Modifications in the Phenolphthalein Method for Spectrophotometric Estimation of Beta Cyclodextrin

Abstract

The phenolphthalein solution used in the assay of P-Cyclodextrin is not stable and undergoes spontaneous decolorization. There is linear decrease in absorbance of phenolphthalein with time. This causes a continuous increase in error with time which is as high as 80% after one hour. A simple procedure is described in the paper to circumvent this problem allowing reproducible and reliable results to be obtained. A single reagent assay was developed by incorporating phenolphthalein in Na,C03 solution. This resulted in stable absorbance readings and increased the linearity of assay up to 200pg/ml of P-cyclodextrin. Modifizierungen der Phenolphthalein-Methode zur spektrophotometrischen Bestimmung von Beta-Cyclodextrin. Die zur Bestimmung von P-Cyclodextrin venvendete Phenolphthaleinlosung ist nicht stabil, sondern unterliegt spontaner EntWrbung. Es gibt eine lineare Verringerung der Absorption mit der Zeit. Diese bedingt eine kontinuierliche Erhohung des Fehlers mit der Zeit, die nach einer Stunde bei 80% liegen kann. Es wird ein einfaches Verfahren beschrieben, urn dieses Problem zu umgehen und reproduzierbare und zuverlassige Ergebnisse zu erhalten. Eine einzelne Reagenzanalyse wurde entwickelt unter Einbeziehung von Phenolphthalein in Na3C03Losung. Diese resultierte in stabilen Absorptionswerten und erhohte die Linearitat der Analyse bis zu 200pg/ml P-Cyclodextrin.

Colorimetric determination of alpha and beta-cyclodextrins and studies on optimization of CGTase production from B. firmus using factorial designs

“…Deviation of linearity could be well explained by the theory of complex formation. Phenolphtalein-β-CD complex was only linear when correlated to β-CD concentration for up to 0.17 mmol/L (Goel and Nene, 1995). The formation of the inclusion complex causes a decrease in absorbance of the methyl orange solution that is only linearly correlated with the cyclomalto hexaose concentration in the range of 0.25 optical density unit and 0.30 mmol/L cyclomaltohexaose, with limited interference by linear oligosaccharides and other cyclodextrins (Lejeune et al, 1989).…”

“…The formation of the inclusion complex causes a decrease in absorbance of the methyl orange solution that is only linearly correlated with the cyclomalto hexaose concentration in the range of 0.25 optical density unit and 0.30 mmol/L cyclomaltohexaose, with limited interference by linear oligosaccharides and other cyclodextrins (Lejeune et al, 1989). The enzyme CGTase hydrolyses starch to produce CDs as its main reaction products and the maltooligosaccharides present in the enzyme hydrolyzates do not interfere in the analysis of CDs by the phenolphtalein method (Goel and Nene, 1995). The experimental data were analyzed in the light of a non-linear relationship that can be easily used with the aid of a statistical package for computers (Figures 1 and 2).…”

“…The concentration of β-CD was analyzed by the decrease in absorbance at 550 nm due to phenolphtalein-CD complex formation (Goel and Nene, 1995 and modifications reported by Matioli, 1997). To 0.2 mL of β-CD solution in 0.05 mmol/L Tris-HCl buffer, pH 8.0, 1.0 mL of the phenolphthalein working solution was added.…”

Enzymatic Characterization of Recombinant Cyclodextrin Glycosyltransferase from Bacillus sp. a2-5a using Sagoo Starch as Substrate

“…The β-cyclization activity of rCGTase was measured by spectrophotometer using phenolphtalein method (Goel and Nene 1995). The reaction mixtures containing 1% (w/v) gelatinized sagoo starch in various pHs.…”

“…Before the β-CD was measured, the reaction was neutralized using 1.2 M NaOH. The reactions were carried out using the rCGtase assay procedure mentioned above (Goel and Nene 1995). The temperature stability of the enzyme was measured by incubating 0.02 UA rCGtase with an equal volume of 50 mM Tris-Cl buffer (pH 7.0) for 30 min, followed by incubation with 1% (w/v) gelatinized sagoo starch for 30 min.…”