PHYSICAL CHEMISTRY OF MILK

Module 12. Nuclear chemistry

Lesson 32
VISIBLE AND ULTRAVIOLET SPECTROPHOTOMETER

32.1 Introduction

The electromagnetic radiation and its spectra are of immense value in developing instruments useful for the analysis of various compounds. Development of spectrophotometer is an outcome of the study of this spectrum. The absorption or reflectance tested using the visible light or near Ultra violet (UV) and Near Infrared (NIR) is referred as absorption spectroscopy or reflectance spectroscopy. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo electronic transitions. This technique is complementary to fluorescence spectroscopy. In fluorescence transition from excited state to ground state is measured, whereas in absorption transition from the ground state to the excited state is measured.

32.2 Principle of Spectrophometers

Molecules containing π-electrons or non-bonding electrons (n-electrons) can absorb energy in the form of ultraviolet or visible light to excite these electrons to higher anti-bonding molecular orbitals. The energetically most favorable
π _>π* excitation occurs from the highest energy bonding pi-orbital (HOMO) to the lowest energy antibonding pi-orbital (LUMO). The more easily the electrons excited the higher wavelength of light it can absorb.

Accurate measurements of light absorption at different wavelengths in and near the visible part of the spectrum are made to understand why some compounds are colored and others are not and also to determine the relationship of conjugation in the compounds to color. Commercial optical spectrometers enable such experiments to be conducted with ease, and usually survey both near ultraviolet and visible portions of the spectrum.

The visible region of the spectrum comprises of photon energies of 36 to72 kcal/ mole, and in the near ultraviolet region this energy increases to 143 kcal/ mole. Ultraviolet radiation having wavelengths less than 200 nm is difficult to handle, and is rarely used as a routine tool for structural analysis. The energies noted above are sufficient to promote or excite a molecular electron to a higher energy orbital. Consequently, absorption spectroscopy carried out in this region is sometimes called "electronic spectroscopy".

Different compounds may have different absorption maxima and absorbance. Intensely absorbing compounds must be examined in dilute solutions, so that significant light energy is received by the detector, and this requires the use of completely transparent (non-absorbing) solvents. The most commonly used solvents are water, ethanol, hexane and cyclohexane. Solvents having double or triple bonds, or heavy atoms (e.g. S, Br & I) are generally avoided. Because the absorbance of a sample will be proportional to its molar concentration in the cuvette, a corrected absorption value, known as the molar absorptivity, is used when comparing the spectra of different compounds.

When sample molecules are exposed to light having an energy that matches a possible electronic transition within the molecule, some of the light energy will be absorbed as the electron is promoted to a higher energy orbital. An optical spectrometer records the wavelengths at which absorption occurs, together with the degree of absorption at each wavelength. The resulting spectrum is presented as a graph of absorbance (A) versus wavelength, as in the isoprene spectrum shown Fig. 32.1. Since isoprene is colorless, it does not absorb light in the visible part of the spectrum and this region is not displayed on the graph. Absorbance usually ranges from 0 (no absorption) to 1 (99% absorption), and is precisely defined in context with spectrometer operation. Because the absorbance of a sample will be proportional to the number of absorbing molecules in spectrometer light beam (e.g. their molar concentration in the sample tube), it is necessary to correct the absorbance value and other operational factors if the spectra of different compounds are to be compared in a meaningful way. The corrected absorption value is called "molar absorptivity", and is particularly useful when comparing the spectra of different compounds and determining the relative strength of light absorbing functions (chromophores). Molar absorptivity (ε) is defined as:

(where A= absorbance, c = sample concentration in moles/liter & l = length of light path through the sample in cm.)

Fig. 32.1 UV-visible spectrum of isoprene showing maximum absorption at 222 nm

(Source: www. pharmaxchange.info)

The UV visible spectrum for isoprene Fig. 32.1 shows maximum absorptivity (λ max) of 222 nm, and as there is no absorbance in the visible spectrum, the compound will not have any colour. Absorption of light in 200 to 800 nm region by molecular moieties is due to the function of pi-electron and hetero atoms having non-bonding valence-shell electron pairs. Such light absorbing groups are referred as chromophores. The oxygen non-bonding electrons in alcohols and ethers do not give rise to absorption above 160 nm. Consequently, pure alcohol and ether solvents may be used for spectroscopic studies. Though UV-Visible spectroscopy can detect the presence of these chromophores in a molecule, most instruments cannot provide absorption data for wavelengths below 200nm. Conjugation will help in moving the absorption maxima to longer wavelength as observed in the isoprene spectra. By UV-visible spetra it is possible to identify these structural features.

Molar absorptivities may be very large for strongly absorbing chromophores (>10,000) and very small if absorption is weak (10 to 100). The magnitude of ε reflects both the size of the chromophore and probability that light of a given wavelength will be absorbed when it strikes the chromophore. The terms used to define shifts in abosorption are shown in Table 32.1. Thus extending conjugation generally results in bathochromic and hyperchromic shifts in absorption. The appearance of several absorption peaks or shoulders for a given chromophore is common for highly conjugated systems, and is often solvent dependent. This fine structure reflects not only different conformations that conjugated systems may assume, but also electronic transitions between different vibrational energy levels possible for each electronic state. Vibrational fine structure of this kind is most pronounced in vapor phase spectra, and is increasingly broadened and obscured in solution as the solvent is changed from hexane to methanol.

Table 32.1 Terminology for absorption shifts

The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of absorbing species in the solution and the path length. Thus, for a fixed path length, UV/ Vis spectroscopy can be used to determine the concentration of absorber in a solution. It is necessary to know how quickly the absorbance changes with concentration. This can be taken from references (tables of molar extinction coefficients), or more accurately, determined from a calibration curve.

32.3 Milk and Dairy Applications

There is increasing awareness among consumers about the quality and safety of milk and milk products. The concentrations of constituents such as water, protein, fat and carbohydrate can in principle be determined using classical absorption spectroscopy Routine analytical methods used for measuring various milk components are sample destructive, expensive and time & labour intensive. Most commonly used instrument for milk analysis is Milko-scan which is based on the infra red spectroscopy.

The analysis of water by near infrared spectroscopy (NIRS) was the first successful application of this rapid technology. NIR spectroscopy is used routinely for the compositional, functional and sensory analysis of food ingredients, process intermediates and final products .Studies carried on NIR spectroscopy have made it possible to analyze milk for adulteration and for several constituents like fat, protein and lactose etc. The greatest advantage of NIR spectroscopy is that it is rapid and accurate. This technique can be used for analyzing various dairy products like milk powders, whey, cheese etc. An additional advantage of NIR spectroscopy is that it could be used online in the process of milking for effective farm management.

Atomic absorptive spectroscopy is helpful in the study of essential micro nutrients such as iodine content in infant milk formulae and milk powder samples.