Duc Anh Nguyen please seek the assistance of someone with professional liquid chromatography (HPLC) experience, on-site. Your statements imply that your method may be invalid and/or you are new to using HPLC and your questions could best be answered by someone on-site who has all of the basic HPLC method and instrument information available to them. Please note the following basic information in troubleshooting :

• *You stated your sample is ~ 70% pure (by what analytical method?). If this is true, then more than one peak may be shown/detected (no information was provided about the method YOU are using so we do not know).
• Column overloading MAY result in split peaks (this is just one sign), but may also result in a large single peak with or without shoulders too (depends on the method used). Overloading the column's sample capacity may result in co-elution of peaks (perhaps this is what you have observed?). Split or shouldered peaks may be caused by injecting your sample in too STRONG a solution (samples should be dissolved in the mobile phase).
• Injection volume amount must be in an appropriate range for the type of injector you are using AND must also be appropriate for the column used (column's volume must be large enough to handle the inj volume).
• You stated that you are using a column with a guard column attached. Damage or voids in either the guard or main column can cause split peaks, shoulders or other changes to the chromatography. Test samples with and w/o the guard column plumbed inline.
• Students are not expected to have the knowledge needed to use complex analytical tools such as an HPLC system without professional help and guidance. It takes the average scientist 5-years of full-time work to achieve just a basic level of proficiency (BASIC, not intermediate). Please seek the assistance of someone with professional liquid chromatography experience, on-site to resolve these issues. You will learn more about what you are doing in this way and increase your chances of acquiring good quality data for your project.

• "HPLC Peak Splitting. Common Reasons For It": https://hplctips.blogspot.com/2016/09/hplc-peak-splitting-common-reasons-for.html
• "HPLC Injection Volume: What Should I Dilute It In and How Much Sample Can I Inject?"; https://hplctips.blogspot.com/2023/07/hplc-injection-volume-what-should-i.html

William Letter Thank you for your reply!

I run a gradient from 100% Water (A) up to 40% ACN (B). I dissolved my Sample prior in 50% ACN and used an injection volume of 5/10 µl which is in the range of my column (4.6mm, 15cm lenght, 100 angstrom, 3 µm pore size). And yes i just started getting into the HPLC for around 6 months and have a lot to learn, simple problems i could solve so far but the main problem is we dont have any experienced hplc scientist in our group. I will study the articles you sent me and try to find the cause of this.

Thanks a lot

• Duc Anh Nguyen wrote: "I dissolved my Sample prior in 50% ACN";

Your injection solution is too strong. Please dissolve the sample in the mobile phase initial condition (100% water is your initial condition. Water with less than 10% ACN may be used).

Your injection volume of 5 to 10ul is fine for a 4.6 x 150mm column. While the volume appears OK, you still may be injecting too HIGH a concentration causing issues. The concentration is important.

What is the flow rate?

What are the detection settings used (e.g. wavelength/bandwidth)?

What is the sample's K prime?

William Letter

Thank you for the kinds tips! I dissolved my sample in 5% ACN and did several runs for 5/10 µL now its a sharp single peak. I use a flow rate of 1ml/min and for the detection i use a bandwith of 10 nm at 4 different wavelenghts (220/250/260/280 nm) which could detect my sample. Regarding the samples K prime i didnt calculate it yet but right now it seems to finally work

That is good news regarding the change to injection solution. Follow good chromatography fundamentals and you will have better results! The greatest danger to using this technique is: that you do not yet know what you do not know... (that is why you can not just figure it out as you go. Training is needed). - - Careful... You should not start HPLC sample analysis or any qualitative analysis until you measure the two most important fundamentals of HPLC. Column Volume and K prime. The K prime of your sample requires that you know the column volume of YOUR column (never assume, measure it). Without such basic data, how do you know that your method retains the sample and chromatography is occurring? This is a BASIC fundamental (taught in the first week) of chromatography and one of the most important key points anyone starting to learn HPLC must know.

• "Determination of HPLC Column Void Volume / Dead Volume, Dead Time (T zero)"; https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html
• "K Prime (also known as: Capacity Factor, Ratio or Retention Factor): One of the Single Most Important HPLC Parameters of All"; https://hplctips.blogspot.com/2015/06/k-prime-also-known-as-capacity-factor.html

The observed behavior is consistent with the principles of chromatography. Increasing the injection volume can sometimes lead to improved peak shapes and resolution, especially when dealing with moderate sample purities. It's important to optimize your injection conditions based on the specific characteristics of your sample and the column you are using.

So i measure my T0 which is around 1.6min (1.75min calculated) and at first my measurements with 5%ACN instead of 50% ACN looked really good. My Kprime is around 5 but now with the same setup and settings/method my sample elutes really weird. It elutes at around 10min at a flowrate of 1ml/min...i am doing some tests now and see if maybe my guard column developed an issue since both the guard column and hplc column are new.

Remove the guard column and repeat.

Are you washing the column after each analysis??? With what.

Do you filter samples and mobile phase?

What column temperature are you using?

Use FRESH solutions each day?

You may still have a fouling issue or insolubility.

William Letter

I removed the guard column and the peakshape is still as bad.

I wash my column with 5 CV 100%ACN and then 5CV 5%ACN after each injection. I use water and acn as a mobile phase and filter them prior use. Our system has an inbuild degasing system, i use both solvents for 2-3 days. I run my method at 30 °C...i will flush my system with the Waters Magic Solution and my column as well and will see if the measurements are improving.

Please do not flush your system with "magic solution" (makes no sense to do so as training is the issue. DO not run misc solvents through the column and system). Do not flush with 100% ACN. Do not flush with 5% ACN.

Flush with an appropriate WASH solution, which will dissolve all material used. This is probably not your mobile phase and may include other solutions (e.g. MeOH. The correct ones to use are the ones that dissolve all materials 100% in solution and are stronger than your mobile phase). The column wash step may be another gradient run (not isocratic) that follows your initial analysis run (never combine the analysis run with a wash and re-equilibration step, all in one method. Create separate analysis, wash and optionally, re-equilibration methods for use.

Will your samples all dissolve in 100% Water? Test.

Will your samples all dissolve in 60% water/40% ACN? Test it.

*Note: Dissolve means 100%, clear solution.

AT this stage you do not have enough training to know what to do so please request help from someone local with professional experience in liquid chromatography. HPLC can not be learned on your own in days, weeks.

Question: In the two chromatograms you posted earlier, are they both of the exact same sample or different samples? Do you have a standard "sample" you can use to check the method's reproducibility with? You need one. If the sample's are different, then you may be looking at "real" changes and not distorted peaks.

William Letter

Thank you for your input, i will go through my workflow more in detail with someone in the department who has experience in HPLC.

In the two chromatograms are shown the same samples and yes i have a standard (another substrate) which runs the same so far and could also identify via Mass to be the substrate i injected.

And regarding my washing/equilibration step i will inject my mobile phase in that case and run the same gradient as a washing step since i cant do a zero injection on my device. Thanks a lot for your input and time

As a little update, i finally got the right method and also the ideal mobile phases for my application. The peaks are now very sharp and reproducible.

Thanks again for all the support

We appreciate the update and are happy to learn improvements have been made. Good luck to you!