Espectrofotómetro UV-VIS

Select the micropipette you wish to use clicking on the option above it.

When you click on a reagent bottle, its cap will be opened and the currently selected micropipettte will move and take a volume of that reagent.

To add an aliquot of sample to a spectrophotometer cuvette:

Click on one of the bottles so the micropipette will be loaded from it.
Select a cuvette clicking on it, or in the option below it.
Click on the pipette and the contants of its tip will be delivered into the cuvette.

To empty a cuvette (and get a fresh one), select it and then click on the . liquid waste container.

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

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

To introduce a cuvette into the spectrophotometer and to take it out:

Select a cuvette clicking on it or on the option below it.
Click on one of the arrows
(Attention: the lid of the compartment must be open)

Action of buttons and icons:

records the current measurement in the external monitor
clears all information displayed in the monitor
captures data displayed in the monitor, in a format that allows to copy them and transfer to a spreadsheet or document
represents the liquid waste container; when you click on it, the cuvette will be emptied
represents the solid waste container; when you click on it, the pipette tip will be discarded (whether it has liquid or not)
is an auxiliary timer, which starts from zero when one clicks on its stopwatch; it is not essential for running the experiment

Micropipettes dispense 1000 or 200 µL aliquots, respectively. The cuvette has a capacity of 3 mL; if you exceed it, the liquid will spill (later on, the cuvette will be replaced so you can continue). To be able to take measurements in the spectrophotometer, the cuvette must have at least 2 mL of liquid.

The absorbance reading shows ##### when it is not possible to measure: if the compartment lid is open, or if the volume in cuvette is insufficient.

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Determination of gamma-glutamyltranspeptidase activity in a urine sample

Materials:

R1 and R2: components of the chromogenic reagent; when mixed in 1:1 ratio, the final reagent will contain:
- 100 mM glycylglycine
- 3 mM γ-glutamyl-3-carboxy-4-nitroanilide
- 100 mM Tris buffer, pH=8.25
Urine sample in which the enzymatic activity is to be determined.

Preparing the experiment:

Add to the three cuvettes 1 mL of R1 and 1 mL of R2, and different volumes of water into each:

cuvette:	1	2	3
volume of R1:				mL
volume of R2:				mL
volume of water:				µL

Calibrate the spectrophotometer after introducing cuvette number 1: set the wavelength to 405 nm and click the “A=0” button. Take the cuvette out.

Measuring the course of the enzymatic reaction :

Absorbance of each cuvette must be measured at one-minute intervals for a total of 10 min, starting from the moment you add the urine. It is possible to alternate measuring both cuvettes (2 and 3, one every 30 s), in a way that the whole experiment will last 10 min. If you prefer, you may preparae first cuvette number 2 and take all its readings, after which you will do the same with number 3; in this case, you will need 20 min.

Prepare in your lab notebook a table where you record times and absorbance readings, similar to the one displayed here:

time (min:s)	cuvette 2	cuvette 3
0:00	add 200 µL of urine
0:30		add 400 µL of urine
1:00	A= ...
1:30		A= ...
2:00	A= ...
2:30		A= ...
etc.

The procedure will be:

Add the planned amount of urine to cuvette 2 (time zero, reaction starts); start your chronometer; introduce the cuvette in the spectrophotometer.

After 30 s, add the planned amount of urine to cuvette 3 (reaction will start when you add the second aliquot completing 2.4 mL).

At t = 1 min, click the button to record the absorbance reading in the monitor below. Take out cuvette number 2 and introduce number 3.

Repeat measurements every 30 s alternating the cuvettes. If time is not exactly the one intended, write down its exact value in your table.

When the 10 min are finished, click the button and copy the listing of absorbance data.

Analysis of results:

Write down in your lab notebook the code assigned to your experiment (in this particular case it is

Make a graph plotting the series of absorbances obtained for both cuvettes against time of reaction. Fit the data to straight lines (one for each cuvette). From the slope of each line, calculate the concentration of enzyme activity in the urine sample.

Rationale:

Reaction catalysed by the enzyme, that allows to detect its activity:

Molar extinction coefficient of 5-amino-2-nitrobenzoate: ε = 9.25·10³ mol⁻¹·L·cm⁻¹