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Difference between spectrocopy

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Difference between spectrocopy

Postby carbonp » Mon Nov 16, 2009 10:34 pm

Hi,
I am confused between Ultraviolet-visible spectroscopy and fluorescence spectroscopy?

Fluorescence is produced when compound absorbs UV light and re-emits visible light. Thus does it mean both are same technique because UV light produces fluorescence. If different what are the specifc difference, i mean in methodology, instrument, results. My topic is on UV-VISIBLE SPECTROSCOPY IN DRUG-PROTEIN BINDING.
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Postby JackBean » Tue Nov 17, 2009 5:28 am

In my opinion, in UV-VIS you measure, what is NOT absorbed and passes through, while in fluorescence you measure 'light' emited by the substance after excitation ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby JackBean » Tue Nov 17, 2009 5:30 am

The difference in instruments is probably such, that in UV-VIS you usually (at least with nowadays spectrophotometers) scan the whole spectrum, whereas in fluorescence you use only some wavelengths (one range for excitation and second for emited light, which should be quite specific;)
http://www.biolib.cz/en/main/

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Re: Difference between spectrocopy

Postby jonmoulton » Tue Nov 17, 2009 6:38 pm

In a UV-Vis spec (I have one on the bench in my lab) you pass a beam through a sample and straight into a detector. In this case you are measuring the light that the sample transmits (or absorbs, it's the same process but a different way of reporting the quantity). A motorized diffraction grating rotates in the light path, determining what wavelength of light is passing through the sample.

With a spectrofluorometer (I have on in the other corner of my lab) you can excite a sample with one beam of light, then measure the light emitted at 90 degrees angle. The "spectro" part means that you can scan through a spectrum of light, either by:
(1) setting the emission detector to a specific wavelength (actually a narrow passband) and scanning though a spectrum of excitation light, or by
(2) setting the excitation beam to a particular wavelength (again, actually a narrow passband) and scanning through the spectrum of emitted light.
This machine has two diffraction gratings, one controlling the wavelength of excitation light passing through the sample and the other controlling the wavelength of emitted light passing into the detector. Data is generally displayed as excitation and emission spectra, like these for Morpholino oligos linked to either carboxyfluorescein or lissamine (sulforhodamine B):
http://www.gene-tools.com/files/fluores ... ofiles.pdf
As the excitation spectrum is gathered, the emission sample is taken in a fixed passband; as the emission spectrum is gathered, the sample is excited by a fixed passband of excitation light.

These two instruments use different cuvettes. The UV-Vis machine uses a cuvette with two opaque sides and two transparent (quartz) sides. The light beam enters though one quartz window, passes through the sample and exits the other side through another quartz window. The spectrofluorometer uses a transparent cuvette and the light path is different: the light bean enters through one side, passes through the sample (exciting fluorescence as it passes) and exits through the opposite side -- and this light is discarded, it is not collected by a detector. Fluorescent light that leaves the cuvette sideways, at 90 degrees to the excitation beam, is collected by a detector.

There are simpler instruments for measuring absorbance (colorimeters) which use fixed-wavelength filters instead of motorized diffraction gratings. There are similar simple fluorometers that use filters to select the wavelengths of the excitation light beamed onto the sample and the emission light allowed into the sensor (I used one of these in grad school for chlorophyll measurements).
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