I'm developing a method for a related substance analysis. I'm using a gradient that goes from 95%water, 5% MeoH to 100% MeOH in 10 minutes using a C18 4.6x150 3.5 microns column. I'm getting a real good separation of my analytes, however, a relatively large peak at approx. 14 min. shows up in every run. At first I thought it could be dirty equipment or an unaccounted impurity, but after cleaning the equipment, changing the water and methanol bottles the peak remained. Also I injected water, methanol, and even air and the peak shows up everytime. I tried four different columns and three different equipments (Waters and HP) an still have the peak. I even changed the gradient to run from 5%ACN and 95% Water for Injection to 100% ACN (injecting water)and still have the peak at the same rention time.
I made the gradient to run instead of in 10 minutes in 60 minutes, and still the peak shows up (now at 45 minutes).
Do you have any suggestion how I could get rid of this peak?
Thanks in advance for your advice.

Are you sure it is not the "gradient peak"? When solvent composition is suddenly changed at the end of the gradient, one typically sees a peak probably due to change in refractive index! Just a thought!

It may be the water (or other additives in it if you use any). You can demonstrate this by running the same gradient with different equilibration times in the starting mobile phase. If the peak increases with increasing equilibration time in the starting mobile phase, you are accumulating junk on the column.

Thanks to both of you for your responce!

I continue with the same problem. I think is has to do something with the gradient since even when I'm not injecting anything I see the peak.
Also I have tried the following (on top of what I mentioned above):
I made the gradient to run instead of in 10 minutes in 60 minutes, and still the peak shows up (now at 45 minutes).
I also used a PDA detector (usually I use at UV detector at 305 nm) an got an UV of the peak and shows absorption from about 200 to 320 nm with max peak absorption at approx. 250 and 290 nm.
I have also run the gradient on columns that has never seen my analytes (straight out of the box) change the water and methanol bottles and the peak is still present.

Thanks again for your advice

If you haven't already done it, you might try Uwe's suggestion from June 1. Set up your system in your normal manner and run a blank run. Allow it to equilibrate the "normal" amount of time and run a second blank then at least double the "normal" equilibration time between runs and run a third blank. If there is something from the water builiding up on the column that doesn't elute until you get the organic composition high enough, you should see an increase in the peak size as you increase the equilibration time. Don't know what your water source is (purchased, distilled on site, Milli-Q, etc) but you may have something that is consistently present in your water source. Are you using straight water or are there any additives (buffers for example)?

I'm using water only with no additive (no buffer), and I have tried changing the water to Water for Injection (we are an injectables company) and still saw the peak. The water I normally used is MilliQ.
I'll try what Uwe suggested.
Thank you

Do you filter your water? I assume you do so check a run without filtering.
We have had tremendeous problems with some brands of Nylon filters,They leaked out an impuriry with molecular weight 226 and multiples thereof.Now we filter our solvents using aluminum oxide based filters and problem has dissappeared.

Good luck!

I once had a similar problem due to in-house distilled, deionized water. The problem only occurred in the early summer and was due to fungal metabolites in the water supply. Try bottled HPLC grade water in place of MilliQ.

I agree with the two guesses, contaminated eluent or the refractive problem. 1.I think you trap and enrich these contaminant on top of your column. 2.What is the end phase of your gradient? Do you hold 100% organic or do you step down to your starting conditions immediatly? There is a good chance that a sudden change in the eluent composition lead to "ghost peaks".
By the way, it's not a good idea to run such radical gradients, because they are always a source of trouble.

Try purchased water and a different lot of organic solvent. Impurities in the mobile phase will build up on the column durring equilibration and then elute durring the run. To check if this is the case, vary the equilibration or post run time and see if the area of the unknown peak changes. If it does, the mobile phase is the problem.

I had a very similar problem a few years back and the source of the problem was a phthalate being dissolved in mobile phase B (the organic) and column loading onto the column during the equilibrium stage of the gradient. Changing between ACN and MeOH did not help as the phalate was soluble in both. The experiment where you increase the time of equilibration also give a larger peak with time but is was not related to the water purity. I eventually solved the problem by Running the organic through the system or part of the system (I can't remember) then concentrated the organic. Then using this concentrated organic (as mobile phase) saw the peak had grown. I then further concentrated the organic and detected the phalate by GC-MS. A further expt of refluxing the organic with the mobile phase tubing showed the presence of phthalate. This is a long shot possibility.

Very good point by the last anonymous!

I too have seen this problem more times than I would like to admit. Every time there is a different problem source. Some of the sources I have found are;
Contaminant dissolved in acetonitrile and in methanol, depositing on the column with high water concentration mobile phase, washing off when the organic strength of the eluent permitted.
Contaminant in water depositing on the column, washing off when the organic strength of the eluent permitted.
Contaminant leaching out of the organic solvent transmission tubing, valves and seals depositing on the column with high water concentration mobile phase, washing off when the organic strength of the eluent permitted.
Bacterial growth in the HPLC water supply bottle and lines depositing on the column with high water concentration mobile phase, washing off when the organic strength of the eluent permitted.
The worst was when my external water supply system failed. I was using a Milli-Q unit with a light bulb system indicating the resitance. When operating, the bulb was lit indicating better than 18.2 megaohms. I was having problems with both HPLC and ion chromatography. The Dionex service and technical people suggested my water as the source of the problems. I told them they were nuts! They came in with some of their own water, used it on my I.C. and all problems dissapeared. Having to eat one's own words does not make a very satisfying snack. The Milli-Q detector was not working. When turned on, the 18.2 light came on, even if salt water was running through it. Since the system indicated all was well, none of the packs had been changed for so long, the system was full of bacteria. That is how my HPLC got contaminated. I replaced the old Milli-Q with a new Elga that had a TOC and electrochemical detection system. The ghost HPLC peak which I had named FBP on all of my previous chromatograms dissapeared. FBP was my attempt at viewing this peak with some humour. FBP stood for Free Bonus Peak.