Please provide the exact gradient method (Time Table of Composition). Please provide the exact flow rate used. This basic information is needed to understand your actual method.

You wrote: "I see my peaks are sinking down under it". *I do not see any "sinking" peaks at all. ???? I see a method that is not finished yet as you still have a few peaks which are not baseline resolved. You need to work on improving that gradient.

You are running at very low UV wavelength settings, so some noise and drift are normal and will be seen with these types of methods.

Please remove the 're-equilibration' and 'return to initial conditions' settings that may be part of your gradient analysis method (they do not belong there and will interfere with the integration!). Proper analysis methods end with a hold time and do not revert back. Optimize the integration settings for your analysis.

Next, TURN OFF that Reference Wavelength (360, 100) software feature (you have not had training in how to use the DAD yet). It is providing no value to your separation and may in fact be subtracting data from your sample. Please read this article to learn more: "

REFERENCE WAVELENGTHS (as used in HPLC UV/VIS)"; https://hplctips.blogspot.com/2011/03/reference-wavelengths-as-used-in-hplc.html

As you are a beginner, please seek out some professional local help with this technique in learning to use the HPLC system and also run a gradient method. It takes many years to be proficient at a basic level with this so use your time efficiently and have someone with professional experience at your school help you.

Please provide the exact gradient method (Time Table of Composition). Please provide the exact flow rate used. This basic information is needed to understand your actual method.

You wrote: "I see my peaks are sinking down under it". *I do not see any "sinking" peaks at all. ???? I see a method that is not finished yet as you still have a few peaks which are not baseline resolved. You need to work on improving that gradient.

You are running at very low UV wavelength settings, so some noise and drift are normal and will be seen with these types of methods.

Please remove the 're-equilibration' and 'return to initial conditions' settings that may be part of your gradient analysis method (they do not belong there and will interfere with the integration!). Proper analysis methods end with a hold time and do not revert back. Optimize the integration settings for your analysis.

Next, TURN OFF that Reference Wavelength (360, 100) software feature (you have not had training in how to use the DAD yet). It is providing no value to your separation and may in fact be subtracting data from your sample. Please read this article to learn more: "

REFERENCE WAVELENGTHS (as used in HPLC UV/VIS)"; https://hplctips.blogspot.com/2011/03/reference-wavelengths-as-used-in-hplc.html

As you are a beginner, please seek out some professional local help with this technique in learning to use the HPLC system and also run a gradient method. It takes many years to be proficient at a basic level with this so use your time efficiently and have someone with professional experience at your school help you.

Thank you for this valuable information,as I'm beginner I have to be patient and tolerant til I learn.

The missing data is : flow rate 1 ml/min

That gradiant

T. A. B.

0. 90. 10

1. 90. 10

7. 10. 90

7.5 10 90

8. 90. 10

10 90. 10

"Super-resolution" in the left part produces a wrong structure.

A few hints to help you, but you really need to work with someone who is experienced as this is no place to teach you a complex technique, and how to use the hardware and the importance of all those software settings too.

(1) Your last peak elutes at 7.5 minutes and your gradient program start to ramp back to initial conditions at that same time. Do you see an obvious problem (actually two problems)? You should. Hold the % composition of ACN for a longer time period (or increase the % a bit more; i.e. 95%) and get rid of the "return to initial conditions section of the method. The column washing and re-equilibration should be a separate method run AFTER the analysis method (as noted earlier).

(2) Nothing happens in the first 4 minutes of your initial gradient and your sample needs better resolution in the latter half (When there is more ACN present). You have only started to 'screen' this sample so next you need to fine tune the method for your sample. Look at the peaks, note where they elute and at what % composition your mobile phase is. Change the gradient parameters to reflect the new information observed. Perhaps start the gradient slow and shallow at 50% ACN, then slowly ramp up. You can start to evaluate this by simply modifying your original gradient to start at 50% ACN and run to 95% in the same 7.5 minutes, then hold for 2 more minutes. Then END the analysis at 9.5 minutes.

@wiliam letter , wonderful, you are great

1) actually I thought that it has no effect on the last peak as at 7.5 my analyt supposed to be already eluates to the detector that why I had no worry about ramping it up at 7.5 (what do you think?)

* I already have a separate method for washing, I did the requilibration inorder to more prepare for the next injection in the sequence

2) yes you are Right, I forgot to improve it after the screening trail as I was focusing only on the middle zone

(1) How do you know that ALL of the "peaks" have eluted if you stop the analysis at 7.5 minutes (while a peak is still eluting off the column!) unless you ramp up the % composition a bit more... and here is the important part.... HOLD the ratio steady for a period of time and wait. *This is one of the basic fundamentals of chromatography. You must (not optional) wait a period of time after what you think is the last peak before stopping, every time. Even if you are sure (and who really is?), you still would wait a few minutes more (it is good science and proper technique).

Re-equilibrating as "part of the analysis method" is a common novice mistake, but now you know better. So, next time you set up the method, do so as I described before. Your integrator will thank you (the less baseline disturbance, the quality of the integration of the peaks improves).

(2) Always look at the "big" picture. When changing composition %'s Or Times, everything can change. Make small changes (one thing at a time), then evaluate. This process takes time. Again, the scientific method guides us in our approach.

That was more than wonderful,

1) actually this aimed to be quantitative analysis for a certain group of 7 compounds I know them and made previous single compound detection at the same conditions so I have their RT already .. that's why I don't want to detect anything else after

2) I will try to improve this to make it shorter and without the requilibration step

3) but still a question, in this photo above how to reduce or to lower the baseline a bit more? after following these hints

(1) "that's why I don't want to detect anything else after". *I do understand what you are saying. However, that is unscientific. We always monitor the remaining time to make sure things have not changed and nothing new has eluted out. Not optional.

(3) The baseline rises because you are changing the mobile phase condition over time. The change in mobile phase composition changes the light transmission (via chemical and physical properties of the liquids used). It is 100% normal to see this change. Once you start doing a better job of column washing, include slightly longer equilibration times, and remove the "return to initial conditions" step (add a HOLD at the end), your overall baseline will have a much better, even rise over time. If you want to make it look even better, then the best option is to use "Baseline Subtraction". Do Not Use the Reference Software Feature! To correctly use baseline subtraction with you DAD, please read the article I referenced earlier (it is explained in there).

That is all I can do for free to help you now. Please study the suggestions made, read the classic texts on the topic and find local help.