Chromatography Forum

So I’m kinda new to HPLC. I am using Waters 2695 equipped with PDA 2998. I have several issues with / questions about the equipment:

1. When I tried to make the calibration curve of atrazine, I got a noisy/drafty baseline. I’ve attached images to this post. Any ideas of how to fix that?

2. When I tried to make the calibration curve of 2,4-D at different concentrations, there were no peaks at some concentrations! Any thoughts?

3. What is the correct procedure for turning off the equipment? Just to flush the column with the more organic solvent and then turning it off?

I appreciate your responses.

Of course you have a noisy baseline! You have ‘blown up’ the baseline to 0.00025 AU. Try 0.01 instead.

Depending on the length of your column and flow rate, the solvent front could be 1.00-5.00 minutes so these peaks are irrelevant! What is your void volume? You may have to inject neat Acetone.

For your try injecting ~100 ug/mL (ppm) to be sure you have a peak. What are your chromatographic conditions?

For your injecting 2,4-D try injecting 100 ug/mL….

HPLC chemist wrote:
Of course you have a noisy baseline! You have ‘blown up’ the baseline to 0.00025 AU. Try 0.01 instead.

Depending on the length of your column and flow rate, the solvent front could be 1.00-5.00 minutes so these peaks are irrelevant! What is your void volume? You may have to inject neat Acetone.

For your try injecting ~100 ug/mL (ppm) to be sure you have a peak. What are your chromatographic conditions?

Thank you for your reply. How should I try 0.01? You mean by increasing the concentration of the sample? And this noise is normal in very low concentrations?

Yes, I have irrelevant peaks, and I don't know the reason. I just injected analytical standard of atrazine into water. I don't know about void fraction. Why would acetone help?

My conditions were:
Column: Nova-Pak (R) C18 4um (3.9x150mm)
Mobile phase: 55% methanol, 45% water
Flow rate: 1 uL/min
Wavelength: 254 nm
Temperature: 40 oC
Injection volume: 10 uL

Acetone (in general) is not retained by most columns and thus elutes in the void volume.

0.01 is just a guesstimate (you have to use your own common sense). A peak is only relevant if it is 0.05% of the main peak you are using (as per ICH Q3 on related substances). Thus, if the main peak has an area of 50,000 units only peaks above 2.5 (0.05%) are relevant.