WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Venkateswara et al. SJIF Impact Factor 6.041 Volume 5, Issue 11, 1643-1647 Research Article ISSN 2278 4357 DETERMINATION OF λ max OF ANTIBIOTIC F-40 PRODUCED BY Streptomyces sulfonensis sp.nov., Ellaiah P. 1, Sri Venkateswara Rao V. 1 *, Ramakrishna S. V. 2, Venkateswarulu Y. 2, Annapurna J. 2 and Venkatesam U. 2 1 Pharmaceutical Biotechnology Division, Department of Pharmaceutical Sciences Andhra University, Visakhapatnam, A.P, India. 2 Indian Institute of Chemical Technology, Hyderabad, Telangana, India. Article Received on 21 Sept. 2016, Revised on 12 Oct. 2016, Accepted on 01 Nov. 2016 DOI: 10.20959/wjpps201611-8110 *Corresponding Author Sri Venkateswara Rao V. Pharmaceutical Biotechnology Division, Department of Pharmaceutical Sciences Andhra University, Visakhapatnam, A.P, India. ABSTRACT A total of 359 actinomycetes were isolated from 8 different natural substrates collected from different locations of Indian state of Andhra Pradesh. The taxonomic studies and literature survey indicates that the potent antibiotic producer was belongs to a new streptomycete species. The antibiotic F-40 was isolated from the culture filtrate of S.sulfonensis by extraction with organic solvents and it was purified and separated by TLC and column chromatography methods. As a part of its characterization, UV λ max was determined using dichloromethane as solvent. The λ max was found to be 232 nm. The data indicates the aromatic nature of the compound and it was used along with other physico-chemical properties in the identification of antibiotic F-40. KEYWORDS: Actinomycetes, Streptomycete, S.sulfonensis, chromatography, UV, λ max. INTRODUCTION Ultraviolet spectrophotometric methods finds extensive use in the identification of various hydrocarbons, vitamins, steroids, heterocycles and conjugated aliphatics. UV absorption spectroscopy can characterize those types of compounds which absorb UV radiation. These compounds contain either unbonded electrons or the conjugated double-bond system such as aromatic compounds. An unknown compound can be identified by comparing its spectrum with the known spectra. If the two spectra are identical (especially in the strong absorption www.wjpps.com Vol 5, Issue 11, 2016. 1643
region), the compound would have similar structure in accordance with Hartley s rule. [1] This rule states that compounds having similar structures would have similar absorption spectra. The structure of compound sometimes changes with the change in the solvent. Hence, UV absorption method is less useful and it should be used along with other IR & NMR methods in identification of unknown compounds. [1] The term λ max is used in UV-Visible spectroscopy where one looks at electronic transitions between the HOMO level and the LUMO level. Molecules absorb light over a range of wavelengths and when this absorbance is plotted as a function of wavelength one obtains an absorption spectrum. An absorption spectrum is simply a plot of absorbance vs wavelength. This plot gives us a range of wavelengths within which a molecule absorbs. [2-3] Lambda max refers to the wavelength in the absorption spectrum where the absorbance is maximum. Generally molecules absorb in a wavelength range centred around the lambda max. It acts as a single quantitative parameter to compare the absorption range of different molecules. Various groups of antibiotics exhibit characteristic ultraviolet absorption typical to that of functional groups. [4-6] Sometimes spectral shifts occur due to changes in solvent polarity, changes in molecular structure etc. All such spectral shifts (i.e., blue shift or red shift) can be quantified with shift in lambda max. For example increase in conjugation length in a molecule shifts the lambda max to the red. One can calculate lambda max for some conjugation systems such as conjugated dienes and polyenes using Woodward-Fieser rules (empirical rules) and this parameter can also be used in the structure determination. [7] There are different types of spectroscopy based on the technique and use. Spectroscopy is suitable for both qualitative analysis and quantitative analysis. Qualitative spectroscopy This is the technique to know the type of sample molecule there by one can tell what the sample is and its chemical nature after comparing the obtained analysis curve peaks with that of standard sample from official books like Pharmacopoeias or books on chemical standards etc. www.wjpps.com Vol 5, Issue 11, 2016. 1644
A sample is subjected to scanning over entire range of UV or visible radiation. The point or wavelength where the sample shows maximum absorbance is noted as its λ max. This λ max is fixed for every sample and there by any unknown sample can be identified by knowing its λ max after comparing with standard. Quantitative spectroscopy: This is a method to determine the exact concentration of a substance in a given sample. At a specified wave length (λ max ) when a given sample is analyzed by spectroscopy, the concentration in the sample can be known by plotting it against a standard substance graph. The aim of the present work was to demonstrate the procedure to find the wavelength of maximum absorbance for the antibiotic F-40 solution. The process involves recording the absorbance over the range of 200 nm to 400 nm, usually in intervals of 25 nm. The data can be graphed to visualize the highest absorbance. MATERIALS AND METHODS Instrument and materials Instrument used was Schimadzu UV-240 double beam UV/Visible recording Spectrophotometer (Graphicord). The antibiotic F-40, pure compound was isolated from the culture filtrate of S.sulfonensis by solvent extraction and chromatographic methods. All chemicals and reagents used were of analytical grade. Methodology Preparation of stock solution The antibiotic F-40 solution was prepared by dissolving 10 mg antibiotic in 5 ml dichloromethane in 10 ml volumetric flask and volume was made up to the mark with the same solvent to obtain stock solution of 1 mg/ml concentration. Procedure The above solution was further diluted and observed the absorbance at different wavelengths against blank. The experiment was repeated two to three times and a graph was recorded which indicates the peaks and absorbance. The peak which showed maximum absorbance gave λ max of the compound present in the solution and it was found to be 232 nm (Fig.1). www.wjpps.com Vol 5, Issue 11, 2016. 1645
RESULTS AND DISCUSSION Solvents plays an important role in UV spectroscopy. The solvents may cause salvation of the solute, ionic character, hydrogen bonding and salt effect hence, the solvent for a sample should be selected in such a manner that it should neither absorb in the region of absorption nor affect the absorption of the sample. The antibiotic F-40 exhibited maximum peak at 232 nm as shown in the Fig.1, which indicates the aromatic nature of the compound. CONCLUSION The unique property of maximum absorption at a particular wavelength is known as λ max and this data was used along with other physico-chemical properties and spectral data of the antibiotic F-40 and it was characterized and published else where. ACKNOWLEDGEMENT We thank the University Grants Commission, New Delhi for providing the financial assistance to V.S.V.RAO. REFERENCES 1. H. Kaur, Pragati s Spectroscopy, Pragati Prakashan, Meerut, Second Ed, 2004-2005. 2. Silverstein, R.M, Bassler, G.C and Morril, T.C. Spectrometric identification of organic compounds, 1991; 5 th ed., John Wiley &Sons Inc., Singapore. 3. http://www.quora.com/what is lambda max and why is it important. www.wjpps.com Vol 5, Issue 11, 2016. 1646
4. Rao, C.N.R, Ultraviolet and visual Spectroscopy, Chemical Applications, Butterworths, London, 1967; 193. 5. Oroshnik, W., Vining, L.C., Mebane, A.D and Taber, W.A., Polyene antibiotics, Science, 1955; 121: 147-149. 6. Ball, S, Bessell, C.J and Mortimer, A. The production of polyenic antibiotics by soil streptomycetes. J. Gen. Microbiol., 1957; 17: 96-103. 7. Glagovich, N. Woodward Fieser Rulesfor Dienes. [Online]http://www.chemistry.ccsu.edu/glagovich/teaching/316/uvvis/diene.html. www.wjpps.com Vol 5, Issue 11, 2016. 1647