HILIC is not a column (though some vendors may place a HILIC label on it, they are only doing so to fool you), it is a mode of chromatography.

Column length alone has nothing to do with suitability for LC-MS analysis. First, develop the HPLC method and make sure it is compatible with the detector type and ion mode desired (e.g. ESI pos/neg; highest purity volatile additives and mobile phase ). For most types of MS systems, highest sensitivity will be obtained with the lowest flow rates. If sensitivity or LOD are not critical, then even 1.00 ml/min rates will be fine, otherwise, low flow rates (depending on the exact MS type) are preferred so the use of narrow ID columns helps to achieve this by maintaining linear velocity (e.g. for conventional methods) without loss of efficiency. You should get some help selecting the column when you first start to develop the actual method used. If LC-MS (ESI) is the eventual goal and sensitivity is important, then start with a column type and dimension which result in the needed separation (Have someone experienced in LC-MS method development help you). You will not know what the ideal column dimensions are until AFTER you start to develop the method. Work out the method on an HPLC system, then scale it ) to the LC-MS system so it works well (change column dimensions, flow rate, gradient conditions, inj volume and so on, as needed).

• Column ID matters: Example. Use of 2.1 mm ID column will reduce the scaled flow rate (linear) down from 1.00 ml/min on a 4.6 mm ID column to ~ 200 ul/min.
• Simple article on scaling for columns used in HPLC/UHPLC: https://hplctips.blogspot.com/2011/07/hplc-to-uhplc-conversion-notes.html

HILIC is not a column (though some vendors may place a HILIC label on it, they are only doing so to fool you), it is a mode of chromatography.

Column length alone has nothing to do with suitability for LC-MS analysis. First, develop the HPLC method and make sure it is compatible with the detector type and ion mode desired (e.g. ESI pos/neg; highest purity volatile additives and mobile phase ). For most types of MS systems, highest sensitivity will be obtained with the lowest flow rates. If sensitivity or LOD are not critical, then even 1.00 ml/min rates will be fine, otherwise, low flow rates (depending on the exact MS type) are preferred so the use of narrow ID columns helps to achieve this by maintaining linear velocity (e.g. for conventional methods) without loss of efficiency. You should get some help selecting the column when you first start to develop the actual method used. If LC-MS (ESI) is the eventual goal and sensitivity is important, then start with a column type and dimension which result in the needed separation (Have someone experienced in LC-MS method development help you). You will not know what the ideal column dimensions are until AFTER you start to develop the method. Work out the method on an HPLC system, then scale it ) to the LC-MS system so it works well (change column dimensions, flow rate, gradient conditions, inj volume and so on, as needed).

• Column ID matters: Example. Use of 2.1 mm ID column will reduce the scaled flow rate (linear) down from 1.00 ml/min on a 4.6 mm ID column to ~ 200 ul/min.
• Simple article on scaling for columns used in HPLC/UHPLC: https://hplctips.blogspot.com/2011/07/hplc-to-uhplc-conversion-notes.html

Mel wrote: "Does peak capacity increase in LCMS compared to HPLC UV?"

"Peak Capacity". (?) Peak capacity can increase at higher flow rates due to narrower peak widths (and for longer columns), but I think you are referring to Capacity Factor (aka: K prime). Understanding what K prime is, and its role in chromatography is one of the most fundamental aspects of the technique. Please review this short article on K prime: https://hplctips.blogspot.com/2015/06/k-prime-also-known-as-capacity-factor.html

MS and UV are detection methods. They have nothing to do with K prime. K prime is related to retention which is related to the actual LC method and column used. Please review one of the many great introductory books on High Pressure Liquid Chromatography (HPLC) to gain an understanding of the fundamentals of this technique (e.g. "Introduction to Modern Liquid Chromatography").

Massive thanks to Bill Letter. This is a massive resource for beginners.

I looked up that peak capacity always comes before resolution but how do you optimise peak capacity?. Especially if I tried all parameters along the optimisation process. I'm guessing thay if separation of peaks is optimal at 95% acetonitrile. Then another pH could be worth trying.

I didn't find anything on optimising peak capacity except for increase temperature to increase mass transfer. The resolution is 3 to 5 but my peak capacities are not at the regulatory standards of 1.5 to 2 minimum.

So I think trying different stationary phases to get a high peak capacity first at different mobile phases is better than a useless method at the end.

The resolution is 3 to 5 but my peak capacities are not at the regulatory standards. I think trying different stationary phases at different mobile phase and % is better than a useless method at the end for real samples?.

I advocate Snyder and Dolan as it is a free pdf for every beginner. Definitely good for the job interviews 🤔

Mel,

Usually, the smaller the particle size of the column's packing material the higher the peak height. However, the back pressure will increase substantially. This particularly true as the column becomes longer.

Thus, one of the benefits of UPLC/UHPLC is the shorter column and smaller particle size, which can now perform many analyses that cannot be done before (like HILIC).

In any case, it is your target molecule (API in pharmaceutical lingo), the formulation, and the concentration that determines the separation. What is you target molecule?

Hi Mel: My recommendation for anyone learning about liquid chromatography is to forget about peak capacity for now.

The formulas that everyone needs to have a solid understanding of are: Determining Column Void Volume (Column void volume allows you to calculate the Tzero time); Capacity Factor (aka: K prime which lets you know if the compound has been retained on the column); Resolution (Rs =ΔtR/w) and Selectivity (α) tell you how well separated (resolved) the peaks are. *Understand and use these basic concepts effectively in Method Development and you will be technically ahead of most working chromatographers!

HPLC Method Development is all about optimizing column choice and mobile phase to provide a scientifically valid and reproducible method which is selective for the sample or compounds being analyzed.

First, identify the best column for the sample/separation. Pick the wrong column type and you could spend years trying to develop a method that never really works or works poorly. This can be the most difficult part of method development as there are thousands of columns to choose from (though many are clones of each other). Only hands-on practical experience speeds up this selection process, so for now get some help and do your research to pick a column type and mode of chromatography that seems appropriate for the sample. Next, develop the method. Measure the void volume of the column before you start (you will need this info to measure your results). Again, method development is all about changing one thing at a time. Use the fundamentals of chromatography to guide you (BOOKS). Address solubility, ionization, pH, temperature, mobile phase composition and other aspects of the method as needed. If applicable, start with broad gradients to scout for information about retention.

Goals:

• Keep it Simple. Simple methods and mobile phases are preferred, always.
• See how many compounds appear to be present, then find a method which retains them ALL on the column. First real peak on chromatogram should have a K prime ~ 2 to 10;
• Baseline resolve the peaks away from each other and the T zero time (Two peaks next to each other should have Resolution > 1.5, >2.0 is best). Selectivity is the relative difference in separation from one peak to another (α > 1).
• Optimize mobile phase and conditions to meet acceptable guidelines. In general, we are also trying to develop methods which make efficient use of materials consumed (i.e. solvents, energy, wear & tear) and can be completed in a reasonable amount of time (w/o sacrificing too much).

I can not possibly post an entire book on HPLC method development here, but luckily, plenty of others have written many on the topic. This is just to help you focus on what is important when you start out during the first few years.