Public Lab Research note

Survey of different kinds of fluorescence spectrometry, optimizing intensity

by warren | August 12, 2014 13:58 12 Aug 13:58 | #11039 | #11039


Learning more about different types of fluorescence spectroscopy -- a common type for oil analysis being (Synchronous) Scanning Fluorescence Spectroscopy (a real mouthful) or SFS, which involves illuminating the sample with monochromatic light at ~5-20 nanometers shorter wavelength than where you're reading it. The light source and sensor both move up (or down?) the wavelength scale at a fixed distance, hence "synchronous scanning".

I believe the type we're doing is called LIFS, or laser induced fluorescence spectrometry. I'll be looking at LIFS literature more to see what we can learn despite the intense jargon!

There's also three dimensional excitation- emission matrix (EEM) spectroscopy, apparently also a form of fluorescence spectroscopy. All three, according to Elfrida Carstea in "Fluorescence Spectroscopy as a Potential Tool for In-Situ Monitoring of Dissolved Organic Matter in Surface Water Systems", can be used "to estimate water pollution and to probe the composition of DOM in watersheds." DOM apparently means Dissolved Organic Matter. Interesting! We're looking for oils, but good to know -- both as a possible future alternative use and as a possible false positive if there is organic matter in our samples.

Data types

Each type of spectroscopy outputs a different sort of graph; the type we're using (laser induced fluorescence spectroscopy) has a characteristic spike where the laser light is read, because it's so bright. You can see that in a) in the below diagram, from the Carstea paper:


Synchronous scanning fluorescence, in b), lacks the spike because the light is never emitted at the same wavelength as light's being measured -- it's always just 5-20 nm out of "view".

Total synchronous fluorescence is 2d, I believe because it measures an entire spectrum for each narrow wavelength of light that's used to illuminate the sample. So you light it up with 400, 405, 410nm, and take a whole spectrum each time. Lots of data! We haven't used monochromators (which are like a reverse spectrometer and can generate any color of light) so we haven't tried this. It sounds like a lot of work, but building a monochromator wouldn't be hard.

Fluorescence intensity

Sometimes it's tough to know what's "common knowledge" among practitioners -- Carstea mentions that:

The fluorescence response is highly affected by solution temperature, composition, concentration, pH and salinity.

Whoa -- we've never tried cooling or heating samples -- we should definitely try it. And adding an acid. And Carstea indicates later that it's low temperature that increases intensity.


Carstea, Elfrida M. "Fluorescence Spectroscopy as a Potential Tool for in-situ Monitoring of Dissolved Organic Matter in Surface Water Systems." Water Pollution 1 (2012).,22


Page 50 also discusses filtering effects -- where the color of the sample itself (yellow, brown) can filter out some wavelengths of fluorescence. This can be seen in this post by @eustatic:


It suggests that "the fluorescence intensity must be multiplied by a correction factor (Valeur, 2001)."

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p.51 has a discussion of pH and salinity on spectra: "intensities increase with higher pH until 10, as observed by Reynolds and Ahmad (1995) at raw sewage samples" -- but it's unclear how this relates to oil samples, since oil does not have pH. Maybe this'd be an issue if we used alcohol to try to dissolve samples.

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Aha! Line 7 of p54 says:

The Raman scatter line can be used to check for instrument stability and to quantify the degree of contamination from a water sample by using the normalised fluorescence intensity to the Raman peak.

This relates to the conversation @ygzstc and @bsugar are having about how to normalize spectra, in this thread:!topic/plots-spectrometry/5hM3w7FQ72w

And my notes in this research note about normalizing spectra:

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So the Carstea paper is great for one particular reason - we've suspected that organic material could fluoresce and potentially interfere with our oil testing, and page 54 includes an overview of the organic material -- of which primarily proteins and humic substances fluoresce. Fluorescence from proteins is apparently an indicator of bacterial growth, while humic substances result from the breakdown of plant matter. Interestingly, Carstea cites research indicating that one fluorescence peak correlates with total organic carbon!

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p56 mentions "polycyclic aromatic hydrocarbons (PAHs), pesticides, environmental hormones" as contaminants that can be detected with fluorescence spectroscopy in water. Great quote on the cost-effectiveness and speed of this technique:

The standard techniques for pesticides and PAHs detection are gas and liquid chromatography, which require tedious extraction or separations procedures and expensive equipments. Fluorescence spectroscopy is a rapid and cost effective alternative, since PAHs and many pesticides are naturally fluorescent (Jiji et al., 1999). When dealing with more components, the use of SFS or EEM techniques is recommended.

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Interesting -- specific PAHs are (as we've seen in other literature) out of the 400-1000nm range of our device:

monoaromatic compounds (benzene, toluene, xylene and phenols) emit fluorescence between 250 – 290 nm. Two aromatic ring compounds, like naphthalene, show a fluorescence peak at 310 – 330 nm, phenanthrenes (three aromatic rings) between 345 – 355 nm and so on (Pharr et al., 1992; Abbas et al., 2006). Pharr et al. (1992) has shown that, only by using standard fluorescence emission spectra, distinguishing between 2 brands of gasoline would be impossible.

However, Cartea cites Patra and Mishra (2002a), Cristescu et al. (2009) and Carstea et al. (2009a) to mention that:

heavy oil, Diesel and engine oil, show fluorescence maxima in the longer emission wavelength region (420 – 550 nm) while lighter oils, petrol and kerosene, present peaks mainly in an intermediate wavelength region (310 – 400 nm).

Super duper! Time to read those articles!

Re: aging of samples, also some great leads on p57:

Deepa et al. (2006) studied the fluorescence signal of transformer oil during its aging process, which was thermally induced at 100 o C for 31 days. They observed a sudden dramatic decrease in fluorescence intensity after only 17 days, for raw oil, followed by a slight increase until day 31. Also, the excitation and emission maxima increased starting with the 20 th day. Deepa et al. (2006) explained that when transformer oil was degraded, its acidity increased, resulting an increase in C-O band and C=C double bands.

Ooh, finally, laser-induced fluorescence spectroscopy (LIFS) -- our technique -- is cited:

Some authors (Burel-Deschamps et al., 2006; Pascu et al., 2001) used absorption spectroscopy and laser induced fluorescence for pesticide monitoring in water. Organochlorurate pesticides in water, crude oil and oil components in water and soil with detection limits of 10 −1 –10 −2 ppm were obtained.

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OK, links to some citations. I'm visiting an MIT building now so it's hard to tell which are open access; i'll try to check on this later:

Jiji et al 1999:,22 (not sure about access, but got a copy)

Cristescu, L., Pavelescu, G., Carstea, E.M. & Savastru, D. (2009). Evaluation of petroleum contaminants in soil by fluorescence spectroscopy. Environmental Engineering and Management Journal, Vol. 8, No. 5, pp. 1269-1273, ISSN 1573-2959. (closed, can't get it)

Carstea, Elfrida M., et al. "Continuous fluorescence assessment of organic matter variability on the Bournbrook River, Birmingham, UK." Hydrological processes 23.13 (2009): 1937-1946. (think it's closed, but got a copy)

Deepa, S., Sarathi, R. & Mishra, A.K. (2006). Synchronous fluorescence and excitation emission characteristics of transformer oil ageing. Talanta, Vol. 70, No. 4, pp. 811-817, ISSN:0039-9140.,22 (closed, couldn't get it)

Re: pesticides:

Burel-Deschamps, Laure, et al. "Laser–Induced Fluorescence Detection of Carbamates Traces in Water." Journal of fluorescence 16.2 (2006): 177-183.,22, (got a copy)

Pascu, Mihail-Lucian, et al. "Pesticide monitoring in water using laser-induced fluorescence and absorption techniques." ROMOPTP'94: 4th Conference on Optics. International Society for Optics and Photonics, 1995.,22 (closed)

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Is this why the "microdrop" style is so popular? could we reduce the filtering effect by totally saturating the sample with laser light?

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Maybe -- the paper describes different kinds of filtering, one from when the laser enters the liquid, one from when the fluorescence exits the liquid. Seems like we can minimize them with geometry (whee!) by scanning close to the edge of the sample jar, and keeping the laser close to the leading surface of the jar.

@natalie -- this is definitely a vote for smaller sample containers, though. If we could get 1 cubic centimeter or less, that'd be great, and for shipping costs too!

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Do any of the samples I have sent thus far match this criteria?

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if we're locking in alignment for the laser, and settling on mineral oil for a solvent, I don't actually think a container-less cuvette is a crazy idea. I linked to a few expired patents on the issue in @gaudi's nanodrop research note.

This one is just a cone with a hole in the bottom that holds a droplet:


this one makes the perpendicular orientation difficult, but its dead simple. just a hole in a plate that is matched to the surface tension of the liquid.


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Well, ideally, much smaller would be great -- i think the ones you've found are mostly 15ml?, but the reality may be that nail polish bottles just aren't sold that small. I've seen 5ml and 3ml on occasion, but not many square sided ones.

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Very true, re: alignment -- is there any way to do a tiny tiny amount like a nanodrop while keeping the (possibly toxic) sample material sealed somehow? "micro cuvettes" or "ultra micro cuvetttes" narrow down to a slide-like width, but all square cuvettes seem to leak :-(

Here's an interesting one -- a round-topped, plastic, UV-friendly cuvette at $84 per hundred pack, $372 per 500: or here on Amazon:

We'd need to buy really good sealing caps, of course.


It'd be too bad to use a specialty material, but we're already getting really particular about the jars, so they're not universally available either.

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