Application Note No. 300/2017. Loading capacity of silica flash cartridges

Transcription

1 Application Note No. 300/2017 Loading capacity of silica flash cartridges Quality in your hands

2 1. Introduction Optimizing flash chromatography requires finding the ideal cartridge and conditions for a given sample. As the goal is to prepare the largest possible amount of pure sample in the shortest possible time and with a minimum use of solvent, it is crucial to know how high loadings affect the separation for different cartridges. Unlike analytical chromatography, preparative separations are usually carried out under overloaded conditions, i.e. with a sample amount that is so large that it causes additional peak broadening. This is acceptable in preparative chromatography, as long as the peaks in the sample do not overlap. The maximum feasible loading for a given separation therefore depends not only on the loading capacity of the column, but also on the initial separation of the compounds of interest. Spherical silica particles provide multiple benefits as a column material over irregularly shaped particles. They can be packed more tightly into the column, leading to more homogeneous flow, reduced peak broadening and thus higher resolution. The low content of fine particles also reduces back pressure, a property that is especially important when using smaller particle sizes. This application note gives an example of how loading affects preparative separations for a specific sample. FlashPure cartridges with particles of different sizes are compared, as well as spherical and irregular packing materials. 2. Equipment Reveleris Prep (equivalent results can be obtained on the Reveleris X2) Silica cartridges FlashPure ID, FlashPure ID HP and FlashPure Select, 24 g Table 1: Properties of the flash cartridges used in this application note. Cartridge FlashPure ID FlashPure ID HP FlashPure Select Amount of Silica (g) Particle size (µm) Morphology Irregular Irregular Spherical 3. Chemicals and Materials Chemicals: n-hexane, technical, ECSA Chemicals AG, Flawil, Mp L0015 Ethyl acetate, technical, ECSA Chemicals AG, Flawil, MP L0021 Diethyl phthalate, Fluka, Dibutyl phthalate, Sigma-Aldrich, For safe handling, please pay attention to all corresponding MSDS! Samples: Mixture of phthalates at a volumetric ration of 1:1 Dilutions of this sample to 50 % by volume and 20 % by volume in hexane

3 4. Procedure 4.1. Separation method The separations were carried out using the parameters listed in Table 2. All tests on a given cartridge type were performed on the same cartridge. In subsequent tests, the equilibration step was skipped. Table 2: Parameters of the chromatographic separation Parameters Flow rate 36 ml/min Equilibration time 3 min Solvent composition Hexane : Ethyl Acetate, 4:1 UV Wavelength 254 nm, Sensitivity Low Injection type Liquid, 1 ml 4.2. Evaluation The resolution (R) is calculated based on the peak width at half height (W 50) and the distance between the peak maxima ( t r) (Equation 1) [1]. This calculation assumes Gaussian peak shape, which is typically not the case at high loadings and can therefore only be considered as an approximate indicator of quality. Visual inspection of the chromatograms is key in judging the quality of an overloaded preparative separation ,, 1 Application Note 300/2017 May /7

4 5. Results Figure 1: Chromatograms obtained from loading the sample diluted to 20 % on FlashPure ID (A), FlashPure ID HP (B), and FlashPure Select (C). Figure 1 shows the chromatograms obtained when loading 1 ml of the sample diluted to 20 %. All cartridges still give good separations at these conditions; however, differences in peak width are clearly apparent. Peak width essentially follows the expected order with smaller silica particles giving sharper peaks. In addition, peak shape differences are noticeable. On the FlashPure ID cartridge, peaks are broadened Gaussian peaks, indicating that the inherent broadening column dominates. In contrast, on the FlashPure Select cartridges, which give much less inherent peak broadening, overload is the dominant broadening mechanism. Accordingly, the peaks have the characteristic trigonal shape of overloaded peaks [2]. Application Note 300/2017 May /7

5 Figure 2: Chromatograms obtained from loading the sample diluted to 50 % on FlashPure ID (A), FlashPure ID HP (B), and FlashPure Select (C). Figure 2 shows how the chromatograms change at higher loading. While all peaks are still baseline separated, the decrease in resolution is visible. Figure 3 shows the result obtained when injecting the undiluted sample. On two out of three cartridges, the peaks are no longer baseline separated. Only FlashPure Select cartridges provide sufficient separation at the highest loading. Based on the chromatogram, even higher loadings can be applied while still providing sufficient separation. Application Note 300/2017 May /7

6 Figure 3: Chromatograms obtained from loading the undiluted sample on FlashPure ID (A), FlashPure ID HP (B), and FlashPure Select (C). Figure 4: Plot of the resolution as a function of loading for different cartridge types. Figure 4 shows the resolutions obtained as a function of loading for the different cartridges. As expected, resolution at the lower loadings is essentially a function of particle size. For higher loadings, the resolution decreases strongly. Application Note 300/2017 May /7

7 6. Conclusion The optimum loading for an efficient and economical separation of a mixture of substances depends on many factors such as the chosen solvent system and gradient, the amount of the target compound and the impurities in the sample and their resolution at lower loading. The better the initial separation, the more sample can be loaded while still obtaining satisfactory separations. For standard separations at medium loading or less challenging separations at high loadings, bigger particles (e.g. FlashPure ID or FlashPure EcoFlex) are the most economical solution. Medium particles, such as used in FlashPure ID HP cartridges, excel at separarating challenging samples at low to medium loading. FlashPure Select cartridges provide the best performance for any application, due to small and spherical particles. Especially for complex separations and at high loading this material allows highest efficiency. Choosing the right cartridge for your separation problem will help you get the most of your chromatography setup and obtain the best cost-benefit ratio in each separation. 7. References [1] Talamona, A.: Laboratory Chromatography Guide, Büchi Labortechnik AG, Second Edition 2012 [2] Snyder, L.R., Kirkland, J.J., and Dolan, J.W.: Introduction to Modern Liquid Chromatography, John Wiley & Sons, Inc., Third Edition 2010 Application Note 300/2017 May /7