Chromatography Forum

My colleague and I test erythromycin according to EP5.4. Now we meet a very harsh problem: water shows an obvious peak 15 mins after injection. It would be reasonable to see the peak within 5mins after injection. but so long a time is very confusing. What is the way the problem?
EP testing method is below
Column:
— size: l = 0.25 m, Ø = 4.6 mm,
— stationary phase: styrene-divinylbenzene copolymer R(8 μm) with a pore size of 100 nm,
— temperature: 70 °C using a water-bath for the column and at least one-third of the tubing preceding the column.
Mobile phase: to 50ml of a 35 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 9.0 ± 0.05 with dilute phosphoric acid R, add 400 ml of water R, 165 ml of 2-methyl-2-propanol R and 30 ml of acetonitrile R, and dilute to 1000 ml with water R.
Flow rate: 2.0ml/min.
Detection: spectrophotometer at 215 nm.

What´s the problem? This is SEC?

— temperature: 70 °C using a water-bath for the column and at least one-third of the tubing preceding the column.

Why do we use waterbath? What if there is leakage at column-tubing connection? We cannot detect small leakage in the water. I feel it is better to use a column oven...

Thanks HW Mueller and Syx. In fact we do use a column box as Syx said. The question is why there is a water peak with the retention about 15mins. By the way what does "SEC" mean?

By the way what does "SEC" mean?

SEC: Size exclusion chromatography...

Are you sure your peak is water?

Water shouldn't absorb - at least in "visible" amounts - over 165 nm and under 1000 nm... If you were working with RID, maybe could be an issue...

You are probably getting some very little organic molecule - such as an organic acid.

Are you sure your peak is water?

yes! So it is strange

SDB has much more rentention capacity than C-18, it will retain very polar compounds, and sometimes, it's hard to elute them. Could be the water peak some OH-containg small molecules as Refael suggested? It make no sense to be water.

If this is what it seems to be, namely SEC, than everything is ~normal. See that chain on RI about water peaks.

The problem is that our friend is using UV at 215 nm... That's why I am finding very hard to believe it is water!

If it was a RID, I wouldn't be that surprised, although I never experienced that myself...

Why do we think it is a water peak? Water would not be retained on this column, and a peak at 15 minutes under your conditions (2 mL/min) is definitely retained.

You have a peak at 15 minutes. Is it negative? Or does it happen when you inject water?

Hello

I must agree with the previous comments; I don't think it is water.

You do not mention anything about your routines for injector or needle wash between injections... Could it be that you have some kind of memory effects from the injection system?

/Peps

Is it SEC or not? Refractive index rears its head in UV also (though in my experience this seems to be mostly a positive-negative affair, regarding water)
Uwe is thinking about a disequilibrium peak (none SEC)?

As an off-the-wall possibility, what about dissolved air? O2 shouldn't absorb at that long a wavelength, but an RI disturbance is possible. Should be easy to check by degassing both mobile phase and diluent.

Or by willfully injecting air, though sometimes this behaves differently (spike or immediate baseline rise due to bubble, if you inject so much that it doesn´t dissolve in the mobile phase).