*4.2.2 Derivative spectrophotometry*

In derivative spectrophotometry the absorbance (A) of a sample is differentiated with respect to wavelength (λ) to generate the first, second or higher order derivative [18].

In the context of derivative spectrophotometry, the normal absorption spectrum is referred to as the fundamental, zero order or <sup>0</sup> D spectrum.


The first derivative (1 D) 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. The second derivative (2 D) spectrum is a plot of the curvature of the <sup>0</sup> D spectrum against wavelength.

The first order derivative spectrum of an absorption band is characterized by a maximum, a minimum and a crossover point at λmax of the absorption band. This bipolar function is characteristic of all odd-order derivatives.

The second derivative spectrum is characterized by two satellite maxima and an inverted band of which the minimum corresponds to the λmax of the fundamental band.

A derivative spectrum is therefore gives better resolution of overlapping bands than the corresponding fundamental spectrum and may permit the accurate determination of the λmax of the individual bands. Secondly, it discriminates in favor of substances of narrow spectral band width against those with broad bandwidth. And consequently, substances with narrow spectral bandwidth display larger derivative amplitude than those with broad bandwidth [15].

These advantages of enhanced resolution and band width discrimination found in derivative spectrophotometry permit the selective determination of certain

absorbing substances in samples in which non-specific interference may limit the application of simple spectrophotometric methods. Ephedrine hydrochloride in ephedrine hydrochloride elixir is assayed by second derivative spectrophotometry, which eliminates the broad band absorption of the excipient.

Derivative spectrophotometry has found significant application in clinical, forensic and biomedical analysis. It has been widely applied in the analysis of different pharmaceutical dosage forms. It solves the problem of analysis associated with drug combination, stability studies of drug and degradation products, drug impurities and interference of excipient in drugs [19, 20]. It also solves the problem of analysis of drugs in biological fluids.

### *4.2.3 Difference spectrophotometry*

Both selectivity and accuracy of spectrophotometric analysis of samples, which contain absorbing interferons, may be greatly improved by the technique of difference spectrophotometry. In difference spectrophotometry assays the measured value is the difference in absorbance (∆A) between two equimolar solutions of the analyte, in different chemical forms which exhibit different spectral characteristics. It is sometimes referred to as differential spectrophotometry.

Certain criteria are required for applying difference spectrophotometry for the analysis of a substance in the presence of other absorbing substances:


The simplest and most commonly used techniques for altering the spectral properties of the analyte is the adjustment of the pH of the solution by means of aqueous solution of acids, alkali or buffers [21]. The measured value (∆A) in a quantitative difference spectrophotometric assay can be proportional to the concentration of the analyte and so it obeys Beer's law. A modified equation may be derived.

$$
\Delta \mathbf{A} = \Delta \mathbf{a} \mathbf{b} \mathbf{c} \tag{2}
$$

**33**

*Drug Analysis*

ary phase.

*4.3.1 Liquid chromatography*

*DOI: http://dx.doi.org/10.5772/intechopen.88739*

should be carefully monitored and assessed.

**4.3 Chromatographic methods (HPLC and TLC)**

raphy (GC) where the mobile phase is a gas [15].

d.Size-exclusion chromatography.

ary phase and liquid mobile phase.

(LSC), partition (LLC), ion exchange and size-exclusion.

tical analysis) are subdivided to:

liquid mobile phase.

*4.3.2 Thin-layer chromatography (TLC)*

There are four classifications for liquid chromatography:

meric stationary phase containing replaceable ions.

a.Adsorption chromatography: liquid solid chromatography (LSC).

b.Partition chromatography: liquid-liquid chromatography (LLC).

c.Ion exchange chromatography: an ionic liquid mobile phase and a solid poly-

The adsorption and partition chromatography (most widely used in pharmaceu-

a.Column chromatography and thin-layer chromatography (TLC): solid station-

b.Column, paper and thin-layer chromatography: liquid stationary phase and

High performance liquid chromatography (HPLC) belongs to the category of column chromatography and it covers four classes of chromatography: adsorption

Thin-layer chromatography has developed into a very sophisticated technique for identification of compounds and for determination of the presence of trace

shifted to the visible region.

methods. This can be achieved by selectively transforming a drug, its degradation product or its impurity into a derivative so that the spectrum of the derivative is

There are several parameters, which require careful and critical consideration in colorimetry. Firstly, the color reagent should be selective for the drug molecule itself, discriminating degradation products which might be present. Secondly, the effect of any parameters which can affect the development and stability of the color should be established. Moreover, the time required to establish the chromophore

Chromatography is essentially a group of techniques for the separation of the compounds of mixtures by their continuous distribution between two phases. One of the two phases is the fixed (stationary) phase, which can be solid or a liquid supported on a solid. The other phase is a moving (mobile) phase which can be gas or a liquid that flows continuously around the station-

According to the nature of the mobile phase, chromatography is subdivided into liquid chromatography (LC) where the mobile phase is a liquid, and gas chromatog-

Where ∆a is the difference absorptivity of the substance at the wavelength of measurement.

The accuracy and selectivity of the method was found to be increased by conversion of normal zero-order or differential UV spectra into higher order [21, 22]. Therefore, the application of difference spectrophotometry is expected to have the totality of advantages of both derivative spectrophotometry (first, second, etc.) combined with delta spectrophotometry [23].

On the other hand, the stability-indicating property, coupled with the selectivity and simplicity of application, of the derivative spectrophotometry (first, second, etc.) and ∆D1 make these methods more preferable to use for drug analysis than the costly HPLC methods, especially in developing countries.

#### *4.2.4 Colorimetric method*

Colorimetric methods, although are generally dependent on functional group in the drug molecule (NH2, OH, SH), are sometimes utilized as stability-indicating

## *Drug Analysis DOI: http://dx.doi.org/10.5772/intechopen.88739*

*Pharmaceutical Formulation Design - Recent Practices*

of analysis of drugs in biological fluids.

addition of one or more reagents.

combined with delta spectrophotometry [23].

costly HPLC methods, especially in developing countries.

such reagents.

measurement.

*4.2.4 Colorimetric method*

*4.2.3 Difference spectrophotometry*

which eliminates the broad band absorption of the excipient.

It is sometimes referred to as differential spectrophotometry.

analysis of a substance in the presence of other absorbing substances:

analyte and so it obeys Beer's law. A modified equation may be derived.

absorbing substances in samples in which non-specific interference may limit the application of simple spectrophotometric methods. Ephedrine hydrochloride in ephedrine hydrochloride elixir is assayed by second derivative spectrophotometry,

Derivative spectrophotometry has found significant application in clinical, forensic and biomedical analysis. It has been widely applied in the analysis of different pharmaceutical dosage forms. It solves the problem of analysis associated with drug combination, stability studies of drug and degradation products, drug impurities and interference of excipient in drugs [19, 20]. It also solves the problem

Both selectivity and accuracy of spectrophotometric analysis of samples, which contain absorbing interferons, may be greatly improved by the technique of difference spectrophotometry. In difference spectrophotometry assays the measured value is the difference in absorbance (∆A) between two equimolar solutions of the analyte, in different chemical forms which exhibit different spectral characteristics.

Certain criteria are required for applying difference spectrophotometry for the

2.The absorbance of the interfering substances is not altered by the addition of

The simplest and most commonly used techniques for altering the spectral properties of the analyte is the adjustment of the pH of the solution by means of aqueous solution of acids, alkali or buffers [21]. The measured value (∆A) in a quantitative difference spectrophotometric assay can be proportional to the concentration of the

Where ∆a is the difference absorptivity of the substance at the wavelength of

The accuracy and selectivity of the method was found to be increased by conversion of normal zero-order or differential UV spectra into higher order [21, 22]. Therefore, the application of difference spectrophotometry is expected to have the totality of advantages of both derivative spectrophotometry (first, second, etc.)

On the other hand, the stability-indicating property, coupled with the selectivity and simplicity of application, of the derivative spectrophotometry (first, second, etc.) and ∆D1 make these methods more preferable to use for drug analysis than the

Colorimetric methods, although are generally dependent on functional group in the drug molecule (NH2, OH, SH), are sometimes utilized as stability-indicating

∆A = ∆abc (2)

1.Reproducible changes are induced in the spectrum of the analyte by the

**32**

methods. This can be achieved by selectively transforming a drug, its degradation product or its impurity into a derivative so that the spectrum of the derivative is shifted to the visible region.

There are several parameters, which require careful and critical consideration in colorimetry. Firstly, the color reagent should be selective for the drug molecule itself, discriminating degradation products which might be present. Secondly, the effect of any parameters which can affect the development and stability of the color should be established. Moreover, the time required to establish the chromophore should be carefully monitored and assessed.
