Introduction:

The major use of ultraviolet and visible spectroscopy is in quantitative analysis. Most of the plasma constituents, drugs and other substances are assayed by techniques that are based on the measurement of the absorption of a solution of the substance at a specified wavelength in the UV/Visible regions. The other applications comprise determination of the pKa of a molecule where a pH-dependent UV shift is generated and as well for finding out the physico-chemical properties of drug molecules prior to formulation and for measuring their discharge from formulations (that is, dissolution studies).

Application of UV/Visible Spectroscopy in Quantitative Analysis:

Most of the organic compounds and inorganic complexes might be found out through direct absorptiometry by employing the Beer-Lambert Law. The computation of the concentration is made either through direct substitution of the suitable quantities in the equation for the Beer-Lambert law if the compound follows the law or through plotting a calibration curve of the concentrations of solutions of known strength against their absorbance at a particular wavelength and reading the concentration of the unknown solution from the graph after measuring its absorbance (figure shown below). The latter can be taken out automatically via the spectrometer that can as well comprise an internal standardization routine. Even if the compound doesn't strictly follow the Beer-Lambert law the concentration of a solution can still be obtained by employing a calibration curve provided an adequate number of points are plotted.

Fig: Typical Calibration Curves

The Beer-Lambert law is followed in (a) but not in (b). 

It is significant to recognize that for most of the accurate work or determination of trace amounts, three criteria should be noticed.

The absorptivity of the species to be determined should be reasonably big. Whereas it is possible to find out metals like copper or cobalt in water as the aqua complex, this will provide accurate outcomes only down to around 1% as ε ~ 10m²mol^-1. Though, for anthracene, C₁₄H₁₀, which consists of three fused aromatic rings, ε = 18000 m² mol^-1and therefore, even a solution of around 0.5 ppm will give an absorbance of approximately 0.1 in a 1 cm cell.

These species should be stable in solution. It should not oxidize or precipitate or change throughout the analysis (except the analysis intends to study that change).

Calibration should be carried out over the range of concentrations to be determined. Agreement by the Beer-Lambert law should be established.

In complex matrices, it is not possible to examine for all the species present by using a few spectra. It is essential to separate the components by employing a chromatographic method.

It must be noted that it is possible to find out two (or more) species in an analytical sample via measuring the absorbance at several wavelengths. Calibration and measurements at two wavelengths allows two components to be found out simultaneously, however if more wavelengths are measured, a better fit of the experimental data is accomplished.

Example:

The two organic components X and Y encompass absorption maxima at 255 and 330 nm, correspondingly.

For a pure solution of X, ε(255) = 4.60; ε(330) = 0.46

For a pure solution of Y, ε(255) = 3.88; ε(330) = 30.0

For a mixture of X and Y in a 0.01 m cell, A(255) = 0.274 and A(330) = 0.111

Compute the concentrations of X and Y in the mixture.

By using the Beer-Lambert law at each and every wavelength: 

A = ε_xc_xl + ε_yc_yl

At 255nm: 

0.274/0.01 = 4.60c_x + 3.88c_y

At 330 nm: 

0.111/0.01 = 0.46c_x + 30.0c_y

Solving these simultaneous equations provides: 

C_x = 5.71 molm^-3 = 5.71 x 10^-3 M

C_y = 0.288 molm^-3 = 2.88 x 10^-4 M

Applications of UV/Visible Spectroscopy in Pharmaceutical Quantitative analysis:

The pharmaceutical industries heavily rely on simple analysis via UV/Visible Spectrophotometry to find out the active ingredients in the formulations. These methods are generally based on the use of standard A (1%, 1cm) value for the active ingredient being assayed and this relies on the instrument being precisely calibrated. There must be no interference from excipients (preservatives, colorants and so on) present in the formulations and the sample must be free of suspended matter that could cause light scattering.

Example:

A typical illustration of a straightforward assay is the analysis of the furosemide tablets:

Tablet powder having ca. 0.25 g of furosemide (frusemide) is shaken by 300 ml of 0.1 M NaOH to extract the acidic furosemide (frusemide). The extract is then formed to 500 ml by 0.1 M NaOH.

A part of the extract is filtered and 5 ml of the filtrate is made up to 250 ml by 0.1M NaOH.

The absorbance of the diluted extract is computed at 271 nm. The A (1%, 1cm) value at 271 nm is 580 in the basic solution.

From the data below compute the % of stated content in a sample of the furosemide tablets: 

Stated content per tablet; 40 mg of furosemide (frusemide)

Weight of 20 tablets = 1.656 g

Weight of tablet powder taken for assay = 0.5195 g

Absorbance reading = 0.596

Calculation:

Expected content in the tablet powder taken = (0.5195/1.656) x 40 x 20 = 251.0 mg

Concentration in diluted tablet extract = (0.596/580) = 0.001028 g/100 ml = 1.028 mg/100 ml

Concentration in original tablet extract = 1.028 x 50 = 51.40 mg/100 ml

Volume of original extract = 500 ml

Thus, 

Amount of furosemide (frusemide) in original extract = 51.40 x 5 = 257.0

Percentage of stated content = (257.0/251.0) x 100 = 102.4 %

Application of UV/Visible Spectroscopy in the determination of pKa Values:

This is possible to make use of UV/Visible spectroscopy to find out the pKa of the ionisable group responsible whenever a pH-dependent UV shift is produced. In case of phenylephrine, the pKa value of the phenolic group can be found out conveniently from the absorbance at 292 nm, as the absorbance of the molecular species where the phenolic group in unionized is negligible at this wavelength. This is not the case for all the molecules. A general equation for the determination of pKa from absorbance measurement at a specific wavelength is illustrated below. The equation below can be employed for an acid (for a base the log term is subtracted) where increasing pH generates a bathochromic or hyperchromic shift:    

pKa = pH + log (Ai-A/A-Au)

Here, 

A = Measured absorbance in a buffer of known pH at the wavelength selected for analysis: 

• Ai = Absorbance of the completely ionized species
• Au = Absorbance of the unionized species

The wavelength employed for the analysis is one where there is great difference between the ionized and unionized species. An approximate knowledge of the pKa value is needed to choose an appropriate pH value, within ± 1 of the pKa value, for measurement of A. For precise determination; the measurement is made at a number of closely spaced pH values. It must be noted that if the acid or base undergoes a shift to the lower absorbance and shorter wavelength with increasing pH the log term above is subtracted; this condition is less common in drug molecules.

Example:

The absorbance of a fixed concentration of phenylepherine at 292 nm is found to be 1.224 in 0.1M NaOH and 0.02 in 0.1M HCl. Its absorbance in buffer at pH 8.5 is determined to be 0.349.  Compute the pKa value of its acidic phenolic hydroxyl group.

pKa = 8.5 + log [(1.224-0.349)/(0.349-0.02)] = 8.5 + 0.402 = 8.902

Application of UV/Visible spectroscopy in pre-formulation and formulation of drugs

The Physico-chemical properties of drug molecules prior to formulation and studying of the discharge of drugs from formulations can be found out by UV/Visible spectroscopy. The kinds of properties that can be helpfully found out by UV method are as follows:

Partition Coefficient:

The partition coefficient of a drug between water and an organic solvent might be found out via shaking the organic solvent and the water layer altogether and finding out the amount of drug in either the aqueous or organic layer via UV spectroscopy. If buffers of different pH values are employed, the variation of partition coefficient with pH might be found out and this gives another means of finding out the pKa value of a drug.

Solubility:

The solubility of a drug in, for example, water might be simply determined through shaking the excess of the drug in water or buffer until equilibrium is reached and then by employing UV spectroscopy to find out the concentration of the drug that has gone into solution. The solubility of an ionisable group present in the drug can be found out by dissolving varying concentrations of the salt of the drug in water and then adding surplus acid to a solution of the salt of an acidic drug or excess base to a solution of the salt of a basic drug, therefore converting the drugs into their un-ionized forms. Whenever the solubility of the un-ionized drug in water is exceeded, a cloudy solution will yield and UV Spectrophotometry can be employed to find out its degree of turbidity via light scattering, which can be measured at nearly any wavelength example: 250 nm.

Release of a Drug from a formulation:

UV Spectrophotometry is routinely employed to examine in vitro release of active ingredients from formulations (that is, drug dissolution studies).

Identification of Chromophores in Qualitative Analysis:

UV and visible spectra are employed to recognize chromophores in the qualitative analysis.  Identification is taken out by comparing the spectrum of the unknown compound to those of the known chromophores via consulting appropriate source books, like Organic Electronic Spectral Data (published by Wiley). If, in experimental error, the spectrum of the unknown compound matches that of a chromophore in the source book, it is taken as proof that the chromophore is found in the structure of the unknown compound. The procedure is identical to that employed to recognize a person from their fingerprints. The process follows no set rules and it is principally a matter of experience.

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