Chromatography Forum

Hi everyone!I have a big problem while i am doing Paracetamol analysis.
My MP includes 3 lines
Line A 17.9 g Na2HPO4/1l H20

Line B 10.14 g NaH2PO4.2H2O/1l H2O

Line C 22,4ml n tetra butyl amoni hydroxit/1l CH3OH

My MP incluce 3 line with ratio A,B,C=37.5:37.5:25
My dissolvent is MP.
My batch injection is
Dissovent(x3)
Standard sample(x3)
Sample(x2)
Standard sample(x1)
The RT is alright as usual until the sample injection(the 2nd injection). The RT in the last Standard changes dramatically. But when I run it again,the RT return to normal and after the 2nd sample injection,the RT changes again.
I run my HPLC with below conditions
Inject volume 20 microlitre
Column temp 35 C.
Wave length 245 nm and flow rate 1.5ml/min.
I am really confused since this thing happen continuously.Please help me!

Did you adjust your phosphate buffer to a specific pH? I is possible that at 35°C and flow 1,5ml/min and if your MP bottels are not at the same temperature as your column, that you are running a temperature gradient. Are you running a gradient or is the ratio of your 3 lines all the same over the run time? Please check also if flow of every "line" is ok and constant. Is you back pressure constant? What is the particle size of your column packing material and what is the dimension of your column? I guess it is 4,6 x 250mm!

if you run your standards 10 times then the RT stays the same?
if you run the samples twice, your RT of the next standard changes?
and if you run the samples twice and the standards 3 times after that, from which injection does the RT comes back to normal?
to me it looks like your samples have something in them that is changing the column and that after a while it goes off and the column behaves normal again

can you say more on the nature of your product?
is it a syrup maybe? or something else?
what's in it specifically?

Did you adjust your phosphate buffer to a specific pH? I is possible that at 35°C and flow 1,5ml/min and if your MP bottels are not at the same temperature as your column, that you are running a temperature gradient. Are you running a gradient or is the ratio of your 3 lines all the same over the run time? Please check also if flow of every "line" is ok and constant. Is you back pressure constant? What is the particle size of your column packing material and what is the dimension of your column? I guess it is 4,6 x 250mm!

No, my buffers are adjusted pH but it is very stable except the recent injections. Moreover, I don't use gradient. My back preesure is normal but my baseline is drift and my peaks's shape become short, broaden a bit. About my column, your guess is totally correct. This morning I did the analysis once again with a new column but same properties. I also prepared new buffers.
By the way, I forgot telling that I use the buffer two days in line without filtering nor sonicating. Hope that I gave you enough information to figure out my trouble and give me a suitable solution.
I will tell you my result I did with all everything is new.

Your baseline is drifting? It shouldn't with an isocratic, isothermic method. However, I don't think that this is related to your specific retention time problem, as this phenomenon seems too reproducible. I'd share the opinion that something in your samples is temporarily changing the column until it is washed off.
You may try to add a washing step (high methanol content) at the end of each run and see if this helps.

By the way, with an isocratic method I would ALWAYS premix the eluent. This helps keeping away a whole lot of problems. Even if it may not cure your retention time shifting, I'd try to use a premixed mobile phase on a single channel. Might at least keep the baseline from drifting... By the way, if I understood correctly, you do NOT adjust the pH of those two buffers? Then, why not combine them into one - it's the same salt but only different concentrations of it, right? Some maths may lead you to a single buffer...

...ups, just saw that there are indeed different salts in those two buffers - forget my last comment on combining them into one buffer.
Nevertheless, try the premixing!