**15. Types of spectrophotometers**

#### **15.1 Single-beam system**

UV radiation is delineated by using the source. A convex lens accumulates the beam of emission and focal point it on the inlet splinter. The inlet splinter allows light from the source to bypass, however blocks out stray radiation. The light then reaches the monochromator, which splits it up consistent with wavelength. The exit splinter is positioned to permit mild of the required wavelength to skip thru. The chosen radiation passes through the pattern cells to the detector, which measures the depth of the radiation attaining it.

Next, to differentiate the depth of radiation preparatory to stop after it passes through the pattern, it is feasible to degree several radiation is absorbed by the pattern on the unique wavelength used. The output of the detection is commonly recorded on graph paper.

The drawback is that estimates the whole quantity of mild accomplishing particular detector, as opposed to particular proportion wrapped. The source of intensity may vary with changes in line voltage. For example, when the line voltage decreases, the intensity of the light coming from the source may decrease. The single-beam spectrophotometer is depicted in **Figure 17**.

#### **15.2 Double-beam system**

The radiation from the supply is authorised to skip thru a reflect device to the monochromator. The activity of the monochromator is to permit a slender variety of wavelengths to skip continuously an go-out slit. The radiation popping out of the monochromator through the go-out slit is received via the rotating zone which divides the beam into, one glancing through the reference and the opposite through the sample cellular. After glancing through the sample and reference mobile, the light beams are focussed onto the detector.

The yield of the detector is hooked up towards a development touchy amplifier which reciprocates to any trade-in transmission through sample and reference. The segment empathetic amplifier transmits the indicators to the recorder that is accompanied with the aid of the motion of the pen or chart. The chart drive is to integrate the rotation of the prism and for this reason, the optical density or transmission of the pattern is set down as a characteristic of wavelength.

The advantage is not necessary to continually replace the blank with the sample or to zero adjust at each wavelength. The ratio of the powers of the sample and

**Figure 17.** *Single beam spectrophotometer.*

*UV-Visible Spectroscopy for Colorimetric Applications DOI: http://dx.doi.org/10.5772/intechopen.101165*

**Figure 18.** *Double beam spectrophotometer.*

reference beams is constantly obtained and used. Any error due to variation in the intensity of the source and fluctuation in the detector is minimised (**Figure 18**).

#### **16. Applications**

#### **16.1 Detection of conjugation**

It enables to identify the relationship between the exceptional groups, especially with appreciate to conjugation can be among or extra carbon–carbon (double or triple) bonds, between carbon–carbon and carbon–oxygen double bonds and between double bonds and at an aromatic ring [11].

#### **16.2 Detection of geometrical isomers**

The trans isomers exhibit λmax at slightly longer wavelength and feature larger extinction coefficients than the Cis isomers. Examples Stilbenes in trans isomers show λmax at 294 nm, while the λmax Cis isomer has 278 nm. Detection of functional groups: to detect the presence of certain functional groups is possible, like conjugation, carbonyl group, and benzene ring.

Molecular weight determination: the molecular weight is determined. For example, the molecular weight of any amine is converted into amine picrate. Then a known concentration of amine picrate is dissolved in a litre of solution and its optical density is measured at λmax at 380 nm.

#### **16.3 Dissociation constants of acids and bases**

Consider an Acid (HA), it undergoes dissociation in water to form H3O+ and A�, i.e.,

HA þ H2O ! H3O<sup>þ</sup> and A�

#### **16.4 Tautomeric equilibrium**

To determine the percentage of various keto and enol forms present in a tautomeric equilibrium. Example: Ethyl acetoacetate in keto form has λmax 275 nm and ε = 16. This has only weak n ! π\* band of the isolated carbonyl group. The enol

form has λmax 244 nm and ε = 16,000, we can measure the proportions of tautomers present in ethyl acetoacetate.

## **16.5 Detection of impurities**

The presence of impurities can be determined by the additional peaks and can be compared with that of standard raw material.

#### **16.6 Structural elucidation**

The presence or absence of unsaturation, the presence of heteroatom like S, O, N or halogen can be determined.

It is used to find out the percentage purity of samples from the formulations or raw material.
