Organic Synthesis

Fluorescein dye synthesis

Theory:

Fluorescein is a synthetic dye that is comercially available as a dark orange powder slightly soluble in water and alcohol. It has found application mainly in fluorescent microscopy and biology/biochemistry due to its fluorescent properities. The compounds is photostable and very sensitive with very high value of molar attentuation coefficient at the wavelength of the argon laser (488 nm) [1]. The wavelenght of the fluorescence photons ranges from 490 nm up to 570 nm causing the green colouring of the solutions [2].

Fluorescein can exist in seven prototropic forms that are strongly dependent on pH value of the enviroment. The neutral form is told to be a mixture of the lactone (70%), zwitterionic (15%) and quinoid (15%) forms (scheme 1) [3,4]. Other forms – cationic, anionic and dianonic – are shown on the scheme 2.

 f3Scheme 1 Neutral forms of fluorescein – quiniod (a), lactone (b), zwitterion (c)

f2

Scheme 2 Fluorescein forms possesing a charge – dianion (a), monoanion (b,c), cation (d)

Fluorescein is both at industrial and laboratory scale prepared by Friedel-Crafts reaction of resorcinol with phthalic anhydride in the presence of an acid catalyst (Lewis or Brønsted) (scheme 3). The mechanism of the fluorescein formation is quite complex and it is composed of two  and elimination steps.

f1

Scheme 3 Reaction scheme of fluorescein preparation

Procedure:

  1. Brønsted acid used as a catalyst

Phthalic anhydride (0.5 g, 3.4 mmol), resorcinol (0.75 g, 6.8 mmol) and three drops of concentrated sulfuric acid was added to the round bottom-flask. The mixtured was heated on an oil-bath (170 °C) during 10 minutes. Dark red solid left in the flask was washed with 10 mL of hot water and 5 mL of concentrated ammonia solution was added under the constant stirring. Dark green solution was poured into 200 mL of distilled water. Concentrated hydrochloric acid was added drop-wise to the solution until the orange precipitate started to form. The precipitate was filtered with suction and washed thoroughly with cold water.

  1. Lewis acid used as a catalyst

Anhydrous zinc chloride (0.1 g, 0.7 mmol) was placed to the test tube and heated preventively on an oil-bath. Then resorcinol (0.2 g, 1.8 mmol) and phthalic anhydride (0.14 g, 0.95 mmol) was added. The test tube was heated for 5 minutes at the temperature 180 °C while occasionally shaking. The mixture was cooled to about 100 °C and 2 mL of water and 0.2 mL of concentrated hydrochloric acid. While heating the mixture at oil-bath the solid on the bottom of the test tube was grinded and the content was vigorously stirred with a glass rod. An orange precipitate that started to form after few minutes was filtered off with suction, washed with a little portion of water and dried in the air.

Photo 11-04-16 09 52 20

Literature:

[1] Sjoback, R. Nygren, J. Kubista, M. Spectrochimica Acta Part A. 1995, Vol. 51, Iss. 6, p. L7-L21.

[2] Blum, H.F. Spealman, C. R. J. Phys. Chem. 1933, Vol. 37, No. 9, p. 1123-1133.

[3] Diehl, H. Talanta. 1989, Vol. 36, No. 3, p. 413-415.

[4] Klonis, N. Sawyer W.H. Journal of Fluorescence. 1996, Vol. 6, Iss. 3, p. 147-157.