Peaks in the Blank

[LC_Trouble]

Hi Everyone,

I am running the reverse phase HPLC,for the past few days i am encountering a problem with the peaks in my Blank run.My Blank is Methanol,I usually purge the HPLC completly for an hour before starting the blank run.Mobile phase is Bottle A(1% formic acid) and Bottle B(99% MeoH +1% formic acid) i cannot change either mobile pase or use water for cleaning,because of my sample.Purging is done by first flushing 1% of bottle A and 99% bottle B for 30 mins and then the vice versa for 30mins with a flow rate of 3ml/min.Can you please give suggestions on how to avoid peaks in the blank.

Thanks,

[philippem]

Hello LC_trouble,

Most probably you are concentrating impurities of the 1% aqueous formic acid onto your column.
You flush first with 1% [1%formic acid], and 99 % [99% MeOH+ 1% formic acid],so all impurities are flushed off the column.
Then you change your composition 99 % [1%formic acid] and 1%[99% MeOH+ 1% formic acid]. so now all impourites of the auqeous solution are concentrated onto your column. Its Reversed phase chromatography

You shouldflush the column only with the strongest solvent ( organic solvent=MeOH with 1 % formic acid) to get rid of the impurities of previous injections

btw Do you use a gradient for your analysis ??

hope this helps

Philippe

[JM]

Pure methanol injection will give you peak near your void volumn ( 1to3 min). If you are geting peak after this time that indicate carry over from sample ( if your blank peak at your analyte RT) or you have column washing issues.

JM

[LC_Trouble]

Hi Pillipe and JM,

Thanks for the reply,So should i use only 99%MeoH for 30 mins for purging and no 1 % of Formic acid.I shall try this and let you know about the result.I am going to use a new methanol and try that also.What do you think about changing the flow rate for purging ,intially i used about 3ml/min and shall i increase it.

Thanks.

[LC_Trouble]

Dear Phillipe,


Yes i am using a gradient anlysis ,but i cannot change that because it will affect my compounds that come first .The Concentration of my methanol at 20 mins is 42% when i use the blank run and this is where i get the peak.if you wish i can send the grap of my blank.

Thanks

[gbalock]

LC,
Can you set up your HPLC fora zero volume injection? Sometimes the peak really isn't a peak. Perhaps it is a gradient peak. Also, some systems have a problem when trying to mix methanol water around the 50% range.

[Bruce Hamilton]

I assume this is a new problem, the analysis worked fine before, the peaks are at the same retention time, are sharp, and are not carry over from sample injections, or new types of samples.

I also assume that when you say " purge at 3 mls/min ", you are flushing the column, and not just purging the degasser. 3 ml/min sounds rather high for flushing modern instruments with water methanol mixes, given the relatively high viscosity changes that occur.

What happens with a sequence of just multiple blanks - do the peaks sizes get smaller with each injection, or stay the same size?.

If you are running a gradient, what happens if you extend the final time by 15 minutes and then run a blank. Do the peaks change in size or retention?.

If the assumptions are OK, the most likely causes are:-

One of your reagents is contaminated - assuming you are using the same grades and sources as before, and haven't opened a new bottle of formic acid, I'd first look at the water, then the methanol, then the formic.

You can test each major component out by running 99:1 of A:B for 15, 30, 60 mins, and performing two blank runs after each time period. Repeat the process with 1:99 of A:B for 15, 30, 60 mins. If the longer periods show bigger peaks, you've found your source.

You can check the formic by halving the concentration in both A and B, and seeing if the peaks get smaller.

Peaks in the second blank should significantly decrease in size. If not, you may have contamination of the blank, the column, or the injector. You may be able to evaluate those by changing the size of your blank injection volume, and using a different source of blank solvent.

Another possibility ( of many ) is that you samples are dropping out of solution in the injector or guard, so ensure your samples are dissolved in the initial mobile phase.

I'd flush the column at the normal flow rate, and I slightly disagree with Philippe's comment about flushing the column with only the strongest solvent - because that assumes all the impurities behave similar to the analyte.

If you start seeing peaks where they shouldn't be, then either change the gradient, or flush with solvents that remove them. In some cases, a high % water flush may be needed to clean out unwanted material that deposits on the column or injector. If it's a regular problem, you should modify the method.

Please keep having fun,

Bruce Hamilton

[LC_Trouble]

Thanks for the reply,I am not using water in my mobile pase.I cannot change the mobile phase also,I am gonna try both of your suggestions for purging,and lets see which works.
Bbalock: I dont really understand what do you mean by zero volume injection?
Thanks.

[LC_Trouble]

Hi Bruce,

When i run the blank for multiple times ,the peaks are reducing and each morning when i run te blank the retention time of the peak is diffrent!..I want to fix this somehow,.I cannot run blank for multiple times every day before starting the analysis its a waste of time.

Once agains Thanks!

[Bruce Hamilton]

When i run the blank for multiple times ,the peaks are reducing and each morning when i run te blank the retention time of the peak is diffrent!..I want to fix this somehow,.I cannot run blank for multiple times every day before starting the analysis its a waste of time.

If you're not using water in your mobile phase, what is the 1% formic acid dissolved into, besides methanol?.

Obviously, I'm not suggesting multiple blank runs for the rest of your life, I'm suggesting a set of experiments that would help you locate the source of your problems. However, it's probably unnecessary, as you tell us above the peaks are variable in retention time and reducing in size.

That suggests there is junk in injector or column, and you are not flushing it out. I'd recommend changing your method to ensure all junk stays in solution, and elutes off the column in the same run as it's injected on. The easiest way may be to ensure that your samples are in the initial mobile phase, the gradient is modified, and the column is at a slightly elevated temperature compared to samples.

Please keep having fun,

Bruce Hamilton

[LC_Trouble]

yes i use HPLC grade water for 1% formic acid,Thanks for your suggestion i will fix it!