The issue might be due to the degradation of the antibiotic sample, potentially stemming from the stability of the stock solution (e.g., storage conditions). My first thought is related to the stability of the column. A longer analysis time than 8 minutes might be necessary, as it could be challenging for the gradient program to reach equilibrium at the initial ratio within 8 minutes. It might be beneficial to allow the system to reach equilibrium by conditioning the column for 10 minutes with Mobile Phase A (PBS + 2% MeOH, pH 7.0) and Mobile Phase B (0.1% TFA in Acetonitrile) before proceeding to the next analysis. This could potentially explain the differences observed between peaks.

The second issue seems to be related to the commands entered or the operations performed within the software.

The issue might be because of decrease in concentration as you have not mentioned about its absorbance (any property which is measured) and it may be because of degradation of solution. (signal is proportional to concentration, first nullify this reason by injecting that antibotic in higher concentration)

The cause of second issue seems to be with software or command given.

You are using a very strange method and it doesn't make sense---mobile phase 1 is PBS, with pH 7.0, while mobile phase 2 is acetonitrile with TFA (pH ~3). So during the run your pH is constantly changing, and your column never gets equilibrated. At the end of the run depend on if you allow enough "post run" time, your column may not reach the original pH. Are you following a publication? Sometimes "the students did the work and the prof wrote the paper" and lots of details got lost or distorted. You should spend some time on the method development.

For the 2nd problem, please go through all the settings in the method. If you want help you should post the method (use "print method" command to export the method as PDF).

Well, First of all, I want to know, you worked with using pharmacopeia method or in house method, what were names of two different antibiotic molecules, did you develop method previously, did you find the maximum / exact wavelength, columns chemistry, chromatographic conditions

What was the name of your HPLC & software, please inform me.

Gang Chen if I wanted to stick to the method as close as possible, would you recommend I lower the pH of the PBS to more closely match the pH of the acetonitrile with TFA?

I would try un-buffered saline or saline with 0.1% TFA, or just plain water with 0.1% TFA.

Rachel Evans: As Gang Chen has suggested, the methods/information for machine one do not appear to make any sense. We do not even know what the instrument make/model/configuration/software are for machine two, but it sure sounds like the system is doing exactly what it has been programmed to do (re-check ALL of the method and sequence settings that you saved. You will find that one of them has instructed the system to do exactly what you are witnessing. Should be an easy fix). It is irrational to provide advice over the web for most HPLC method questions, but especially when the proposed method in the first example does not appear to follow good chromatography practices. Wanting to "stick to the method as close as possible", when it is likely to be declared invalid (and any data generated from it, invalid) also makes no sense. Don't stick with something just because someone else used it. It may not be correct. You would not believe how many HPLC and LC-MS methods we review for the journals and companies that are completely invalid and unscientific, yet they get published.

Is there someone at your school (or whom they can hire) with the needed years of practical, industrial HPLC experience who can come on-site to review your system and issues? It takes many years of professional training just to learn the basic of HPLC. Many more years of training for method development and troubleshooting. You can NOT learn these analytical techniques from taking classes or watching videos. There is no short-cut. You need someone with training, in-person, on-site to assist you in using the HPLC system AND insure the data you collect will be acceptable.

I’ve recently published a short research paper titled:

" A Numerical Simulation of Spherical Tank Drainage Using the Modified Euler Method: A Physics-Based Approach "

It presents a simplified yet insightful Modified Euler’s method for solving differential equations and an physical applications on it.

Feel free to check it out, and if you find it useful, I’d really appreciate your recommendation on ResearchGate.

[https://www.researchgate.net/publication/390947672_A_Numerical_Simulation_of_Spherical_Tank_Drainage_Using_the_Modified_Euler_Method_A_Physics-Based_Approach ]