Virtual UV-VIS spectrophotometer

Micropipettes are used to take liquid from reagent bottles and transfer it to test tubes (so preparing reaction mixtures). A Pasteur pipette is used to take all liquid from a tube and transfer it to the spectrophotometer cuvette.

What are Pasteur pipettes and how they are used (video)

Select the micropipette you wish to use by clicking on it (the one currently selected will have an orange-coloured spot; the other one will have it gray ). If you click on the Pasteur pipette, both micropipettes will have their spot grayed out.

Pipettes must not be dragged, instead you should just click on the target where you want the pipette to move to (reagent bottle, test tube, cuvette or solid waste basket).

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.

When you click on a test tube:

  • if one of the micropipettes is selected and it is loaded with sample, this will be added to the tube;
  • if the Pasteur pipette is selected, it will aspirate the whole contents of the tube.

To add the contents of the Pasteur pipette to the spectrophotometer cuvette, click on the cuvette.

General procedure:

  1. Adjust the volume setting of the micropipette, clicking on the left- or right-hand sides of the wheel, or type the value in the dial.
  2. Select the micropipette clicking on its body.
  3. Click on one of the bottles so the micropipette will be loaded from it.
  4. Click on one of the tubes so that the contents of the micropipette tip is released into the tube.
  5. Prepare mixtures with several reagents, following that procedure, in all tubes.
  6. Click on the Pasteur pipette to select it.
  7. Click on a tube to collect its contents with the Pasteur pipette.
  8. Click on the cuvette to deliver the sample into it.

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, click on one of the arrows

  1. (Attention: the lid of the compartment must be open)

Action of buttons and icons:

  • records the current measurement in the external monitor
  • clear clears all information displayed in the monitor
  • photo 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 or the Pasteur pipette will be discarded (whether they have liquid or not)

To empty the cuvette (and get a new one) click on the liquid waste container.

The Pasteur pipette delivers 2 mL aliquots. 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 sample.

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.

Disponible también en español

Determination of glucose concentration using a colorimetric enzymatic method

Composition in tube

Materials:

  • Two samples in which we wish to determine concentration of glucose
  • Standard solution of glucose, 10 mg/dL
  • Saline solution: 0.9 g/L NaCl
  • R: chromogenic reagent, containing:
    • glucose oxidase (EC 1.1.3.4)
    • peroxidase (EC 1.11.1.7)
    • phenol
    • 4-aminophenazone (PubChem CID: 6009; InChIKey: RMMXTBMQSGEXHJ-UHFFFAOYSA-N)
    • 92 mM Tris buffer, pH=7.4

Preparing the experiment:

Connect the spectrophtometer and set the wavelength to 505 nm.

Using variable volume micropipettes P200 and P1000, prepare in the test tubes the mixtures described in the table: (you may likewise decide using other volumes, but the total in each tube must be 1 mL)

tube glucose
standard 		unknown
sample		 saline
solution
0 0   1000 µL
1 30 µL   970 µL
2 90 µL   910 µL
3 150 µL   850 µL
4 300 µL   700 µL
5 500 µL   500 µL
6   80 µL of sample A 920 µL
7   40 µL of sample A 960 µL
8   20 µL of sample A 980 µL
9   200 µL of sample B 800 µL
10   400 µL of sample B 600 µL
11   700 µL of sample B 300 µL

When they are all finished, add 1 mL of the chromogenic reagent to each tube and for the reaction to develop (15 virtual minutes).

Example of results in a real experiment.

Use the Pasteur pipette to transfer the contents of tube 0 to the spectrophotometer cuvette. Take the cuvette into the spectrophotometer and click on the “A=0” button. Take the cuvette out and discard its contents.

Transfer with the Pasteur pipette the contents of tube 1 to the cuvette, put this into the spectrophotometer and write down the absorbance value in your lab notebook. You may click the  button to record the absorbance reading in the monitor below.

Repeat the procedure for the remaining tubes.

Make a table with the concentration of glucose in the mixture of each tube (tubes 0 to 5) and the respective absorbance value. You may click the  button to copy the listing of absorbance values.

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 absorbance obtained for each tube prepared with the glucose standard (tubes 0 to 5) against their glucose concentration. Fit the data to a straight line.

From that line, calculate:

  • glucose concentration in each tube, from 6 to 11;
  • glucose concentration in each of the two starting samples (A and B).

Rationale:

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

Determination of glycemia (glucose concentration in blood)

Composition in tube

Materials:

  • Two serum samples in which we wish to determine glucose concentration
  • Control solution of glucose (or standard), with certified concentration 100 mg/dL
  • Saline solution: 0.9 g/L NaCl
  • R: chromogenic reagent, containing:
    • glucose oxidase (EC 1.1.3.4)
    • peroxidase (EC 1.11.1.7)
    • phenol
    • 4-aminophenazone (PubChem CID: 6009; InChIKey: RMMXTBMQSGEXHJ-UHFFFAOYSA-N)
    • 92 mM Tris buffer, pH=7.4

Preparing the experiment:

Connect the spectrophotometer and set the wavelength to 505 nm.

Important notes:

  • The control will be analysed in triplicate (tubes 1, 2, 3).
  • Dilutions of serum cannot be performed in this virtual laboratory, but they will be applied internally: although the pipette will take undiluted sample from the bottle, what will be added to tubes 7 and 10 will be assumed as diluted 1/2 and diluted 1/5 for tubes 8 and 11.

Using variable volume micropipettes P200 and P1000, prepare in the test tubes the mixtures described in the table:

tube glucose
control		 unknown
sample		 saline
solution
0 0   20 µL
1 20 µL    
2 20 µL    
3 20 µL    
       
6   20 µL of serum A  
7   20 µL of serum A, diluted* 1/2 (read the note)
8   20 µL of serum A, diluted* 1/5 (read the note)
9   20 µL of serum B  
10   20 µL of serum B diluted* 1/2 (read the note)
11   20 µL of serum B diluted* 1/5 (read the note)

When they are all finished, add 2 mL of the chromogenic reagent to each tube and for the reaction to develop (15 virtual minutes).

Use the Pasteur pipette to transfer the contents of tube 1 to the spectrophotometer cuvette. Take the cuvette into the spectrophotometer and click on the “A=0” button. Take the cuvette out and discard its contents.

Transfer with the Pasteur pipette the contents of tube 1 to the cuvette, put this into the spectrophotometer and write down the absorbance value in your lab notebook. You may click the  button to record the absorbance reading in the monitor below.

Repeat the procedure for the remaining tubes.

Make a table with the measurements. You may click the  button to copy the listing of absorbance values.

Analysis of results:

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

Calculate the concentration of glucose in the control tubes (1 to 3) and calculate the average of their absorbances.

Using Lambert-Beer law: A = ε × L × c, by comparison of each unknown sample with the control, calculate:

  • glucose concentration in each tube, from 6 to 11;
  • glucose concentration in each of the two starting sera (A and B).

Rationale:

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

Determination of protein concentration using a colorimetric method: Lowry's method

Composition in tube

A A
B1 B2
F-C

Materials:

  • Two samples in which we wish to detemine the concentration of proteins
  • Standard solution of bovine serum albumin, 2 g/L (BSA)
  • Reagent C: prepared by mixing A, B1 and B2 reagents in ratios 100 : 1 : 1 (volume)
    • Reagent A: 2% Na2CO3 in 0.1 M NaOH
    • Reagent B1: 1% CuSO4·5H2O (i.e., 1 g / 100 mL)
    • Reagent B2: 2% sodium-potassium tartrate (2 g / 100 mL)
  • Folin-Ciocalteau reagent, diluted 1/4 in water (main component: phosphomolibdotungstic acid)

Preparing the experiment:

Connect the spectrophtometer and set the wavelength to 580 nm.

Using variable volume micropipettes P200 and P1000, prepare in the test tubes the mixtures described in the table: (you may likewise decide using other volumes, but the total in each tube must be 400 µL)

tube albumin
standard		 unknown
sample		 water
0 0   400 µL
1 30 µL   370 µL
2 60 µL   340 µL
3 90 µL   310 µL
4 120 µL   280 µL
5 150 µL   250 µL
6   120 µL of sample A 280 µL
7   200 µL of samplea A 200 µL
8   120 µL of sample B 280 µL
9   200 µL of sample B 200 µL

When they are all done, add 2 mL of reagent C to each tube and for the first reaction to develop (10 virtual minutes).

Add 200 µL of Folin's reagent to each tube and for the second reaction to develop (15 virtual minutes).

Example of results in a real experiment.

Use the Pasteur pipette to transfer the contents of tube 0 to the spectrophotometer cuvette. Take the cuvette into the spectrophotometer and click on the “A=0” button. Take the cuvette out and discard its contents.

Transfer with the Pasteur pipette the contents of tube 1 to the cuvette, put this into the spectrophotometer and write down the absorbance value in your lab notebook. You may click the  button to record the absorbance reading in the monitor below.

Repeat the procedure for the remaining tubes.

Make a table with the concentration of proteins in the mixture of each tube (tubes 0 to 5) and the respective absorbance. You may click the  button to copy the listing of absorbance values.

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 absorbance obtained for each tube prepared with the protein standard (tubes 0 to 5) against their protein concentration. If the behaviour is linear, fit the data to a straight line.; if you appreciate some curvature (saturation of the reaction), you will get more exact results fitting data to a 2nd order polynomial or to a rectangular hyperbola.

From that line, calculate:

  • protein concentration in each tube, from 6 to 11;
  • protein concentration in each of the two starting samples (A and B).

Rationale:

Alkaline medium induces protein denaturation, and their unfolded chains stabilise thanks to nitrogen atoms in the polypeptide backbone coordinating Cu2+ ions. In this way, tyrosine rsidues become exposed and may react with Folin's reagent, which oxidises phenol groups, molibdenum and tungsten (wolfram) becoming reduced into a dark blue coloured compound.

Determination of creatinine concentration using a colorimetric mnethod

Composition in tube

Materials:

  • Two urine samples in which we wish to detemine concentration of creatinine
  • Standard solution of creatinine, 30 mg/L
  • Saturated picric acid solution (PubChem CID: 6954; InChIKey: OXNIZHLAWKMVMX-UHFFFAOYSA-N)
  • 1 M NaOH

Preparing the experiment:

Connect the spectrophtometer and set the wavelength to 520 nm.

Using variable volume micropipettes P200 and P1000, prepare in the test tubes the mixtures described in the table: (you may likewise decide using other volumes, but the total in each tube must be 1 mL)

tube creatinine
standard		 unknown
sample		 water
0 0   1.00 mL
1 0.25 mL   0.75 mL
2 0.50 mL   0.50 mL
3 0.75 mL   0.25 mL
4 1.00 mL   0
5      
6   20 µL of sample A 980 µL
7   40 µL of sample A 960 µL
8   80 µL of sample A 920 µL
9   20 µL of sample B 980 µL
10   40 µL of sample B 960 µL
11   80 µL of sample B 920 µL

When all are done, add 0.75 mL of picric acid and 0.25 mL of NaOH to each tube and for the reaction to develop (15 virtual minutes).

Example of results in a real experiment.

Use the Pasteur pipette to transfer the contents of tube 0 to the spectrophotometer cuvette. Take the cuvette into the spectrophotometer and click on the “A=0” button. Take the cuvette out and discard its contents.

Transfer with the Pasteur pipette the contents of tube 1 to the cuvette, put this into the spectrophotometer and write down the absorbance value in your lab notebook. You may click the  button to record the absorbance reading in the monitor below.

Repeat the procedure for the remaining tubes.

Make a table with the concentration of creatinine in the mixture of each tube (tubes 0 to 4) and the respective absorbance. You may click the  button to copy the listing of absorbance values.

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 absorbance obtained for each tube prepared with creatinine standard (tubes 0 to 4) against their creatinine concentration. Fit the data to a straight line.

From that line, calculate:

  • creatinine concentration in each tube, from 6 to 11;
  • creatinine concentration in each of the two starting samples (A and B).
  • the value of creatinine coefficient* for the individuals from whom samples were taken.

Rationale:

Detection of creatinine is possible by a reaction based on the original method by Max Jaffe:

The product, coloured, belongs to a family of compounds called Janovsky's complexes.

(*) Creatinine coefficient is defined as the amount of creatinine, in mg, excreted per day and per kg of body weight.
  • M. Jaffe (1886) Zeitschrift für Physiologische Chemie 10: 391-400.
  • J.V. Janovsky, L. Erb (1886) doi:10.1002/cber.188601902113