**3. Spectrophotometric techniques fundamentals, important, and its applications in pharmaceutical analysis**

#### **3.1 Fundamentals of spectrophotometric techniques**

Ultraviolet–visible spectrophotometry indicates the absorption spectrum in the region between 200 and 800 nm. The absorption in the ultraviolet and visible region depended on the molecules that contain π electrons and non-bonding electrons pairs, which can absorb the energy of ultraviolet or visible light to rise to a higher antibonding molecular orbital. The more easily excited the electrons, the longer the wavelength of light they can absorb.

This technique is one of the spectroscopic methods based on the interaction of electromagnetic radiation with the material. Electromagnetic radiation (**Figure 2**) is considered as waves of energy propagated from a source in space and consists of oscillating electric and magnetic fields at right angles to each other. Each

**Figure 2.** *Electromagnetic spectrum.*

Electromagnetic radiation has characteristics of wavelength (λ), frequency (ν), or wave number, ν [<sup>1</sup> <sup>λ</sup>]. Molecule or ion may absorb energy from Electromagnetic radiation of suitable wavelength (or frequency) resulting in: (a) Electronic excitation caused by absorption of UV–visible radiation leading to UV–visible spectroscopy. (b) Molecular rotation by absorption of microwave radiation leading to microwave. (c) Vibrational excitation is caused by the absorption of infrared radiation leading to infrared spectroscopy.

The UV–visible spectral method involves UV–visible spectroscopy. This arises due to the absorption of ultraviolet (UV) or visible radiation with the sample resulting in an electronic transition within the molecule or ion. The relationship between the energy absorbed in an electronic transition, the frequency (ν), wavelength (λ), and wave number (ν) of radiation-producing transition is:

$$
\Delta E = hv = h\ \frac{c}{\lambda} = hvc\tag{5}
$$

where h is Planck's constant, c is the velocity of light. Δ*E* is the energy absorbed during an electronic transition in a molecule or ion from a lower-energy state (E1) (ground state) to a high-energy state (E2) (excited state). The energy absorbed is given by

$$
\Delta E = E \mathbf{2} - E \mathbf{1} = hv \tag{6}
$$

Potentially, three types of ground state orbitals may be involved: (i) σ (bonding) molecular as in C – C, (ii) π (bonding) molecular orbital as in C = C, and (iii) n (nonbonding) atomic orbital as in C – Br, C – OH. In addition, two types of antibonding orbitals may be involved in the transition, σ\* (sigma star) orbital and π\* (pi star) orbital. A transition in which a bonding σ electron is excited to an antibonding σ orbital is referred to as σ – σ\* transition. In the same way, π – π\* represents the transition of one electron of a lone pair (non-bonding electron pair) to an antibonding π orbital. Thus the following electronic transitions can occur by the absorption of ultraviolet and visible light: σ – σ\* , n – σ\*, n – π\* , π – π\* . The energy required for various transitions (**Figure 3**) obeys the following order: σ – σ\* > n – σ\* > π – π\* > n – π\* .

σ – σ\* transition: This transition can occur in compounds in which all the electrons are involved in the formation of single bonds (σ-bond only) and there is no lone pair of an electron, such as saturated hydrocarbon like methane, ethane, etc. which requires radiation of high energy with short wavelength (less than 150 nm). The usual

**Figure 3.** *The types of the transition electrons.*

measurement cannot be done below 200 nm. Thus the region of transition below 200 nm is called the vacuum ultraviolet region. Methane which contains only C – H, σ-bond can undergo σ – σ\* transition exhibiting absorption peak at 125 nm. Ethane has an absorption peak at 135 nm which also must arise from the same type of transition but here electrons of C – C bond appear to be involved. Since the strength of the C – C bond is less than that of C – H bond, less energy is required for excitation, as a result, absorption occurs at a lower wavelength. Thus organic molecules in which all the valence shell electrons are involved in the formation of σ-bonds do not show absorption in the normal ultraviolet region, that is, 180–400 nm. n – σ\* transition: This type of transition takes place in a saturated compound containing one hetero atom with unshared pair of electrons. Examples of such transitions are saturated alkyl halides, alcohols, ethers, amines, etc. which are commonly used as a solvent because they start to absorb at 260 nm. However, these solvents cannot be used when measurements are to be made in 200–260 nm. In such cases saturated hydrocarbons which only give rise to σ – σ\* transition must be used. However, the drawback is that these are poor solvating agents. π – π\* transition: This transition is available in compounds with unsaturated centers of the molecules. Examples of such transitions are alkenes, alkynes, aromatics, carbonyl compounds, etc. this transition requires lesser energy, and hence, the transition of this type occurs at a longer wavelength within the region of the UV-spectrophotometer. In unconjugated alkenes, the absorption band is around 170–190 nm. In carbonyl compounds, the band due to π – π\* transition appears at 180 nm and is more intense, that is, the value of the molar extinction coefficient is high. The introduction of the alkyl group to the olefinic linkage shifts the position of the band to a longer wavelength by 3–5 nm per alkyl group. The shift depends on the type of the alkyl group and the stereochemistry of the double bond. n – π\* transition: This type of transition occurs in unsaturated bonds containing at least one hetero atom like O, N, S, and halogen with n electron. Examples of such transitions are aldehydes and ketones, etc. Saturated aldehydes (C = O) show both types of transitions, that is, low energy n – π\* and high energy π – π\* occurring around 290 and 180 nm, respectively. In aldehydes and ketones n – π\* transition arises from the excitation of a lone pair of electrons in a 2p orbital of an oxygen atom with the anti-bonding π orbital of the carbonyl group. When hydrogen is replaced by an alkyl group as in ketone, this results in the shift of the band to a shorter wavelength. Besides the above transition,

high energy but quite intense π – π\* transition also occurs in carbonyl compounds. However, the molar extinction coefficient (ε) values associated with n – π\* transition are generally low and range from 10 to 100 while values for π – π\* transition, on the other hand, normally fall in the range between 1000 and 10,000.

The quantitative analysis using UV–visible spectrophotometry is based mainly on the Beer-Lambert law, which explains the relationship between the absorbance of analyte under analysis and its concentration:

$$A = \log \mathbf{I}\_0/\mathbf{I} = \mathbf{e} \mathbf{C} \mathbf{x} \tag{7}$$

where ε is molar absorptivity, x is the path length, and C is the concentration of analyte.
