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Spectrophotometry
Chapter 2

Basis of Derivative Spectrophotometry

2.1

The Main Law of Light Absorption by a Substance

Photobiological processes occur under the influence of light of ultraviolet (UV), visible, and near infrared spectral regions. Generally, values of light flux intensity, I, and wavelength, l are used in optical measurements. The frequency index  n is also considered to characterize an absorbed light. Frequency is expressed in reciprocal seconds [cÀ1] and presents itself as the ratio of a radiation velocity c to a wavelength l [cm or nm]:
 ¼ c=l ¼ 3Á1017 =lnm ; n where c ¼ 3 Â 1010 cm Á cÀ1 or 3 Â 1017 nm Á cÀ1, the velocity of light. The frequently used index is the wavenumber  equal to the number of waves in 1 cm. n The wavenumber is a reciprocal wavelength expressed in reciprocal centimeters:
½cmÀ1 Š :  ¼ 1=lcm ¼ 107 =lnm n Thus,  ¼ 40; 000 cmÀ1, when l 250 nm. A number of spectrophotometers have n wavenumbers on the scale dials. For example, the wavelength of the cadmium red
˚
line, as accepted by international agreement, is equal to 6438.4696 A (angstroms).
˚ is 1/6438.4696 part of cadmium red line.
So 1 A
Only absorbed quanta can realize photochemical impact and the reader should remember that a spectral region of a photobiological process is conditioned by the absorption spectrum of a substance involved in this process.
The extinction law for actinic monochromatic light absorbed by a substance layer can be written in the exponential or logarithmic form:
I ¼ I0 Á 10ÀeCd ;

V.S. Saakov et al., Derivative Spectrophotometry and Electron Spin Resonance (ESR)
Spectroscopy for Ecological and Biological Questions,
DOI 10.1007/978-3-7091-1007-2_2, # Springer-Verlag Wien 2013

(2.1)

5

6

2

lgðI0 =IÞ ¼ eCd;

Basis of Derivative Spectrophotometry

(2.2)

where I0 and I are light intensity before and after passing through a layer of substance, of solution, or a leaf, e is a coefficient depending on wavelength



References: 69 Schreiber U, Bilger W (1987) Rapid assessment of stress effects on plant leaves by chlorophyll Schreiber U, Bilger W, Neubauer C (1997) Photosynthesis: a comprehensive treatise. Cambridge University Press, Cambridge, pp 320–336 Semenov IN, Perfilova IL (2000) Chemistry ( in Russian – Khimiya). Khimizdat, SPb., P 656 Shabalin II, Petrova LP (1969) Comparison of absorption spectra measured in different devices SP-4. (in Russian). Zavodskaya Lab 30:551–552 Shaklee KL, Rowe JE (1970) Wavelength modulation spectrometer for studying the optical properties of solids. Appl. Optics. 9:627–632 Sharma VK, Aulakh JS, Malik AK (2003) Fourth derivative spectrophotometric determination of Shibata S, Furukawa M, Goto K (1969) Dual-wavelength spectrophotometry. Part 1. General method Shibata S, Furukawa M, Goto K (1973) Dual-wavelength spectrophotometry.Part IV. Qualitative and quantitative analysis by means of first-derivative spectra Shibata S, Furukawa M, Nakashima R (1976) Dual-wavelength spectrophotometry. Part VI. Shibata S, Goto K, Ishiguro Y (1972) Dual-wavelength spectrophotometry. Part III. Determination of arsenazo I in arsenazo III Shlyk AA (1971) Determination of chlorophylls and carotenoids in green leaves. (in Russian). In: Biochemical methods in plant physiology. Nauka, Moscow, pp 154–170 Shtern E, Timmonis K (1974) Electronic absorption spectroscopy in organic chemistry. (in Russian). In: Pentin YuA (ed) Mir, Moscow, p 296 Siek TJ, Rieders F (1984) Determination of carboxyhemoglobin in the presence of other blood hemoglobin pigments by visible spectrophotometry. J Forensic Sci 1:39–54 Singleton F, Collier GL (1956) Infra-red analysis by the derivative method 6:495–510 Skujins S (1986a) UV Instruments at work Skujins S (1986b) UV Instruments at work. Varian AG.. No UV-31. Pt. 1:1–33 Smirnov BS, Badu EI (1967) The way of differentiation of random time functions with Sneddon J, Bezur L, Michel RG, Ottaway JM (1982) Square-wave wavelength modulation system for use in atomicspectrometry Snellman W (1968) An a scanning method with increases sensitivity in atomic absorption analysis using a continuum primary source Snellman W, Pains TC, Yee KW et al (1970) Flame emission spectrometry with repetitive optical scanning in the derivative mode Spitsyn PK, Korepanov VE (1980) Modernization of registrating spectrophotometer SP-8. (in Russian). Zhurn Analit Khim 35:2441–2444 Spitsyn PK, L’vov ON (1985) Derivative spectrophotometry of rare-earth elements Zhurn Analit Khim 40:1241–1248 Stauffer FR, Sakai H (1968) Derivative spectroscopy Such V, Traveset J, Gonzalo R, Gelpi E (1980) Stability assays of aged pharmaceutical formulas for thiamine and pyridoxine by high performance thin-layer chromatography and derivative ultraviolet spectrometry. Anal Chem 52:412–419 Sverdlova OV (1973) Electron spectra in organic chemistry Talsky G (1994) Derivative spectrophotometry. Law and high orders. VCH Verlaggesellsch. Talsky G, Mayring L (1978) Uber die analoge – Differentiation hoher Ordnung zur Feinlauflosung von UV-Visible-Spektren und anderen elektrischen Meßsignalen Talsky G, Mayring L, Kreuzer H (1978a) High-resolution, higher-order UV VIS –derivative spectrophotometry Talsky G, Mayring L, Kreuzer H (1978b) Derivativespektrophotometrie h€her Ordnung zur o Tarasov KI (1968) Spectral devices. (in Russian). Mashinostroenie, Leningrad, p 237 Taulier A, Levillain P, Lemonnier A (1986) Advantage of spectrophotometry in derivative for the dosage plasma and urinary hemoglobin – Comparison with the method using Allen’s correction. Comparison with the method using Allen’s correction. Ann Biol Clin (Paris) 44:242–248 Tereshin GS (1959a) Accuracy of spectrophotometry in spectrophotometer. (in Russian). Zhurn Analit Khim 14:388–395 Tereshin GS (1959b) Accuracy of spectrophotometry optimal spectrophotometric conditions. (in Russian). Zhurn Analit Khim 14:516–522 Twyman F, Lothian GF (1933) Conditions for securing accuracy in spectrophotometry Soc 45:643–662 Udovenko GV, Baranov AA, Rutman GI, Saakov VS et al (1974) Method of derivative spectrophotometry as the way of assessment of plastid apparatus reaction on extremal influence. Udovenko GV, Saakov VS (1976) Resistenz der getreidepflanzen gegen unguenstige Bedingungen des Milieus: physiologische und genetische Aspekte Univer. zu Berlin, Math Naturwiss Reihe 25:776–786 Vierordt K (1873) Die Anwendung des Spektralapparates zur Photometrie der Absorptionsspectren und zur quantitativen chemischen Analyse Wahbi AM, Ebel S (1974) The use of the first-derivative curves of absorption spectra in quantitative analysis. Anal Chim Acta 70:57–63 Whitten WB, Nairn JA, Pearlstein RMW (1978) Derivative absorption spectroscopy from 5—300 K Williams JGM (1959) An oscillating-plate differentiator for spectrophotometry. J. Scientific Instrum Williams DT, Hager RN Jr (1970) The derivative spectrometer. Appl Optics 9:370–373 Williams BL, Willson K (eds) (1975) Principles and techniques of practical biochemistry

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