[nexisrocks]

Suppose one wishes to use a simpler filter fluorescence spectrometer to determine quinine.
The light source will use a mercury lamp coated with a phosphor with an emission band from
340 to 450 nm and the quinine emission will be measured at 450 nm.  Which of your filters
would you use for the excitation side of the cell and which for the emission side of the cell?
     
     I'm not really sure how to answer this question... would you just use a bandpass filter on the   
     excitation side of the spectra, to only allow the wavelengths you want to go and excite the sample? 
     But then, wouldn't the monochromator select the wavelength anyways?  So why would you use
     one?  Same with the emission side...


Using the wavelength of maximum absorbance, what is the lowest concentration of
anthracene one could determine, assuming that the minimum absorbance is 0.0044?

     The concentration of a solution is reflected in the intensity of the peak measured in an
     absorbance spectrum.  The stock solution of anthracene was diluted to about 14μg/mL, and
     the absorbance spectrum was taken.  The maximum wavelength here was 249 nm, with an
     intensity of 2.51662.

     The ratio of minimum absorbance to absorbance at the maximum wavelength must be
     calculated, as the intensity is directly proportional to concentration.  Thus, knowing how
     the two absorbances relate to each other, one can calculate the lowest concentration.

                         Ratio = 2.51662/0.0044
                                     = 571.959

                         Lowest Concentration = (14μg/mL)/Ratio
                                                                       = (14μg/mL)/571.959
                                                                       = 0.02448μg/mL
                                                                                 OR 24.48ng/mL


This question was given in the lab, however I've never seen a question like it before.  All I had to work with was the absorption spectrum of anthracene and the data given in the question...


Also, we ran several transmittance spectra on the anthracene sample, and held a different filter in front each time.  Afterwards, we had to determine which type of filter it was that each spectrum belongs to.  The types of filters are: neutral density, cutoff, bandpass and interference.  Below are my graphs, and I've attempted to designate a type of filter to each of them, but I have no idea whether I did them correctly or not - especially the bandpass ones.  I'm not sure what else they could be, but there are two peaks, so...  (I put what I thought were the filter types in the title).

#1:


#2:


#3:


#4:


#5:


#6:


#7:


Thanks!

[Rabn]

I would base the filter selection on whether or not the wavelength of max absorbance is present in the spectrum...i.e. if anthracene's max absorbance occurs at 249nm I only see one spectrum with data at 249nm; filter 3.

[nexisrocks]

I'm not sure I understand... what difference does that make?

[Arkcon]

OK, to sum up, you're doing fluorescence, analate is excited at one wavelength, emits at another, shorter incorrect, should be longer (closer to the red), wavelength.  You don't have gratings or prisms with slits, you're going to use phosphors for emission, and colored bits of glass to block out extraneous wavelengths, letting the detector "see" only the emitted fluorescence.  Can you select the correct ones for your application.

[nexisrocks]

So would you just want a bandpass filter on each side, to only allow the wanted wavelengths through?  Or maybe a bandpass on the excitation side and an interference filter on the emission side?

Because the bandpass lets a larger amount of light through than an interference filter, so you have more wavelengths of light going to excite the sample, but a smaller wavelength of light making it to the detector?

[Arkcon]

You question is, where do you want a filter that passes more wavelengths -- think about how fluorescence works to see which type of filter goes where.

[nexisrocks]

Well, the phosphor emits at 340 nm to 450 nm; I'm assuming we use that because somewhere in those wavelengths is the best wavelength to excite quinine, so would you just want an interference filter there to pass only the maximum wavelength?  Or would you want to pass all of the wavelengths there (in which case you would use a bandpass filter)?

It also says that quinine emits at 450 nm - a very narrow band - so would you want an interference filter for the emission to get rid of any stray light which is coming out around the quinine emission band?

[Arkcon]

It also says that quinine emits at 450 nm - a very narrow band - so would you want an interference filter for the emission to get rid of any stray light which is coming out around the quinine emission band?

See, that's the bit I wanted you to figure out for yourself, the phenomena of fluorescence itself is a very narrow filter, a rather tight wavelength is emitted, you need a less rigorous filter after the sample, but before the detector, just to remove the scatter.

FWIW, it's pretty exciting that you're learning this stuff.  Most recently, I've used spectrophotometers, with high quality gratings and slits for both excitation and emission.

But my first job was working for a company trying to build a laser fluorescence scanner.  Gratings and prisms would have been too expensive, and take too much space inside the instrument, and pose other engineering problems.  So it was all filters, ND filters for excitation attenuation, bandpass for wavelength selection, etc.  Sorry, it was long ago, and I don't recall the specifics.

[ARGOS++]

Dear Nexisrocks;

Slowly you are moving in right direction.

But it will be easier if you take a view on how such Fluoro-/Fluori-meters are different to normal Spectrometers on: "Excitation Spectrum”.

Also the picture in the Attachment will be of a big help, because it describes your situation very precise, except that you have to shift the wavelength scale that the maximum of the “Emission” will come to the 450nm.

A help for what the job of both filters has to be you can find also on: "Filter Fluorometer”.

With all these helps you should end in only one, maybe two Filter combinations.
You should also remember that the “Emission” has most a ‘much’ lower Quantum Yield.

[Edited:]
(Dear Arkcon:  Closer to Red (= bathochrome) means: longer wavelengths!)


Good Luck!
                    ARGOS⁺⁺