Why I've peak in gradient eluition?

[DR]

Do the extra peaks get larger if you allow a longer run time at initial conditions before injecting a blank?

[alemaggot]

Do the extra peaks get larger if you allow a longer run time at initial conditions before injecting a blank?

Yes DR! They increase there width.

[DR]

You may need to clean up your water. Search these forums for "Empore extraction disk" for details on how to do this. If you have a high pressure mixing system, use of a C18 guard column in between your "A" pump and mixing chamber may also help.

[alemaggot]

I've try this way already. I've used ultrapure water by sigma aldrich. The peak area was decrease, but it was yet high. I've tryed to use Empore extraction disk without better results.

[DR]

What wavelength are you running at?

Did you do the Empore trreatment on just the water, or on the C7-sulfonic acid solution?

In my experience, nearly all sulfonic acids contain more than a little junk. The lower the wavelength you use, the more of a problem this junk will be.

If you Empore treated the water, you may also need to Empore treat the A phase (prior to addition of any organic solvent) to get rid of salt associated junk.

Sulfonates, low wavelengths... not a good situation to be in.

If you have several bottles of C7-sulfonate to choose from, you could make a small amount of 1% w/w solution from each and do a UV scan of each (300nm to 200nm should do), then use the container showing the lowest cutoff. We have even done this with MeOH bottles for *really* low UV mobile phases...

Best of luck,
DR

[alemaggot]

I work at 254nm of wavelenght!

However DR the extra peaks problems are not give by sulfonic acid, but from a simple phospate buffer solution. When CH3CN enter in column, they elute!

Thank you

[HPLCaddict]

As DR already suggested, try to purify your complete mobile phase A via the extraction disc.

[HPLCaddict]

One more thing: Your gradient starts with 100% aqueous, right? Is your column meant to be used under purely aqueous conditions? This may not be related to your system peaks, but plain C18 columns should be used with at least ~5% organics in the eluent to avoid phase dewetting.

[alemaggot]

Sorry but I don't know what is "dewetting". Can you explain to me? Thanks!

However I don't remember exactly if my column can work with 100 % of buffer. I seem that I can use it with 100% of buffer not for total course, but for few time.

[tom jupille]

Sorry but I don't know what is "dewetting". Can you explain to me? Thanks!

It's at times like this that I really miss Uwe Neue. Look here: viewtopic.php?f=1&t=6797&hilit=dewetting

[alemaggot]

Goodmorning guys,

In this day I'm developing a new method. The first method written in this post now is obsolete.
However I must write here because I've new problem with extra peaks in my chromatogram

The white (only purified water) run is this:



The run condition are:

Injection volume: 20ul
Detector wavelenght: 254nm
Dwell: 2,5
pump (gradient without linear curve)

min A% B%
1.5 100 0
5 85 15
6 80 20

Mobile phase A: KH2PO4 20mM. pH 2,5 + 10 % MeOH
Mobile phase B: 100% ACN

Column: Kinetex C18 100mm * 4,6 mm * 2,6um

Can you help me to eliminate this extra peak?

thank you very much

[alemaggot]

Nobody can help me?

[HPLCaddict]

- How does the baseline of a blank run (no injection) look like?
- Do you allow enough time for reequillibration at the end of the gradient?
- As a side note: Do you really need a ternary eluent mixture (Buffer+MeOH+ACN)? Couldn't you do the trick with only one organic modifier?

[Andy Alpert]

The peak may be from impurities in reagent-grade phosphate that accumulate on the column during reequilibration and then elute during the gradient. These can be removed by stirring a solution of the phosphate salt with a mixed-bed ion exchange resin and then filtering out the resin, but it's easier just to buy an HPLC-grade phosphate salt (or HPLC-grade phosphoric acid and then add some base to adjust the pH).

[alemaggot]

- How does the baseline of a blank run (no injection) look like?
- Do you allow enough time for reequillibration at the end of the gradient?
- As a side note: Do you really need a ternary eluent mixture (Buffer+MeOH+ACN)? Couldn't you do the trick with only one organic modifier?

- I didn't had perform this run
- I perfmorm 5 minute reequlibration
- If I use only about 1% of ACN in mobile phase A I can have retention, but Dwetting problem be imminent!