Peak investigation

Prince

<p><span>Hello All,</span></p><p></p><p><span>Recently we have found peak next to main band at about 0.5% for two different batch at different occassion.</span></p><p></p><p><span>We didn't have this peaks on HPLC.</span></p><p></p><p><span>Is that because UPLC is more sharp and giving good resolution or is that because of contamination or degradation?</span></p><p></p><p><span>Can you please educate as how UPLC does needle wash and sample syringe wash?</span></p><p></p><p><span>Does it do washes between the sample injection? What does it use for wash? Sample itself or mobile wash or strong wash or weak wash?</span></p><p></p><p><span>We don't filter the mobile phase on UPLC and use mobile phase older than a week. Will that make any difference to the run?</span></p><p></p><p><span>your answer will be of great help.</span></p><p></p><p><span>Many Thanks in advance.</span></p>

lizh

Prince:

You are suspicious of carryover, " there's no smoke without fire"...so what happens on a blank injection?

If you have not already done so, try two types of blanks.

(1) The first is to run the gradient without the sample manager making an injection. In Empower , this is "condition column". Just run the gradient without the sample manager dipping the needle, this will tell us if the system/solvents/column outside of the sample is contaminated.

(2)The second type is to run a blank injection, where the injection sequence occurs, simply program a value of "0" in the sample list. This will tell us if the SM adds to the problem or can be eliminated.

Please send the chromatograms and full details on your method and please include the gradient table if there is one and the conditions.

Best regards,

Liz

In answer to your question the washes serve to wash the loop and SM fluidics and the inner and outer surface of the needle. The wash chemistry is important and different considerations are important depending on the injection mode. For example in Full Loop and PLNO mode the weak needlewash is not coinjected. It is coinjected for PLPA mode. In the case of the strong wash, which is never injected, its role is to solvate the sample components to remove them so they do not casue carryover and ensure that they are removed from the SM surfaces and also to remove the weak wash, until replaced with fresh. Therefore, Strong wsah should be very "strong". Without details on the method I cannot make any sensible recommendations and if you have details on the class of your components (polar, non-polar, hyrophobic etc) I cannot suggest what would be strong. For example acid can be added to the strong wash, if the mobile phase conditions are acid this can be helpful, but would not be suitable for a basic mobile phase.

markodjokic

I had the same problem months ago. It is very important what you use as weak and strong needle wash. Weak wash must contain ratio of organic compaund as your

mobile phase without salt. If your mobile phase is for example 60 % buffer and 40 % methanol, your weak needle wash must be 60 methanol and 40 % water (same

if mobile phase contains ACN, weak wash is 60 % water and 40 % ACN). Strong needle wash must contain high precentage of organic compound. I use 95 % ACN

(or methanol). It is very important to change weak and strong needle wash every time depending on the mobile phase.