#HPLC_method_development

Lead Changes in selectivity brought about by modifying the composition of the mobile phase have been discussed up until this point. This method is typically the most practical and economical way to alter selectivity. Alternately, relying on changes in column selectivity to control the separation is a strategy that is typically reserved until all other mobile phase alternatives have been exhausted. As with alterations to the selectivity of the mobile phase, chromatographers should make substantial …

Controlling selectivity-Additional factors-3.0 – Column selectivity Read More »

Lead In the earliest posts we saw the how solvent concentration and type are powerful tools for controlling separation and also discussed these variables alone may not be sufficient for achieving the desired sample resolution. In such cases chromatographers may seek to employ other parameters to adjust selectivity viz.. In the last post the discussion was focused on the influence of pH on selectivity. In this blog the focus will be on the effect of …

Controlling selectivity-Additional factors-2.0 – temperature Read More »

Lead In the previous post the influence of solvent concentration and solvent type on the selectivity of liquid chromatography (LC) separations was discussed. We saw by systematically altering the solvent concentration, chromatographers can achieve dramatic changes in selectivity and retention. Changing the solvent type during method development, from acetonitrile to methanol, for instance, provided additional selectivity leverage. In some instances, it is advantageous to combine multiple organic solvents. Although varying solvent concentration and type are …

Controlling selectivity-Additional factors-1.0 – pH Read More »

Lead This is in continuation to my earlier post <https://chiralpedia.com/blog/controlling-selectivity-solvent-role-1-0/> where we discussed the role solvent, first approach – varying solvent strength, in controlling retention factor and selectivity of chromatographic separation. In this post the focus is on the second approach, blending of solvents, to arrive at a good separation of difficult-to-separate peak pairs in HPLC method development. Second Approach: Blend solvents Òne can find suitable separation conditions for many samples simply by adjusting the …

Controlling selectivity-Solvent role-2.0 Read More »

Lead Choosing a set of initial conditions that would be likely to produce a successful liquid chromatography (LC) separation was the main topic of discussion in the first post in the HPLC method development series. The majority of samples will benefit from starting with a C8 or C18 column packing based on Type B silica using a mobile phase of acetonitrile or methanol modified with water or a low-pH buffer, even though no single set …

Controlling selectivity-Solvent role-1.0 Read More »

Lead In the last post we saw the tools at your disposal to assess the quality of a chromatogram viz. retention factor, peak asymmetry, peak shape, column plate number, and related aspects.  These chromatogram observations are important, but the main purpose of chromatography is to achieve a separation that is reasonable. To ensure whether the separation is satisfactory one need to add one more measurement to the tool box namely the resolution. In this post …

Measuring quality of chromatogram – Tools 3.0 Read More »

Lead In the previous post, “Measuring quality of chromatogram – Tools 1.0” we talked about two of the separation attributes retention time and retention factor. In current post the discussion is on other features viz. peak asymmetry, peak shape, column plate number. and related aspects. This time let us try to answer the following queries to understand how these attributes also help to assess the quality of chromatogram. Peak asymmetry and peak tailing Peak shape …

Measuring quality of chromatogram- Tools 2.0 Read More »

Lead In the previous post, we talked about choosing suitable initial conditions when developing a new liquid chromatography (LC) method. The possibility of a successful separation is increased by selecting the appropriate column conditions and mobile phase composition. Getting the controls that quantitatively measure the separation’s quality is another crucial step in the method development process. Although the majority of chromatographers are able to assess the separation quality from a chromatogram, it is critical to …

Measuring quality of chromatogram – Tools 1.0 Read More »

Lead Starting out with a sound strategy is one of the most important aspects of minimizing chromatographic separation issues. A good method makes it much simpler to maintain operation within parameters and address issues as they appear. Selecting the right tools is very critical to achieve the best results,. By looking at the cause-and-effect diagram given below one can understand the factors that influence chromatographic separation. Basically the factors falls into two major categories namely …

Selecting the tools Read More »