# UPLC calibration

<p>Hi!!</p><p></p><p>This is again about an SOP to perform calibration and maintenance of the UPLC (it is a Acquity UPLC). We are trying to write the SOP based on the qualification performed by Waters representative and based in that procedure, here comes the questions:</p><p></p><ol start="1"><li>For the Wavelength Accuracy Waters procedure collect spectra twice, one using a range of 200-400 nm and another using 250-400 nm. The caffeine max are at 205 and 273.  Why to collect between 250-400 nm if collecting the spectra between 200-400 nm will cover both max? What will be the reason/advantage in collecting at both ranges?
</li><li>When doing the Sample Manager Heater/Cooler Temperature Accuracy the sugest to select a temperature as low as 4 degrees without exceeding 20 degrees below T ambient. Question is: why is that limit of 20 degrees? What happens if we are, let's say, 21 degrees below ambient T?
</li></ol><p></p><p>Thanks a lot for your answers!!</p><p></p><p>Dorcas</p>

## Answers

• I believe Waters performs wavelength accurracy using two ranges because of how they generate their report.  The report sorts each range by peak height or max absorbance (I forget which), so that the maximum wavelength is at the top.  If you collect it as one range, it will only show the maximum of all wavelengths, not for both peaks found in caffeine.

The 20 degrees below ambient is the typical best result Peltier coolers can achieve.

• Hello:

I got some further clarification from the SQT team at Waters.

The Wavelength accuracy depends on finding the MAX ABSORBANCE for selected wavelengths. The way we designed the custom fields to sort for max requires isolating 273 range from the 205 range. We could just as well done 200-250 and 250-400. Separate ranges is the fundamental requirement.

Hope this is helpful,

Liz

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