UV-VIS sspectrophotometer

To switch the spectrophotometer on and off, click on the switch

When you click on one of the reagent bottles, the cap will open; from that moment, the pipette will draw from that reagent. When you click again on the bottle or its cap, it will be capped.
Trick: while a bottle remains open, it is enough to hover the pointer over the pipette to have this filled up again.

To add an aliquot of a sample to a cuvette:

Select a cuvette clicking on it or on the radio button below it.
Click on the pipette.

To empty a cuvette, select it and then click on the waste vessel.

To access the cuvette compartment, click on the latch or on the cover.

To introduce a cuvette into the spectrophotometer or to get it out:

Select a cuvette clicking on it or on the radio button below it.
Click on one of the arrows
(Beware that the compartment cover must be open)

Action of buttons and icons:

records the measurement in the external monitor
records a spectrum
clears all information off the monitor
captures an snapshot of the spectrum, which can then be copied or saved via the context menu in the browser
represents the waste disposal vessel; when clicked on it, the selected cuvette will be emptied

The pipette delivers 1 mℓ aliquots. Each cuvette has a 3 mℓ capacity; if surpassed, it will spill (the cuvette will then be emptied in order to continue). To be measured in the spectrophotometer, the cuvette must contain at least 2 mℓ of sample.

The absorbance reading displays ##### whenever it is not possible to measure: if the compartment cover is open or if the volume in the cuvette is not enough.

Disponible también en español

Relation between absorbance and concentration

Materials: We will use a solution of para-nitrophenol (pNF), which is a yellow coloured compound in alkaline medium.

Calibration: First of all, fill a cuvette with 3 mℓ water, take it into the spectrophotometer, set the wavelength to 405 nm and click on the “A=0” button. Take the cuvette out and pour it off.

Experiment design: Using para-nitrophenol (pNF) and water, prepare 3 samples in separate cuvettes, containing different volumes of pNF and the same total volume of 3 mℓ.

To help you do this, fill in this table beforehand (or better, a table that you make in your laboratory notebook) with the volumes you are planning to add into each cuvette:

cuvette:	1	2	3
pNF volume:				mℓ
water volume:				mℓ

Once the 3 mixtures are prepared in the 3 cuvettes, put them, one after another, into the spectrophotometer and write down their absorbances.

cuvette:	1	2	3
A₄₀₅ =

Qualitative analysis of results: Observe the relationship between colour in the cuvettes and the value of absorbance. By comparing the 3 measurements, do you perceive any dependence between the absorbance and the higher or lower concentration of pNF in the cuvette?

Quantitative analysis of results: Calculate pNF concentration in the final mixture in each cuvette, knowing that the concentration in the bottle is 80 µM pNF. Prepare a table with the data and draw a graph plotting A versus C. How would you describe what you are observing?

cuvette	C (µM)	A₄₀₅	A₄₀₅ [pNF] (µM)
1
2
3

Fundamentals of a protein quantitation assay

What is known as Bradford's method is a common assay for measuring concentration of proteins in a sample. It is based on proteins binding the Coomassie Blue dye (Coomassie Brilliant Blue G250) and hence altering the visible spectrum of the dye.

Materials: These are available:

Bradford's reagent, containing Coomassie Blue, phosphoric acid and methanol or isopropanol;
proteins diluted in the reagent.

Experimental design: Prepare a cuvette with just Bradford's reagent and two other cuvettes (or more) with different amounts of the protein solution. Add to all the cuvettes the needed amount of reagent for a total volume of 3 mℓ each.

To help you do this, fill this table in advance (or better, a table that you make in your laboratory notebook) with the volumes you are planning to add in each cuvette:

cuvette:	1	2	3
volume of protein:				mℓ
volume of reagent:				mℓ

Calibration: Put the first cuvette (which only contains Bradford's reagent) into the spectrophotometer, set the wavelength to 595 nm and click on the “A=0” button. This is called the blank, or "reagents blank" and serves to compensate the colour of the reagent when measuring the proteins.

Measurement: Then put the other cuvettes into the spectrophotometer, one after another, and write down their absorbances.

cuvette:	1	2	3
A₅₉₅ =

Qualitative analysis of results: Observe the relation between colour in the cuvettes and the absorbance. When comparing the measurements, can you observe any dependence between absorbance and the higher or lower concentration of proteins in the cuvette?

Quantitative analysis of results: Calculate the concentration of proteins in the final mixture in each cuvette, knowing that the concentration in the bottle is 800 mg/ℓ. Prepare a table with the data and draw a graph plotting A versus C. How would you describe what you are observing?

cuvette	C (mg/ℓ)	A₅₉₅	A₅₉₅ C_prot (mg/ℓ)
1
2
3

1 mℓ of an unknown sample is mixed with Bradford's reagent, for a total volumen of 3 mℓ. Calculate the protein concentration in the original sample if the assay has rendered a value A₅₉₅ = reveal the value
Write your result: c = mg/ℓ and check if it is correct.

Fundamentals of a protein quantitation assay

Materials: These are available:

Bradford's reagent (3× concentrated), containing Coomassie Blue, phosphoric acid and methanol or isopropanol;
proteins dissolved in water.

Experimental design: All the cuvettes must contain 1 mℓ of concentrated Bradford's reagent and a totla volume of 3 mℓ. The first cuvette will not receive any protein, while the other two will have different amounts of the protein solution. Add water to complete the volume.

To help you do this, fill this table in advance (or better, a table that you make in your laboratory notebook) with the volumes you are planning to add in each cuvette:

cuvette:	1	2	3
volume of protein:				mℓ
volume of water:				mℓ
volume of reagent:				mℓ

Measurement: Then put the other cuvettes into the spectrophotometer, one after another, and write down their absorbances.

cuvette:	1	2	3
A₅₉₅ =

cuvette	C (mg/ℓ)	A₅₉₅	A₅₉₅ C_prot (mg/ℓ)
1
2
3

Absorption spectrum of haemoglobin

The red colour in haemoglobin is due to it absorbing visible radiation, although it also displays absorption peaks at and near the ultraviolet region.

Experimental design: Using water and the haemoglobin solution, prepare different dilutions and analise their full UV-VIS spectrum. To this purpose, fill a cuvette with haemoglobin and in the other two cuvettes prepare different mixtures of haemoglobin with water. All cuvettes should have a total volume of 3 mℓ.

To help you do this, fill this table in advance (or better, a table that you make in your laboratory notebook) with the volumes you are planning to add in each cuvette:

cuvette:	1	2	3
volume of haemoglobin:				mℓ
volume of water:				mℓ

Measurement: Put the cuvettes into the spectrophotometer, one after another, and click the button to record the spectrum between 200 and 800 nm. Click on the camera icon and copy the resulting image in order to compare all of them at the end of the experiment.

Qualitative analysis of results: Compare the three spectra. Which effect can you observe when the starting haemoglobin is diluted? Does the position (λ) of the maximal absorption peaks change? Describe your observations.

2^nd measurement: Observing the spectrum, write down two wavelengths (in the visible region, not ultraviolet) where there is a peak (a local maximum of absorption). Then, take mesurements of absorbance and write them down, for the 3 cuvettes at each of those two wavelengths. Draw a graph with your results, considering that the starting haemoglobin solution (in the bottle) has a concentration of 200 mg/ℓ.

cuvette	C (mg/ℓ)	λ₁ = A	λ₂ = A	A C_Hb (mg/ℓ)
1
2
3

Quantitative analysis of results: How can you describe the conclusions of what you have observed? Which of the wavelengths would be useful to determine the concentration of unknown samples of haemoglobin?

Disponible también en español