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Quantum Yield of Rhodamine B in Ethanol (Gain Corrected Spectra)

by dhaffnersr | July 12, 2016 15:46 12 Jul 15:46 | #13283 | #13283

Rhodamine B is a chemical compound and a dye


Rhodamine B is a chemical compound and a dye. It is often used as a tracer dye within water to determine the rate and direction of flow and transport. Rhodamine dyes fluoresce and can thus be detected easily and inexpensively with instruments called fluorometers or in this case, a homemade spectrometer. Rhodamine dyes are used extensively in biotechnology applications such as, fluorescence microscopy, flow cytometry, fluorescent correlation spectroscopy and ELISA.

Rhodamine B is used in biology as a staining fluorescent dye, sometimes in combination with auramine O, as the auramine-Rhodamine stain to demonstrate acid-fast organisms, notably Mycobacterium.

Rhodamine B is tunable around 610 nm when used as a laser-dye, its luminescence quantum yield is 0.65 in basic ethanol, 0.49 in ethanol and1.0, and 0.68 in 94% ethanol. The fluorescence yield is temperature dependent.

This research projects goal is open-ended, in that, there are many useful possibilities with this dye, its results are very reproducible and its excitation wavelength can be attained at 510nm, max absorption at 543nm makes it suitable for a 532nm green laser, which I hope to demonstrate here in this paper.

A word of caution is in order, Rhodamine B can be very toxic, especially to the eyes, and can be carcinogenic (controversial.) So very good lab procedures are to be followed, like wearing gloves and safety eye ware and avoiding any spills.

Below is a picture of the pipettor I used in transfering the samples to their respective cuvettes



Next, is the sample of Rhodamine B that I prepared as my reference. 1ml was transfered to the cuvette and mixed with 95% pure lab grade Ethanol.


The next graph is that of the Oregon Medical Laser Center's spectrum of Rhodamine B in Ethanol


This is an important spectra because when calculating "relative" quantum yields, you need a reference that has an already determined and verified quantum yield value to reference against, and this one has a known quantum yield of 70% in Ethanol.

Also, this spectra was captured on a ISA-SpexFluoroMax-2 Spectrometer, upon which I have verified this fact by contacting Dr.Steven L.Jacques Professor, Dept. of Biomedical Engineering & Dermatology at the Oregon Health & Science University, CH13B 3303 SW Bond Ave, Portland, OR 97239 USA (all references are at the end of this research paper.)


The next set of spectra are samples 1 through 5, all of which, have been corrected for gain.


These are the values for all 5 samples, including the reference sample spectra (all spectra captured on Spectral Workbench at Plab and data processed using Spekwin32 capture program.)


Concentration levels for all 5 samples


Quantum Yield values for all 5 samples


This is the graph comparing sample 5 to the reference sample control_and_sample_5.png

As can be seen by the graph, there is a margin of error for sample 5 of 0.068% and 1.72% for the control sample (reference.) I think this can be attributed to the relative error of the pipettor delivery system and some margin of errors in the Rhodamine B stock solution.

This problem will be addressed in the next series of graphs.


Here I plotted both my reference sample (control,) and that of the OMLC's sample, using the Molar Absorption Coefficient calculation of l/[mol*cm] @ wavelength and applied that to the Y axis to approximate the concentration levels of the OMLC's spectra. These are other tolerances I tried to account for also in my error calculations: Analytical Balance/my scale for measuring the Rhodamine B(powdered form) ±0.001g, Volumetric Flasks, 100ml (glass)-±0.08 mL , Transfer Pipettor-0.14%

The percent relative uncertainty of each measurement is determined by the division of the tolerance by the amount measured multiplied by 100. The percent relative uncertainty is useful when propagating error from calculations requiring multiplication or division.

The total uncertainty in a series of measurements is calculated by the following formula, where e represents the individual uncertainties. % total error = √Σ(%e2)

Although I can't know with absolute certainty about the exact procedures the OMLC used to prepare their samples, I can only approximate them, with as much precision as mathematically possible. The Molar Coefficient of the OMLC's sample is 1. Sample 5 was 0.99932, were as, the reference sample(control) was 0.99760?

This is where I believe the error lies either between the transfer of samples to cuvettes, stock solutions or perhaps a more basic problem of spectrometer alignment and light scattering problems. The processing of data may contribute a percentage also I suspect, experience plays a big part in this type of work I am certain.

In conclusion, Rhodamine B is an excellent dye to be utilized here at Public Lab and when working with home built spectrometers, with certain precautions in mind, the compound can be easily adapted for calibration purposes and fluorescent study in the interest of furthering one's knowledge on the subject of UV-vis Spectroscopy.

I have included in my references, a link to Dr.Freidrich Menges doctoral thesis (2008,) caveat, it is in GERMAN! I have translated the entire document for my personal use and have not obtained permission from Dr.Menges for distribution in english, so if you are interested its there for you to work with, the only problem is I find no way to translate the graphics, easy to place them in their proper place but not in english.



2 References Chien, G. L., C. G. Anselone, R. F. Davis and D. M. Van-Winkle. Fluorescent vs. radioactive microsphere measurement of regional myocardial blood flow. Cardiovascular Res. 30:405-12, 1995. Guilbault, G. G. Practical Fluorescence. Modern Monographs in Analytical Chemistry. 3: 1990. Steven L. Jacques Professor, Depts. of Biomedical Engineering & Dermatology mail: Oregon Health & Science University, CH13B 3303 SW Bond Ave, Portland, OR 97239 USA

3 Rhodamine B is tunable around 610 nm when used as a laser dye.[2] Its luminescence quantum yield is 0.65 in basic ethanol,[3] 0.49 in ethanol,[4] 1.0,[5] and 0.68 in 94% ethanol.[6] The fluorescence yield is temperature dependent.[7]







11 Multidimensional fluorescence spectroscopy Friedrich Menges from Schefflenz, Mathematics and Natural Science Section, Department of Chemistry University of Konstanz - Rhodamine B in Ethanol (Plab v2.5 Spectrometer)

“The fluorescence quantum yield φ is defined as the percentage of absorbed photons that are emitted as fluorescent light it describes quantitatively how good (or bad) a compound fluoresces. The fluorescence quantum yield can be dependent on many parameters.”11 (pg.26 fig 3.5, fluorescence quantum yield lifetime) __


Upon proofreeding the post...again, I forgot to include the quatum yield for the reference sample(control) which is:



Dave H

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