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2.2 ULTRAVIOLET AND VISIBLE SPECTROPHOTOMETRY

      Absorption spectroscopy in the ultraviolet and visible region is one of the most useful tools for qualitative and quantitative analysis.

      When a beam of monochromatic radiation traverses a solution containing an absorbing substance, its radiant power is reduced in relation to the distance that it travels through. It also decreases in relation to the concentration of absorbing molecules or ions encountered in that medium. These two factors determine the proportion of the total incident energy that emerges.

The decrease in power of monochromatic radiation is stated quantitatively by Beer’s law:

log (1/T) = A = abc.

      The terms used in connection with spectrophotometric tests are defined as follows:

      Absorbance (A ) is the logarithm, to the base 10, of the reciprocal of the transmittance (T ). (Note Descriptive terms used formerly include optical density, absorbancy and extinction.)

      Absorptivity (a ) is the quotient of the absorbance (A) per litre, of the substance (c) and the absorption path length in cm (b). 

      (Note It is not to be confused with specific absorbance, specific extinction or extinction coefficient [A (1 per cent, 1 cm) or E (1 per cent, 1 cm)]. These are generally used in different pharmacopoeiae, as the quotient of the absorbance divided by the product of the concentration, expressed in g per 100 ml, of the substance, and the absorption path length in cm, therefore: A (1 per cent, 1 cm) = 10 a.)

      Molar absorptivity (ε) is the quotient of the absorbance (A) divided by the product of the concentration, expressed in moles per litre, of the substance (c) and the absorptivity (a) and the molecular weight of the substance. (Note Terms formerly used include molar absorbancy index, molar extinction coefficient and molar absorption coefficient.)

      Transmittance (T ) is the quotient of the radiant power transmitted by a sample (I) divided by the radiant power incident upon the sample (I₀).

      Absorption spectrum is a graphic representation of absorbance, or any function of absorbance, plotted against wavelength or function of wavelength.

      Where a monograph gives a single value for the position of an absorption maximum, it is understood that the value obtained may differ by not more than ±2 nm. 

Apparatus

      All types of spectrophotometer are designed to permit substantially monochromatic radiant energy to be passed through the test substance in a suitable form and to allow measurement of the fraction of energy that is transmitted. The spectrophotometer comprises an energy source, a dispersing device with slits for selecting the wavelength band, a cell for holding the test substance, a detector of radiant energy, associated amplifiers, and measuring and recording devices. Some instruments are manually operated, while others are equipped for automatic operation. Instruments are available for use in the visible region of the spectrum, usually 380 nm to about 700 nm, and in the ultraviolet and visible regions of the spectrum, usually 190 nm to about 700 nm.

      Both double-beam and single-beam instruments are commercially available and either is suitable. Depending on the type of apparatus used, the results may be displayed on a scale, on a digital counter, or by arecorder or printer.

      The apparatus should be maintained in proper working condition. The housing of the optical system should minimize any possibility of errors due to stray light; this is particularly relevant in the short-wave region of the spectrum.

      CONTROL OF WAVELENGTHS Verify the wavelength scale using the absorption maxima of Holmium Perchlorate Solution, the line of a hydrogen or deuterium discharge lamp or the lines of a mercury vapour as shown below. The permitted tolerance is ±1 nm for the range 200 to 400 nm and ±3 nm for the range 400 to 600 nm. 

241.15 nm (Ho)	404.66 nm (Hg)
253.70 nm (Hg)	435.83 nm (Hg)
287.15 nm (Ho)	486.00 nm (Dβ)
302.25 nm (Hg)	486.10 nm (Hβ)
313.16 nm (Hg)	536.30 nm (Ho)
334.15 nm (Hg)	546.07 nm (Hg)
361.50 nm (Ho)	576.96 nm (Hg)
365.48 nm (Hg)	579.07 nm (Hg)

      CONTROL OF ABSORBANCE Check the absorbance using Potassium Dichromate Solution UV at the wavelengths indicated in the following table, which gives for each wavelength the exact value of A (1 per cent, 1 cm) and the permitted limits.      The wavelength scale may also be calibrated by means of suitable glass filters that have useful absorption bands through the visible and ultraviolet regions. Standard glass containing didymium (a mixture of praseodymium and neodymium) has been widely used. Glass containing holmium is considered superior. The exact values for the position of characteristic maxima in holmium glass filters are 241.5±1, 287.5±1, 360.9±1, and 536.2±3 nm. Holmium glass filters are obtainable from some national institutions and from commercial sources. The performance of an uncertified filter should be checked against one that has been properly certified.

      LIMIT OF STRAY LIGHT Stray light may be detected at a given wavelength with suitable filters or solutions. For example the absorbance of a 1.2 per cent w/v solution of potassium chloride at a path-length of 1 cm should be more than 2 at 200 nm when compared with water as reference liquid.

      SPECTRAL SLIT WIDTH When measuring the absorbance at an absorption maximum, the spectral slit width must be small compared with the half-width of the absorption band, or erroneously low absorbances will be measured. Particular care is needed for certain substances and the instrumental slit width used should always be such that further reduction does not result in an increased absorbance reading.

      CELLS Cells usually in the spectral range discussed are 1-cm absorption cells with glass or silica windows. Other path lengths may also be used. The cells used for the test solution and the blank should be matched, and must have the same spectral transmittance when containing only the solvent. If this is not the case, an appropriate correction must be applied.

      SOLVENTS In measuring the absorbance of a solution at a given wavelength, the absorbance of the solvent cell and its contents shall not exceed 0.4 and is preferably less than 0.2 when measured with reference to air at the same wavelength. The solvent in the solvent cell shall be of the same batch as that used to prepare the solution and must be free from fluorescence at the wavelength of measurement. Ethanol, absolute ethanol, methanol and cyclohexane UV used as solvents shall have an absorbance, measured in a 1-cm cell at 240 nm with reference to water, not exceeding 0.10. Statements of concentration and thickness of the solution to be used in the determination of light absorption apply to measurements made with photoelectric instruments.

      SOLUTIONS FOR USE IN THE CALIBRATION OF WAVELENGTHS AND ABSORBANCE

      Holmium Perchlorate Solution Dissolve 40 g of holmium oxide in sufficient 1.4 M perchloric acid to produce 1000 ml.

      Potassium Dichromate Solution UV Dry a quantity of potassium dichromate by heating to constant weight at 130º. Weigh accurately a quantity not less than 57.0 mg and not more than 63.0 mg and dissolve it in sufficient 0.005 M sulfuric acid to produce 1000.0 ml.

Determination of Absorbance

      Unless otherwise prescribed, measure the absorbance, A, at the prescribed wavelength using a pathlength of 1 cm, and the measurements are carried out with reference to the solvent used to prepare the solution being examined. In certain cases measurements are carried out with reference to a mixture of reagents, details of which are prescribed in the monograph.

      When the absorbance is being measured for a quantitative determination, for example, an assay or a limit test, a manually scanning instrument is used. In tests for identification, it is more convenient to use a recording instrument and the concentration of the solution and the path-length are specified accordingly. If these conditions are not appropriate for a particular instrument, the thickness and the concentration of the solution should be varied. A statement in an assay or test of the wavelength at which maximum absorption occurs implies the maximum occurring either precisely at or in the vicinity of the given wavelength.

      When an assay or test prescribes the use of a reference substance, the spectrophotometric measurements are made first with the solution prepared from the reference substance and second with the corresponding solution prepared from the substance being examined. The second measurement is carried out as quickly as possible after the first, using the matched cell and the same experimental conditions.

      The requirements for light absorption in the Pharmacopoeia apply to the dried, anhydrous, or solventfree material in all those monographs in which standards for loss on drying, water or solvent content are given. In calculating the result, the loss on drying, water, or solvent content determined by the method specified in the monograph is used.

SECOND DERIVATIVE SPECTROPHOTOMETRY

      Derivative spectrophotometry involves the transformation of absorption spectra (zero order) into first, second or higher order derivative spectra. A first derivative spectrum is a plot of the gradient of the absorption curve (rate of change of the absorbance with wavelength, dA/dλ) against wavelength. A second derivative spectrum is a plot of the curvature of the absorption spectrum (d² A/dλ² ) against wavelength. If the absorbance follows the Beer-Lambert relationship, the second derivative at any wavelength, λ, is related to concentration by the following equation:

Apparatus

      A spectrophotometer complying with the requirements prescribed above and equipped with an analogue resistance-capacitance differentiation module or a digital differentiator or another means of producing second derivative spectra should be used in accordance with the manufacturer’s instructions. Some methods of producing second derivative spectra lead to a wavelength shift relative to the zero order spectrum and this should be taken into account, when necessary. Unless otherwise stated in the monograph, the spectral slit width of the spectrophotometer, where variable, should be set as described under Spectral slit width above. The cells used should comply with the statements given under the heading Cells.

Resolution

      When prescribed in a monograph, record the second derivative spectrum in the range 255 to 275 nm of a 0.020 per cent v/v solution of toluene in methanol using methanol in the reference cell. The spectrum shows a small negative extremum (or trough) located between two large negative extrema at 261 nm and 268 nm, respectively as shown in the figure. Unless otherwise prescribed in the monograph, the ratio A/B (see the figure) is not less than 0.2.

Procedure

      Prepare a solution of the substance being examined, adjust the various instrument settings according to the manufacturer’s instructions and calculate the amount of the substance being determined as prescribed in the monograph.