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New cuvette holder design and Rhodamine B testing

by dhaffnersr | March 22, 2016 15:38 22 Mar 15:38 | #12875 | #12875

I am getting a much better handle on processing spectral data on my software, so I decided to completely redesign my cuvette holder(it is 90 percent inclosed within the holder,) so I could contain as much reflected fluorescent light back into the cuvette, and direct it twoards the slit with more efficiency.

Rhodamine B sample preparation was done as follows:

1) a 100ml flask was placed on a digital scale and tared, 1g of rhodamine b was measured into flask and filled to the 100ml mark with distilled water and then shaken lightly. I then placed the flask on a magnetic stirrer on high for 2hrs until all the rhodamine b was dissolved.

Using a graduated disposable pipette, I then transferred 1ml of rhodamine b to another 100ml flask and filled to the 100ml mark and swirled it several times, this is the 100ppm dilution.

Then I transferred 5ml from the second flask containing the 100ppm dilution to a 3rd 100ml flask and filled to the mark and swirled several times.

This is the 100ppb dilution.

Now I have my 2 samples marked S1-100ppm and S2-100ppb

Reference sample is distilled water.

3 quartz cuvettes were used/ cleaned with Isopropyl alcohol and rinsed with distilled water.

Light source is a 532nm green laser.


S1- 100ppm rhodamine b before data processing


Reference(distilled water in quartz cuvette) and Sample


Subtraction of Ref and Sample 100ppm


Zoomed view of subtraction data


Excitation and emission graph for rhodamine b/100ppm@532nm

Next set is rhodamine b 100ppb:


Rhodamine b 100ppb before data processing


Ref(distilled water in quartz cuvette) and Sample


Subtraction of Ref and Sample rhodamine b 100ppb


Zoomed view subtraction of ref and sample rhodamine b 100ppb


Excitation and emission wavelength of rhodamine b 100ppb@532nm


Rhodamine B sample used


Samples ready for testing


Newly redesigned cuvette holder and laser operation illuminating sample.


The 2 samples of rhodamine b although differing in solution concentration, appear to both have peak wavelengths of 586nm, predicted peaks vary because of regents used (usually NaOH, predicted peaks are usually 565nm to 573nm) in this case I used distilled water with no Ph adjustment which could account for the higher peak wavelengths.

So experimentally within the parameters that I have set, the peaks for rhodamine b in distilled water with no Ph adjustment appear to be @586nm.


Here are some up close pics of the cuvette holder, yesy it is really ugly but it is in the "proof of concept" phase:

PIC_0096.JPG The way it is designed I can detach it easily.







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Very helpful, thanks Dave! Why a green laser?

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Hey Jeff, I used the green laser (532nm) because of the excitation wavelength of rhodamine b, which is about 543nm, that's why I displayed the excitation and emission data also, to show the excitation at 100ppm which was 536nm and 100ppb was 538nm.

So my experiment was pretty close to predicted excitation wavelengths, plus I am still working on my sample procedures so I get them correct.

Dave H

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