What is the difference between scanning a TLC plate at 550 nm (Visible), 254 nm (UV) and 366 nm (fluorescent)?
Dear Bhavesh,
Here are the answers of the inquired questions:
1. TLC densitometer is an unit of HPTLC which provide the absorbance or fluorescence reflected by the sample. It works within a spectral range of 190-900nm. This range include all wavelengths from visible to ultraviolet. The choice of lamps depends upon the wavelength you selected. The information about the types of lamps and working of densitometer is mentioned in a HPTLC manual.
2. Once you have selected the choice of wavelength then densitometer will automatically start scanning the entire plate. The will store in the software in the form of peak tables. These peak tables consist of Rf values and area absorbed by each spot. From there you can carry forward the calculations.
I hope the discussion will be helpful to you. For any other query feel free to ask.
thanks!
I used the Image Master software. In fact, this software is used in proteomic analysis, to do the spots densitometry, but I could evaluate my TLCs utilized it. However, some softwares used in the translluminators can do this. The wavelenght utilized in the scanning depends on the kind of molecule you are searching for. Sometimes you need some staining methods.
I'm assuming your TLC plates have F254 dye that makes the plates glow green under UV light?
If so, many organic compounds absorb light at 254 nm making a dark spot because the light is blocked from the dye, so the dye can't fluoresce. The greater absorbance by the compound (either by concentration or absorption), the darker the spot.
336 nm- the compound absorbs light and re-emits another wavelength that you can see.
550 nm- I assume the detector is just looking for a colored spot.
Dear all,
it would be grateful if you could kindly suggest me the mechanism of the scanning during densitometry. How we get an Area of a spot during scanning in different modes? Why different sources of wavelength (lamp) being used for scanning? What is their exact role during scanning?
Getting the area of a spot is generally done by finding the edges of a spot, then counting pixels within the area defined by that edge, and relating that to some sort of unit (each pixel = so many square millimeters). The different lamps are used because some work better in UV and some have better output in the visible range of the spectrum.
the most important difference in sensitivity
Scanning densitometry is the most efficient procedure for quantitative evaluation..1–9;25/ In situ, quantitative measurements of TLC plates can be performed in many ways. The reflectance, transmittance, simultaneous reflectance and transmittance, fluorescence, or fluorescence quenching of a spot can be measured by use
of a densitometer. The most sensitive in situ method is fluorimetry.
ref: Planar Chromatography in Clinical Chemistry Raka Jain and Joseph Sherma
in Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) pp. 1583–1603
John Wiley & Sons Ltd, Chichester, 2000
Dear Bhavesh,
Here are the answers of the inquired questions:
1. TLC densitometer is an unit of HPTLC which provide the absorbance or fluorescence reflected by the sample. It works within a spectral range of 190-900nm. This range include all wavelengths from visible to ultraviolet. The choice of lamps depends upon the wavelength you selected. The information about the types of lamps and working of densitometer is mentioned in a HPTLC manual.
2. Once you have selected the choice of wavelength then densitometer will automatically start scanning the entire plate. The will store in the software in the form of peak tables. These peak tables consist of Rf values and area absorbed by each spot. From there you can carry forward the calculations.
I hope the discussion will be helpful to you. For any other query feel free to ask.
thanks!
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