Google

Computer-Interfaced Experiments - Absorbance/Transmittance Measurement

Dyes
Light Absorbance of Cyanidin

Objectives: Determination of the Absorbance Maxima of Cyanidin and its Metal Complexes



Datalogging and data analysis using the Program "Measuring and Evaluating"
and the Analog-Digital-Converter CASSY-E - LEYBOLD DIDACTIC




German version



Chemicals:
ethanol 96 %
1 N HCl
0.1 N aluminium(III) chloride solution
0.01 N iron(III) nitrate solution
0.5 % NH3 solution

Apparatus and glass wares:
photometer fitted with a recorder output
cuvettes


Hazards and safety precautions:

Ethanol is highly flammable.

Safety glasses, gloves and good ventilation required.


Experimental procedure:

Using 50 mL of ethanol the dye from the petals of two red roses is extracted. In each case 5 mL of the filtered extract are pipetted into four test tubes. Drop by drop the solutions are mixed with 1 N HCl until an intensive red coloration occurs. Into the third test tube, drop by drop, 0.5% NH3 solution is added, until the solution turns to a blue. One of the solutions is mixed with iron(III) nitrate or aluminium(III) chloride solution.


Foto
Fig. 1:   1: Extract     2: extract mixed with HCl     3: extract mixed with HCl und NH3
3: extract mixed with HCl und AlCl3 solution



The recorder output of the photometer is connected to the input B of the INTERFACE.























Matching of the program 'Multimeter':
- In the program  'Measuring and Evaluating'  is activated the subprogram  'Multimeter'  and via the menu item  <F3>'Select measur. quantities' ®'Reselect channel B'  is selected the quantity  'Voltage DC'.
- After the program is switched to   <F4>'Automatic/Param./Select formula' ® Enter parameter   wavelength  is entered. The  'Physical symbol' is labelled by  l  and the   'Physical unit'  by  nm.
- Under the menu item  'Enter formula'  the   'Physical symbol'  is labelled by   A. The  'No. decimal places'  is indicated by  4.
According to  A = 2 - logU·100  (CASSY registers a voltage of approx. 1 V at 100% transmittance) the beginning of the formula  A (n, l, U) =   is completed by the input of  
- lnU / ln10  (Fig. 2).


Fig. 2: Menu screen - Matching of the program


Measurement: With the wavelength knob a wavelength of 360 nm is selected. After zero calibration is completed, a cuvette filled with water is placed into the sample compartment. With the sample cover closed, using the light control knob the meter needle is adjusted to "0" on the absorbance scale (100% T). On the measuring screen a voltage of approx. 1 V is displayed. Now the cuvette (filled with water) is replaced by a cuvette, those to 2/3 with the dye solution filled is. After striking the function key  <F1 >  the appropriate wavelength is to be entered. By pressing  ¿  the measured value is confirmed. Afterwards the wavelength is changed by increasing the wavelength by 5 nm. The cuvette filled with water is placed again into the light-tight sample holder. The needle is adjusted to "0" on the absorbance scale. Now the cuvette containing the dye solution is inserted and the absorbance is monitored at the appropriate wavelength.

Extreme wavelengths, in the ultraviolet or infrared ranges, require special filters.

Before storing the data into file, the x-axis is labelled with  l  and the y1-axis with   A  by means of  <F7> 'Select representation'® 'Display' . Under  'Select graph options'  one ensures that the data points are displayed as crosses.


Graphical analysis:

A direct comparison of the measurements is allowed in an overlay mode, which can be activated under  <F8>'Disc operations'®'Multigraph on'. The desired series of measurements are selected individually, in order to represent them together in the main menu under  <F6 >'Evaluate in graph'. By striking the function key   <F3>  and by the input of the number of the appropriate graph, a 'best fit smooth curve' is drawn through the data points  (Fig. 3, 4).



Fig. 3: Multigraph screen
cyanidin chloride:  lmax = 375nm / 530 nm (1)      anionic anhydrobase:  lmax = 375 nm / 560 nm   (2)


Absorptionsspektrum
Fig. 4: Multigraph screen
cyanidin chloride: lmax = 375nm / 530 nm   (1)     iron complex:  lmax = 535 nm  (2)
aluminium complex:  lmax = 560 nm  (3)


Discussion:

·   Anthocyanins localized in the cell vacuole are a type of complex flavonoid, that produce blue, purple or red colors of many blooms and fruits. They have a sugar moiety, most often attached to the 3 position or both the 3 and 5 positions together  (1).


·   The anthocyanins are broken down by acids into sugars and the corresponding dye components (anthocyanidins). By acid hydrolysis flavylium salts (oxonium salts) are formed, whose cations are resonance-stabilized  (2).



·   The three anthocyanidins found in roses are cyanidin, peonidin and pelargonidin.



·   While the mineral acidic salts of the anthocyanidins are more or less red colored, so e.g. cyanidin chloride available in test tube 2, the anthocyanidine structures formed by reaction of oxonium salts with hydroxide ions, show violet or blue colours  (4)



Structures of cyanidin in aqueous solution under varying pH




Degradation of cyanidin in strong alkaline medium


The color of anthocyanins depends on the acidity of the medium. At acidic pH = 1-3, anthocyanidins exist predominantly in the form of the red flavylium cation (2-phenylchromenylium cation). Increasing the pH leads to a decrease in the color intensity and the concentration of the flavylium cation which undergoes hydration to produce the colorless hemiketal or carbinol pseudobase (chromenol). The conjugated 2-benzopyrilium system is disrupted due to a nucleophilic attack of water at the 2-position of the anthocyanidin skeleton. A rapid proton loss of the flavylium cation takes place as the pH shifts higher. Now the equilibrium is shifted toward a purple quinoidal anhydrobase at pH < 7 and a deep blue ionized anhydrobase at pH < 8  (4).  When pH increases further the carbinol form yields, through opening of the central pyran ring, the light yellow chalcone. The color of the alkaline solutions can be reverted by changing the pH back to acidic. The anthocyanidin equilibrium forms shift back to the equilibrium where the red colored flavylium cation predominates. However, if the pH value is too high and unstable ionic chalcones have already formed, recovery of the flavylium form can not be achieved by simple re-acidification. Chalcone is converted to an a - diketone via keto-enol tautomerism. Subsequent breakdown of the chalcone leads to carboxylic acid (i.e. substituted benzoic acid) originating from the B-ring and hxdroxyaldehyde (i.e. 2,4,6-trihydroxybenzaldehyde) from the A-ring of tje parent anthocyanin  (5).

·   Some metals, such as Fe3+ and Al3+ form stable deeply colored coordination complexes with anthocyanidins whose cyanidin components bear ortho-dihydroxyphenyl structure on the B-ring. The experiment above shows that Al3+ is complexed by anthocyanidins of the rose to form a purple color. So far in the petals of roses no metal complexes were found.




Aluminium complex of cyanidin-3-glucoside



References:
Microscale Projection Experiment  Anthocyanins as pH-Indicators and Complexing Agents
  Demonstration Experiment on Video   Red Cabbage - red or blue?
  Demonstration Experiment on Video   Reaction of Leavening Ingredients with the Dye of Hybiscus Tea
Les anthocyanes


Index of CASSY Experiments






Site Meter