Why I've peak in gradient eluition?

[alemaggot]

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).

You think that the problem is only this?

[alemaggot]

I guys,

I've buy and try a new salt and ortophosporic acid HPLC grade. The Peak area is less. But is always present! The area value is about 100000 mAU/s. It's very big!

Have you got other test that I can try? I'm hopeless

Thank you

[alemaggot]

Nobody can help me?

[skunked_once]

I will offer some suggestions based on some of the other replies.

1: It is not a good idea to use 100% aqueous mobile phase on a C-18 column unless the column is designed to allow this. Can you tell us what column you
use? Instead of starting at 100% aqueous buffer, can you start at 5% acetonitrile/95% buffer?

2: First clean the column by flushing with 100% acetonitrile after you have flushed out all buffer salts with water.
Now do two test runs with your gradient using only water and acetonitrile and run the gradient with no injection.
If the peak appears, either your water or you HPLC system is contaminated.
If this blank is clean, inject pure water and run a gradient with water and acetonitrile.
If this blank is clean, add only one of the buffer components to your mobile phase and repeat the gradient.
Test each of the buffer components in this manner to find the one causing the peak.

Good luck and keep us posted on your results!

[Hollow]

guessing you run this at 1ml/min?
this could also be some signal due to changes in refractiv index and/or in the pressure changes. At 10% ACN/90%H2O there is a viscostity maximum.

Beside this, accumulation of impurities from
a) salt
b) pH-Probe (if used)
c) Water
d) tubings
could be possible, but your ACN concentration is not yet really high and the chrom doesn't show really peaks.

Could you collect a trace of the system pressure? See if it correlates with the UV-Signal.