Hi Srikanth,

can you say something about the peak width? Maybe you have too much dead volume in your system. Which tubings (ID) do you use and how long are they? Are the tubings cleanly cut at the ends and are the ferrules seated? I would check that before I turn to the column.

Regards

Markus

Please write more details about your experiments to be able of help you

Your question is not descriptive, but in general, if your peaks are broad then theoretical plates are not achieved if your column is old and deteriorated. Add additive to your mobile phases such as triethylamine or trifluoroacetic acid etc. depending upon you analyte nature. Increase column temperature, decrease column material particle size, select a solvent with lower viscosity.

Hi all

Thanks everyone for your answers. Column is brand new and tubings were perfectly cut which were made of PEEK material. I used high quality reagents but i always got low theoretical plates for R-enantiomer. Mobile phase is mixture of buffer and ACN and diluent is buffer. Column is Chiral AGP.

How flowcell and number of theoretical plates are related ?

Regards

Srikanth

Hi Srikanth,

what scan rate of your detector do you use? Maybe try a higher scan rate for a smaller peak width.

Regards

Markus

Hi Markus

Thanks and am using response time of 2s on VWD detector. What high scan rate should i use on VWD and DAD ?. Can you provide an example since it varies from instrument to instrument .

Regards

Srikanth

Hi Srikanth,

a DAD normally can scan with a rate of 20 Hz. Try it and compare it with your scan rate you are using at the moment.

Regards

Markus

Hi Markus

Thanks.Is there any relationship between flowcell and no of theoretical plates.?

Regards

Srikanth

Hi Srikanth,

if the cell volume of your flow cell is too big than this can lead to peak broadening.

What is your current cell volume? There is a relationship between cell volume and peak width (s. the attached picture). For example your peak width is doubling when you change your cell volume from 2 µl up to 10 µl.

Regards

Markus

P.S.: Have a look to the following link:

http://www.lcresources.com/training/tsbible.html

LC-GC 10(1) 20-25 (1992)

Thanks Marrkus

So using low cell volume will increase plate count in my case .

Reagrds

Srikanth

Most important question not asked or answered. What is the K prime of the peak used to calculate N from? Usually early eluters, with narrow peak shapes will have highest N value. Also, what are you comparing the 'N' number you have too? Is it exactly the same sample/compound using exactly the same method (is it trans-stilbene oxide, the most useless test standard for chiral columns)? IF so, what is the percentage difference between them (what are the actual values)? You have not provided basic HPLC method info with your question. Basic info is always needed to discuss HPLC questions.

Is the Flow Cell volume Important? Yes, it certainly can affect measured value of N. Using low flow cell volume will increase plate count? No, not necessarily. The reason is simple, sample dilution. If the cell is too large relative to the volume and flow, then the peak may broaden due to dilution. Just like all of the other parts, the flow cell must be optimized for the method and application. If the cell is too small relative to the column volume, peak volume and flow rate, then the sample may take a very long time to flow through the cell, increasing the peak width.

Other things that affect plate count, 'N' ? Well, many things. Peak shape and elution time are very important. The exact way YOUR HPLC system is plumbed (PEEK tubing???? That does not tell us anything useful) is almost certainly not the same as the HPLC system which was used to generate the original #. Each HPLC must be optimized for each application and column used. Variations are normal and to be expected. The exact lengths, ID's, types of connections made, flow cells, column dimensions and injection volume (sample concentration), Wavelength and BANDWIDTH used all play a role. High plate #'s come from the use of narrow, Gaussian shaped peaks, eluted quickly, in as little volume as possible using an optimized flow path (of your HPLC).

Sampling rate could be of concern, but 99% of most users are grossly over-sampling, not under-sampling so not something we typically worry about. "Scan rate does not vary from instrument to instrument". It is supposed to be calculated for each and every method/application, not guessed at or using the default value. The operator is responsible for calculating and using the correct value. 2 seconds may or may not be the best rate. There is no possible way anyone could provide you with the correct answer because you have not provided us with any information about your actual method. We need column dimensions, flow rate, the peak width at half height of your sample peak and the peak shape to calculate it.

May I just you start learning about HPLC by reading some of the excellent texts on this topic? Perhaps their is someone you know which industrial HPLC experience whom could help you?

A few free articles which may help you in your studies:

• "Number of theoretical plates (N), Calculation Formulas"; https://hplctips.blogspot.com/2018/06/number-of-theoretical-plates-n.html
• "HPLC Flow Cell Volume & Path Length:" https://hplctips.blogspot.com/2011/03/flow-cell-volume-path-length.html
• "Determining the Data Acquisition Rate (Sampling Rate) For Your HPLC Detector"; https://hplctips.blogspot.com/2012/12/determining-data-acquisition-rate.html
• "Chiral HPLC and SFC Column Screening Strategies for Method Development:"; https://hplctips.blogspot.com/2011/06/chiral-hplc-and-sfc-column-screening.html

Additionally, you may wish to take some time and begin to research the basics of liquid chromatography [e.g. The text, "Introduction to Modern Liquid Chromatography" by: Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan is excellent for all levels of users]. This will provide you with some introductory information on the topics of setting up and using HPLC methods for analysis and the basic concepts and fundamentals of analysis.