Kromasil® Kromasil application guide The way to peak performance in liquid chro

Kromasil® Kromasil application guide The way to peak performance in liquid chromatography K R O M A S I L A P P L I C A T I O N G U I D E Content Guidelines 3 Applications 6 Amino acids 6 Drugs and metabolites 9 Environmental 23 Food and nutrition 26 Natural products 28 Peptides 31 Vitamins 33 Other 34 Literature references 41 Substance index 44 Kromasil availability 47 Cover figure shows a 3D chromatographic separation, with UV absorption at different wavelengths vs. time. Kromasil applications – from our lab and the literature The Kromasil packings and columns have been used over the years for demanding separations all over the world. In this guide we have collected examples of a variety of chromatographic separations, from small synthetic pharmaceuticals, up to peptides and larger molecules. We hope this guide will be a useful tool when developing new HPLC separation methods in your lab. Copyright Copyrighted elements of this publication may not be reproduced in any way without the consent of their respective owner. 2 The Kromasil packings – some hints for the best performance G U I D E L I N E S The Kromasil family of packing materials is developed to be the perfect choice from analytical to process scale. Kromasil is presently available as bare silica, C4, C8, C18, NH2, CN or as Kromasil Chiral for separation of optical isomers. Pore sizes are 60 Å, 100 Å, and 300 Å, and particle sizes 3.5, 5, 7, 10, 13 and 16 µm. Slurry-packed columns are available from analytical up to 2" inner diameter, all with analytical efficiencies. For larger preparative and industrial scale columns bulk packing is provided. To learn more about the properties of Kromasil silica please consult our other technical information. Choice of mobile phase Normal Phase conditions Choose mixtures of hexane or heptane, and polar modifiers like alcohols, ethyl acetate, methylene chloride, etc. to adjust retention. The optimum retention factor range is normally 2 £ k £ 5 for a two-component sample, but can be wider for a multi-component sample. Acidic and basic additives can improve the chromatographic performance. In most cases small amounts of acetic acid or formic acid (0.05 – 0.10%) improve peak shape for acidic or basic solutes. In some cases the combination of acid and an organic amine (e.g. triethylamine) is necessary, for difficult solutes. The acid should always be in excess relative to the amine, in order to operate at a pH where the silanol groups on the silica are protonated. Reversed Phase conditions Choose mixtures of water or buffer, and water misc- ible solvents like alcohols, acetonitrile, THF, etc. to adjust retention factor k to an optimal range. The pH can be controlled by using a buffer, and in order to minimize the ionization of the silica and the solutes. In order to control peak shape for very basic solutes an additive like TEA (triethylamine) can be added, if necessary. Kromasil is a fully hydroxylated ultra-pure silica, making the surface less acidic, resulting in good peak shape also for basic compounds. For the C4, C8 and C18 phases, due to the very hydrophobic nature of the surface, it is important to always keep at least 4 – 5% of organic in the mobile phase, both when flushing or running the chromatographic separation. The reason is that in the case of a 100% aqueous mobile phase there is a risk that the surface within the porous system in the Kromasil particles is “dewetted”, resulting in a total or partial loss of retention. This phenomenon of dewetting is more pronoun- ced for high quality, high coverage materials, where the bonding procedure has been very efficient. This will result not only in higher retention times because 3 Figure 1 | Influence of the mobile phase additives on the separation of mefloquine. Conditions: Column: 4.6 ¥ 250 mm, Kromasil CHI-DMB, 5 µm Flow rate: 2 ml/min. Detection: UV 280 nm Mobile phase: Heptane/2-Propa- nol/HCOOH (90 / 10 / 0.2) Mobile phase: Heptane/2-Propa- nol/HCOOH/Tri- ethylamine (90 / 10 / 0.2 / 0.1) Mefloquine a = 1.19 a = 1.32 CF3 N HC OH N H CF3 Figure 2 | The retention factor, k, vs. organic content. General retention behaviour at low organic content using high density RP phases. 1 0 5 10 % organic in water ln k always keep at least 4 – 5% of organic in the mobile phase 4 of a higher coverage and hence hydrophobicity, but also in a higher hydrolytic stability, and a longer life- time of the column. If a 100% aqueous mobile phase has been used accidentally, and the stationary phase has been dewetted, the column can easily be regenerated. Just flush the column with a mobile phase consisting of 40 – 50% or more of organic for 2 – 5 column volumes. After this the column can be equilibrated again with the mobile phase, and the original reten- tion times should be seen. We also recommend to always filter buffer solu- tions in order to remove small particulates. It is also preferable to premix aqueous/organic solutions, in order to avoid problems with gas formation in the mobile phase, or a temperature increase or decrease as an effect of endo- or exothermic mixing heats. The recommended pH range for our RP phases is between pH 1.5 up to 9.5. However, in some appli- cations mobile phases with pH above 11 have been used for continuous chromatography, for several thousands of column volumes. How to improve speed of separation There is today a strong driving force towards faster separations, and hence smaller particles and shorter columns. A smaller particle will give a higher efficiency at a higher flow rate; for example will a 5 µm particle give twice the efficiency compared to 10 µm, at twice the flow rate. And if the resolution is to be kept constant one can also reduce column length by 50%. Table 1 gives the relation between the critical parameters when going to smaller particles, and shorter columns. It can be seen that the combination of smaller particles, shorter columns and higher flow rate will result in much faster analyses. The only drawback is the back pressure, which will increase significantly as particle size goes down. All in all, one will save a factor 2 in analysis time by going from 5 to 3.5 µm for example, but also experience twice the back pressure. How to improve resolution A good resolution in a short period of time is usually a requirement in analytical HPLC. One has essentially three ways to improve the resolution, as can be seen in the equation below: 1. The separation factor, a, can be increased. This can be done by optimizing stationary and mobile phase, i.e. choosing the best column and mobile phase composition for the specific application 2. The number of theoretical plates can be increased. This can be done by: a. Increasing the column length b. Decreasing the particle size c. Optimizing the flow rate. The optimum for small particles is at a higher flow rate than for larger particles (inverse linear relationship) 3. The retention factor, k, can be increased, if it is too low. This can be done by adjusting the elution strength of the mobile phase K R O M A S I L A P P L I C A T I O N G U I D E Table 1 | Relation between optimal flow rate, analysis time and back pressure, DP, when going to smaller particles, and shorter columns. The comparisons are for a constant resolution, Rs. Column length (mm) 250 125 87.5 Particle size (mm) 10 5 3.5 Flow rate (ml/min.) 0.5 1.0 1.43 Relative time 1 0.25 0.125 Relative Rs 1 1 1 Relative DP 1 4 8 Rs = 1 4 (a – 1) · N · k1 1+k 5 Scale-up The analytical separation is very often the start for a scale-up to semi-prep, prep, or large industrial scale chromatography. In the case of Kromasil there is the possibility of seamless scale-up from analytical to semi-prep and prep, and even large diameter Dyna- mic Axial Compression (DAC) columns. All Kromasil columns independent of diameter are delivered with the same, high efficiency guarantee, and even the large DAC columns can be packed giving the same performance. We recommend that the method development for the preparative separation is performed using analy- tical columns, or possibly 10 mm ID if larger volumes of the sample fractions are needed. A small column will make the development work easier, and since the performance is identical in small and large dia- meter columns, the result in large scale can easily be predicted from the work in small scale. 10 µm particles are recommended, since the efficiency, back pressure, etc. then will be close to the large scale separation. For large scale 10 – 16 µm particles are usually a good choice. However, the performance using a different particle size can easily be uploads/Management/ kromasil-application-guide.pdf

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• Publié le Mai 21, 2022
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