The physical measurement of colour

Measuring Concentrations using absorbance,

Transition Metal Solutions,

Case 1 - Detection of b-glactosidase

For example, when cloning a gene the required genetic information (i.e.DNA) from an original is inserted into the E. coli vector plasmid along with the associated b-glactosidase enzyme. The insertion of this information into the vector is not 100% effective, and therefore an indicator is needed in order to ascertain which E. coli have been successfully encoded. 5-bromo-4-chloro-3- indolyl-ß-D-galactoside (or X-gal) is a good example of a substrate whose  colour properties change upon cleavage by b-glactosidase. 

As shown below, the test solution turns from colourless to indigo if the enzyme is present. When the glycosidic linkage is cleaved two of the free indoxyl molecules join together in a dimmer mechanism, before oxidation occurs to form an insoluble blue precipitate

Figure 11: The action of b-galactosidase.  This image is taken from  Cepko (ref. 12) and is copyright restricted according to the source given (i.e. it is not the authors' own work).

Case 2 - Detection of ions

Figure 12: The structure of a substituted ferrocene ditopic receptor. This image is taken from Miyaji (ref. 7) and is copyright restricted according to the source given (i.e. it is not the authors' own work).

A recent example taken from Miyaji (ref. 7) is the substituted ferrocene-based ditopic receptor shown above, which contains a urea unit and a benzocrown ether unit. This chromogenic receptor if used under appropriate concentrations of anions and cations, acts as a molecular switch. When anions (e.g. F^-) bind to the nitrobenzene unit, the absorption spectra changes from the UV region to visible – a colourless to yellow change is observed. If cations (e.g. K⁺) are added afterwards, their binding to the crown-ether unit turns the solution back to colourless. If the ions are added in the opposite order (i.e. K⁺ then F^-), no colour change at all occurs – that is K⁺ inhibits the action of F^-. Intermolecular interactions which cause these shifts in the Fe d-d spectra band tend to pull electron density away from the urea unit. Click on the above image to have the intermolecular interactions explained in terms of bond strength and formation. 

Case 3 - Detection of 'flu virus

Although Type-A influenza viruses mutate frequently (thereby avoiding human defence mechanisms), their binding affinity for sialo oligosaccharides on host cells remains high. The reaction scheme below shows the synthesis of a Ruthenium complex which carries a disialo oligosaccharide. The strong luminescence of the tris-bipyridine Ru complex comes about due to the saccharide shell isolating the Ru core from outer solvent molecules. However the high binding affinity of lectin in viruses to the YDS-DRu, particularly the bis-adduct, disrupts the shell thereby reducing its’ luminescence.

Figure 13: Synthesis of YDS₁-DRu.  This image is taken from Kojima (ref. 8) and is copyright restricted according to the source given (i.e. it is not the authors' own work).

It is not yet known why there is such a high affinity but some electrostatic and hydrophobic interactions have been detected between the cationic, aromatic complex centre and the amino-acid residues of anionic hemagglutinin on lectin. This highly sensitive sensory system could have huge implications in the near future for the medical world as currently influenza in the elder population can be fatal and therefore quick diagnosis and treatment is essential. This summary of recent research was taken from Kojima (ref. 8).

Case 4

The principle behind Photodynamic Treatment (PDT) is that cytotoxic singlet oxygen, produced by irradiation of the inserted photosensitizer, damages the local tissue which contains an area of abnormal cell growth (or tumours). Hypocrellins (derivatives of hematoporphyrin) are the latest photosensitive pigments to be investigated. On consideration of the window of light absorption of hypocrellins HA and HB compared to that of a tumor, there is little in common. It should also be noted that any potential pigment needs to have good uptake into cells as well as low self-aggregation in blood in order to reach the target tissue - that is it must be amphiphilic (i.e. not HA or HB).  However a more amphiphilic derivative of these hypocrellins (HBO₂H - below) was investigated in terms of the above properties, and in particular for the treatment of some vas capillary conditions which have a shallow tissue depth allowing penetration of light with wavelengths corresponding to the active window of HBO₂H.  Although more than one phototherapeutic agent is going to be required to treat all such diseases in this class, the current results give a promising start in combating a wide range of disorders for future generations.  This summary of recent research was taken from Zhao (ref. 9).