I am trying to assess chlorophyll-a content from coral tissue using spectrophotometry (with SPECTROstar Nano, BMG). However, I always seem to obtain some negative values in my optical density raw data which then affects the chl-a calculations (Ritchie, 2008)
My extraction solvent is 95% etOH (laboratorios Roldan) and I use this as a blank (one blank well at the end of each line of wells filled with the samples)
I have tried different protocol settings in the spectrophotometer but still getting negative values:
Usual protocol settings:
Endpoint
BMG 96-well microplate
wavelengths: 632,649,665,696
Path length corr.ection off
well scan: spiral average (4mm diameter)
settling time 0.5s
No. of flashes: 30
Shaking before plate reading: orbital 500rpm for 5s
Variation:
well scan: orbital
settling time: 0.1
No. of flashes: 21
Thanks so much for your thoughts!!
The NanoStar is an excellent plate reader, but it probably has 0.5 - 1 % stray light in its PDA system, depending on wavelength, which can cause photometric errors. Depending on the source of chlorophylls, there may also be interference from chl b, phycobillins, and carotenoids (latter only at blue and green wavelengths). Another possibility is loss of magnesium from chlorophyll a due to slight acidity, yielding pheophytin with different spectral properties. This can be prevented by adding a small amount of NaCO3 to your Ethanol extraction solvent. You may have better luck with fluorescence detection due to it's higher specificity (Ex 410 nm, EM 675nm).
Macarena Blanco Pimentel It may be due to the fact that some intensity is spilling over to the blank wells due to which the test samples are showing negative results. Generally, negative readings indicate a poor blank measurement.
I'm not familiar with using wells for this type of application. However, by analogy with using conventional cells in a spectrophotometer, it sounds like mismatching and or contamination.
What happens if you fill the wells of a couple of plates with solvent and the wells of a couple more plates with the same chlorophyll extract?
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