There are several solutions with different concentration of a fluorescent material which it's concentration are between 10 to 100 ng/ml. The available method is flourimetery.
What device is available, specifically? I would say under most conditions, yes. It will be important to ensure that the only variation between the solutions is the concentration of the fluorescent compound (i.e., the buffers/solvent should all be the same) and that that volume measured is the same (if path length matters).
It will also be important that there is a standard curve. Preferably at least 4 but ideally 7 or either known concentration samples that you can built a reference against. Without a standard curve it will be difficult or impossible to determine exact concentrations of any unknown samples.
Yes you can measure the concentration of fluorophore by fluorometry.
Fluorescence vs concentration is nonlinear for high absorbance values and linear for low absorbance values. One can measure concentration only in the linear regime i.e., for solutions of low absorbance (A < 0.05).
Yes, if you are in the linear-response range of your detector, which you will see from your calibration curve. For standard fluorimetry it is advised to keep A
In addition to all said. It would be much easier to measure absorption coefficient fro concentration determination. Particularly because absorption cross-section is much less sensitive to physical parameters than quantum yield, and much easier to find in literature. If your absorption is too low just take a long cuvette.
Thak you all.
As Mr. Ravi Kishore mentioned Fluorescence vs concentration is nonlinear for high absorbance values, I interested to know the range which I should do measurements (10 to 100 ng/ml) is high or not?
Hossein: As you said, important is the absorbance, not the concentration. So measure the absorbance at the highest concentration you intended to use and you will get the answer. I would also suggest you to check the linearity of absorption in the concentration region you will use. A non-linear plot may indicate aggregation or a similar thing that will also influence fluorescence. For aggregation of e.g. rhodamine dyes you may check: J. Phys. Chem., 1988, 92, 4286 or Talanta 1999, 49, 99.
That depends on the extinction coefficient of your dye. At the highest concentration ensure that A(=ecl) < 0.05.
Reason for non-linearity in Fluorescence intensity vs concentration curve
Fluo Intensity =k x Quantum Yield x Absorbed Intensity = k x Quantum Yield x (I0-I)
Substitute I = I0 x10-ecl in the above equation
Fluo Intensity = k x Quantum yield x I0 (1-10-ecl)
Expanding the exponential term as Mclaurian series
Fluo Intensity = k x Quantum yield x I0 (2.303ecl -(2.303ecl)2/2! + (2.303ecl)3/3! -....other terms)
Higher order terms make the fluorescence intensity vs concentration curve non-linear at higher absorbance.
When 2.303 ecl < 0.05, the relative error one makes by neglecting all the higher order terms except the first term is 0.13%. Neglecting all the higher order terms one obtains
Fluo Intensity = 2.303 x k x Quantum yield x I0 x ecl
In this equation fluorescence intensity varies linearly with concentration.
If the absorbance increases more than 0.05 then the error also increases. Now it depends on you as to what percentage of error can be tolerated in your experiment / measurement.
- Badges
- Science topic
- Similar topics
- Cell Biology
- Methods