Hello,
Our lab is currently adapting a previous working method (designed for the Turner TD-700) for the quantitative analysis of chlorophyll-a in water by fluorescence to be used with a new instrument (Cary Eclipse Fluorometer). Details in full about the method below. In testing our new method, we were able to produce a reliable standard curve up to 100 ug/L chl-a with a raw fluorescence of 781 (Attached Fig 1.)
In order to test this method with environmental samples, we prepared filters of increasing filtered volume from a single collected sample (e.g. 1 filter @100ml, 1 filter @250ml, 1 filter @400ml, etc). My thought was that we would see a proportional increase in fluorescence, based on filtered volume (i.e. the 400ml filter would have ~4x the fluorescence of the 100ml sample.) Instead, we saw what looked like quenching (or at least reduced fluorescence) around the 400ml mark (raw fluorescence ~500). Our 600ml sample had roughly the same fluorescence as the 400ml sample and our 800ml sample had significantly lower fluorescence than both of them etc. (Attached Fig 2.)
We do not see any similar effect in our standard curve and the fluorescence reading for these samples are well below where our standard curve tops out. Has anyone ever seen a similar phenomenon? Do you know what causes it or how to fix it?
Brief Method Description:
Water samples are filtered on GFF filters and stored with desiccant in a freezer. Samples are extracted in ethanol in a dark environment. Extracted samples are excited @434 and emission is measured at 673 nm.
Thank you.
My guess is that you may be seeing an inner filter effect, whereby the absorbance of excitation and/or emitted light by the sample reduces the fluorescence intensity. To avoid this problem, dilute each sample with solvent so that they all have the same absorbance at the peak absorbance wavelength, preferably less than 0.1. Then correct for the dilution after measuring the fluorescence.
This may be due to the distribution of algae size in the tested samples.
My guess is that you may be seeing an inner filter effect, whereby the absorbance of excitation and/or emitted light by the sample reduces the fluorescence intensity. To avoid this problem, dilute each sample with solvent so that they all have the same absorbance at the peak absorbance wavelength, preferably less than 0.1. Then correct for the dilution after measuring the fluorescence.
Agree with Adam B Shapiro - also are samples filtered in the field or back at the lab? Filtering 600 mL would take longer than 100 mL, and therfore subjected to more light. For example, on the graph you show of sample analysis, are there 2 points that were done in duplicate? If yes - those duplicates have values that are lower - are those lower values associated with samples that were filtered after 1st sample. This may show a trend of degradation with time of exposure.
- Badges
- Science method
- Similar topics
- Chemistry
- General Biochemistry
- Biochemical Techniques
- Analytical Chemistry