate results are obtained than when the different repeat response pulses are integrated individually and the variances are averaged afterwards. It was also found that adding five repeats already leads to a significant improvement of the variance estimate, whereas the addition of 10-20 repeats is needed before the variance estimate converges to a steady value.

Extending the Total Pore Blocking method to normal phase high performance liquid chromatography.

It has been demonstrated that it is possible to extend the Total Pore Blocking (TBP)-method for the determination of the external porosity (ε(e)) of packed bed columns from reversed-phase (RP) chromatography to normal-phase (NP) chromatography conditions by switching the nature of the blocking agent and the interstitial void flushing liquid, i.e., by using a hydrophilic blocking agent (pure or buffered water at pH 3.0 or pH 7.0) and a hydrophobic flushing liquid (linear alkanes such as decane). Several parameters that might influence the accuracy of the method, such as the applicable range of flow rates and the meso-pore size of the particles have been investigated. The influence of several different parameters on the obtained external porosity value has been investigated. From a wide selection of possible t(0)-markers, the class of linear alkanes has been shown to be the single possible one. This brings along the need to use refractive index detection to measure the signal of the linear alkane tracer (e.g., dodecane) in a stream of (another) linear alkane. The results of the newly established NP-TPB method have been compared to the values of the external porosity obtained by ISEC and proved to generate the same results, however with a much smaller read-out error (being only of the order of 0.1%).

High performance liquid chromatography column packings with deliberately broadened particle size distribution: relation between column performance and packing structure.

The effect of the addition of 25%, 50% and 75% (weight percent, wt%) of larger particles (resp. 3 and 5 µm) to a commercial batch of 1.9 µm particles has been investigated as an academic exercise to study the effects of particle size distribution on the kinetic performance of packed bed columns in a magnified way. Comparing the performance of the different mixtures in a kinetic plot, it could be irrefutably shown that the addition of larger particles to a commercial batch of small particles cannot be expected to lead to an improved kinetic performance. Whereas the addition of 25 wt% of larger particles still only has a minor negative effect, a significantly deteriorated performance is obtained when 50 or 75 wt% of larger particles are added. In this case, separation impedance number increases up to 200% were observed. Studying the packing structure through computational packing simulations, together with the experimental determination of the external porosity, helped in understanding the obtained results. This showed that small particles tend to settle in the flow-through pores surrounding the larger particles, leading to very high packing densities (external porosities as low as 32% were observed) and also negatively influencing the column permeability as well as the band broadening (because of the broadened flow-through pore size range).

Experimental investigation of the difference in B-term dominated band broadening between fully porous and porous-shell particles for liquid chromatography using the Effective Medium Theory.

The difference in B-term diffusion between fully porous and porous-shell particles is investigated using the physically sound diffusion equations originating from the Effective Medium Theory (EMT). Experimental data of the B-term diffusion obtained via peak parking measurements on six different commercial particle types have been analyzed (3 porous and 3 non porous). All particles were investigated using the same experimental design and test analytes, over a very broad range of retention factor values. First, the B-term reducing effect of the solid core (inducing an additional obstruction compared to fully porous particles) has been quantified using the Hashin-Shtrikman expression, showing that the presence of a solid core can account for a reduction of about 11% when the core diameter makes up 63% of the total particle diameter (Halo and Poroshell-particles) and a reduction of 16% when the core diameter makes up 73% (Kinetex). Remaining differences can be attributed to differences in the microscopic structure of the meso-porous material (meso-pore diameter, internal porosity or relative void volume). The much lower B-term diffusion of Halo and Kinetex particles compared to the fully porous Acquity particles (some 20-40% difference, of which about 10-15% can be attributed to the presence of the solid core) can hence largely be attributed to the much smaller internal porosity and the smaller pore size of the meso-porous material making up the shell of these particles.

Use of pressure drop profiles to assess the accuracy of Total Pore Blocking measurements of the external porosity of chromatographic columns.

By comparing the pressure drop in a column where the meso-pores of the particles have been blocked using the Total Pore Blocking (TPB) method to measure the interstitial volume of the column with that in the same column when the particle meso-pores are fully open, it could be demonstrated in a very sensitive way that the interstitial volume is completely devoid of any significant amount of remaining pore blocking agent in the final phase of a TPB experiment. Monitoring the pressure signal until it returns to its original value can hence be used as a reliable indicator that all blocking agent has been removed from the interstitial void at the end of the flushing period. As a consequence, any small molecular weight dead volume marker that is employed in this phase can explore the full interstitial volume, so that the value of the latter can be measured without being underestimated by the fact that some fractions of the interstitial void would still be occupied by the blocking agent.

A study of the parameters affecting the accuracy of the total pore blocking method.

We report on a study wherein we investigate the different factors affecting the accuracy of the total pore blocking method to determine the interstitial volume of reversed-phase packed bed columns. Octane, nonane, decane and dodecane were all found to be suitable blocking agents, whereas heptane already dissolves too well in the applied fully aqueous buffers. The method of moments needs to be used to accurately determine the elution times, and a proper correction for the frit volume is needed. Failing to do so can lead to errors on the observed interstitial volume of the order of 2% or more. It has also been shown that the application of a high flow rate or a high pressure does not force the blocking agent out of the mesopores of the particles. The only potential source of loss of blocking agent is dissolution into the mobile phase (even though this is a buffered fully aqueous solution). This effect however only becomes significant after the elution of 400 geometrical column volumes, i.e., orders more than needed for a regular total pore blocking experiment.

Influence of pressure and temperature on the physico-chemical properties of mobile phase mixtures commonly used in high-performance liquid chromatography.

To fulfil the increasing demand for faster and more complex separations, modern HPLC separations are performed at ever higher pressures and temperatures. Under these operating conditions, it is no longer possible to safely assume the mobile phase fluid properties to be invariable of the governing pressures and temperatures, without this resulting in significantly deficient results. A detailed insight in the influence of pressure and temperature on the physico-chemical properties of the most commonly used liquid mobile phases: water-methanol and water-acetonitrile mixtures, therefore becomes very timely. Viscosity, isothermal compressibility and density were measured for pressures up to 1000 bar and temperatures up to 100 degrees C for the entire range of water-methanol and water-acetonitrile mixtures. The paper reports on two different viscosity values: apparent and real viscosities. The apparent viscosities represent the apparent flow resistance under high pressure referred to by the flow rates measured at atmospheric pressure. They are of great practical use, because the flow rates at atmospheric pressure are commonly stable and more easily measurable in a chromatographic setup. The real viscosities are those complying with the physical definition of viscosity and they are important from a fundamental point of view. By measuring the isothermal compressibility, the actual volumetric flow rates at elevated pressures and temperatures can be calculated. The viscosities corresponding to these flow rates are the real viscosities of the solvent under the given elevated pressure and temperature. The measurements agree very well with existing literature data, which mainly focus on pure water, methanol and acetonitrile and are only available for a limited range of temperatures and pressures. As a consequence, the physico-chemical properties reported on in this paper provide a significant extension to the range of data available, hereby providing useful data to practical as well as theoretical chromatographers investigating the limits of modern day HPLC.