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Author Topic: Fluorometric analysis  (Read 2809 times)

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Elly

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Fluorometric analysis
« on: February 09, 2010, 12:24:44 AM »

Hallo everybody,
I'm a biologist and I'm using spectrophluorofotometer for the analysis of neutral lipids.
To identify the optimal wavelenght for the quantitation of my samples, I've submitted my samples to an excitation scan (in literature I found that in different situations the emission wavelenght is almost the same...)
My instrument also has a function that allows the idientification of the optimal wavelenghts (emission and excitation).
The wavelenghts indicated by the instrument as "optimal" are not the same that I can graphycally identify observing the spectra..in some case they are very far from the ones that correspond to the effective maximum intensity.
Is this normal? Is there some explanation for this?
I hope that you'll help me!!

Elly

P.S. I'm sorry for my english...I hope that this message is understandable...
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Golden_4_Life

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Re: Fluorometric analysis
« Reply #1 on: February 09, 2010, 03:31:27 AM »

Welcome to the "real world" of Empiricism, Elly.
There is always some variance between what others have observed and what you observe in your own experiments; however, for the sake of reproducibility we have established that about at 5% to 10% variance is tolerable on a given measured parameter.
I have done numerous quantiations of genomic DNA using spectroflourometry and have used DNA binding dyes such as PicoGreen and SyberGreen.
The excitation wavelengths (wave-X and emission wavelengths (wave-M) do often differ because flourophores are excited at lower waves and emit at higher waves.  What you see in publications for wave-X and wave-M will vary somewhat from what you see in your own work; however, the relative difference ought to be similar.  This is normal, so just document your findings and carryon with the examination of your hypothesis.
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Elly

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Re: Fluorometric analysis
« Reply #2 on: February 09, 2010, 03:38:24 AM »

Thank you for the answer!
I absolute agree with you about the differences among my optimal wavelenghts and the ones reported in literature, but my problem is that the instrument gave me as optimal, two wavelenghts that are not the ones where the excitation peak and the emission peak (registered from the same instrument) reach their maximum values...
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Golden_4_Life

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Re: Fluorometric analysis
« Reply #3 on: February 10, 2010, 02:51:20 AM »

Based on your thread I would conjecture that either the product's operation manualhas a "mis-print" error or the filters that you are using are incorrectly installed.   Usually, there is a filter disc for the excitation and a filter disc for the emission signals installed on chamber that intersects that light source and sample cuvette.  Pls let us know whut you find out. 
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Golden4Life

Elly

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Re: Fluorometric analysis
« Reply #4 on: February 10, 2010, 04:58:12 AM »

My instrument doesn't have any filter, it has only a Xenon lamp, and we've a new lamp with few hours of life...I don't think it's a technical problem...I think that there should be some theoretical principle that I can't undestand...:-(
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Golden_4_Life

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Re: Fluorometric analysis
« Reply #5 on: February 10, 2010, 10:14:48 AM »

This is perplex but interesting problem--yet the answer to it is fairly straightforward to trouble-shoot.  A florometer is a very simple instrument consisting of a light source, a wavelength specific filter, a detector (measuring meter), and a LED display to show the data.  From what your blogging it appears that the problem is not the light source, not the filters, and not the LED display; therefore, this leaves only the Detector.
What florohore are you using in the experiments? And, what are its spectral characteristics (excitation & emission waves)?

Hopefully, the other chrom writers can add some explanation to your quandry.
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Golden4Life
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