Advantages of Spectroscopy

(a) Spectroscopic methods are much more rapid and much less time consuming.

(b) They give information which is recorded in the form of a permanent chart, generally in an automatic or semiautomatic manner.

(c) They require very small amount (at mg and µg levels) of the compound and even this amount can be recovered at the end of examination in many cases.

(d) The structural information gained by spectroscopic methods is much more precise and reliable.

(e) They are much more selective and sensitive and are extremely valuable in the analysis of highly complex mixtures and in the detection of even traces of impurities

(f) With these methods, continuous operation is often possible and this facilitates automatic control of process variable in Industry.

1.3.1 Derivative spectrophotometry 11-14

For the purpose of spectral analysis in order to relate chemical structure to electronic transitions, and for analytical situations in which mixture contribute interfering absorption, a method of manipulating the spectral data is called derivative spectroscopy. Derivative spectrophotometry involves the conversions of a normal spectrum to its first, second or higher derivative spectrum. In the context of derivative spectrophotometry, the normal absorption spectrum is referred to as the fundamental, zero order, or D0 spectrum. The first derivative D1 spectrum is a plot of the rate of change of absorbance with wavelength against wavelength i.e. a plot of the slope of the fundamental spectrum against wavelength or a plot of dA/d? vs. ?. The maximum positive and maximum negative slope respectively in the D spectrum corresponds with a maximum and a minimum respectively in the D1 spectrum.

The ? max in D spectrum is a wavelength of zero slope and gives dA/d? = 0 in the D1 spectrum.

These spectral transformations confer two principal advantages on derivative spectrophotometry. Firstly, an eve order spectrum is of narrower spectral bandwidth than its fundamental spectrum. A derivative spectrum therefore shows better resolution of overlapping bands than the fundamental spectrum and may permit the accurate determination of the ? max of the individual bands. Secondly derivative spectrophotometry discriminates in favour of substances of narrow spectral bandwidth against broad bandwidth substances. All the amplitudes in the derivative spectrum are proportional to the concentration of the analyte, provided that Beer’s law is obeyed by the fundamental spectrum.

Fig 1 F1 shows a hypothetical absorption band and second derivative. Absorption maximum is associated with a negative slope in the first derivative and with a minimum in the second derivative.

The UV-Visible spectra consist of increasing or decreasing absorbance as function of wavelength A= f (?): Zero order.

Fig 1.1 The zeroth (a), first (b), and second (c) derivative spectra of Gaussian

band

In Fig 1 F2 the first or higher derivative spectra of absorbance or transmittance with respect to wavelength is recorded versus the wavelength.

dA/d? = f’ (?): First order (D1),

d2A / d?2 = f” (?): Second order (D1),

d3A / d?3 = f” (?): Third order (D1),

d4A / d?4 = f” (?): Fourth order (D1),

Advantages of derivative spectroscopy

1. Positions of local maxima are precisely defined even if the absorption spectrum is diffuse. As result, minor details of spectrum become enhanced. These details aid in distinguishing between similar spectra of different compounds.

2. Compounds in which absorption spectra overlap and cannot be separated by conventional methods, are easily recorded.

3. In quantitative analysis, selectivity and sensitivity are increased.

Fig 1.2 First (b) Second (c) Third (d) and Fourth derivative Spectrum (e) of Gaussian peak (a)

If an analysis of binary mixture of X and Y is to be carried out by this method, first or second derivative spectra of individual component is generated provided that peaks and valleys of X and Y are dissimilar. Fig 1 F3 represents the overlain derivative spectra of drugs X and Y.

At wavelength of zero crossing of derivative spectra of X, the component Y should show dA/d? or d2A / d?2 and vice versa. Since the values dA/d? and d2A / d?2 also obeys Beer’s Lambert’s law. First or second derivative spectra of various known concentration of mixtures of X and Y are analyzed taking zero crossing wavelength of X to measure Y and vice versa.

Fig 1.3 Overlay spectra of X and Y drugs by first order derivative method

A calibration curve of dA/d? or d2A / d?2 Vs concentration can be prepared for each compound, which can be employed for quantitative estimation.

1.3.2 Instrumentation of UV Visible spectrophotometer

Instruments for measuring the absorption of UV or visible radiation (Fig 1 F4) are made up of the following components;

1. Sources (UV and visible) – Hydrogen or deuterium (UV), tungsten lamp

2. Wavelength selector- Prism or grating monochromators

3. Sample containers – Quartz, glass cuvette.

4. Detector.

5. Signal processor and readout.

Fig 1.4 Schematic Diagram of UV–Visible spectrophotometer

1.4 CHROMATOGRAPHY 15, 16

The techniques through which the chemical components present in complex mixtures are separated, identified and determined are termed as chromatography.

Chromatography can be simply defined as follows:

“It is the technique in which the components of a mixture are separated based upon the rates at which they are carried or moved through a stationary phase (column) by a gaseous or liquid mobile phase”.

Based on the mobile phase this technique can be simply classified into two categories

A. Liquid Chromatography

B.Gas Chromatography

In chromatography, the stationary phase may be solid or liquid and the mobile phase may be liquid or gas. Depending upon the stationary and the mobile phase used, separation occurs because of a combination of two or more factors such as rates of migration, capillary action, extent of adsorption etc. Chromatographic methods can be classified on the basis of the stationary and the mobile phases used(Table 1.1).