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    Application of Spectral Studies in Pharmaceutical Product development: (Basic Approach with Illustrated Examples) First Revised Edition
    Application of Spectral Studies in Pharmaceutical Product development: (Basic Approach with Illustrated Examples) First Revised Edition
    Application of Spectral Studies in Pharmaceutical Product development: (Basic Approach with Illustrated Examples) First Revised Edition
    Ebook274 pages1 hour

    Application of Spectral Studies in Pharmaceutical Product development: (Basic Approach with Illustrated Examples) First Revised Edition

    By Ramalingam Peraman and Y. Padmanabha Reddy

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    About this ebook

    his book compiled with a special emphasis on the application of spectral and analytical data in the development and characterization of pharmaceuticals. This book provided lot of illustrated examples for better understanding of spectral applications, and also it will enhance the knowledge and working skills of the learners. This book also covered advanced techniques of current pharmaceutical industry interests such as XRD, Raman Spectroscopy, LC-MS/MS, LC-NMR, q-NMR, SEM/TEM etc. Thus, this book is formulated for the first time on the concept of spectral interpretation based on illustrated applications in real time research, and it will definitely attract young researchers, scholars, graduate students belonging to the disciplines of pharmaceutical, life and chemical sciences.
    Contents:
    1. UV?Visible Spectroscopy 
    2. Infra Red (IR) Spectroscopy 
    3. H–1 -NMR Spectroscopy 
    4. Electron Spin Resonance Spectroscopy 
    5. Mass Spectrometry and Interpretation 
    6. Raman Spectroscopy 
    7. Differential Scanning Calorimetry (DSC) 
    8. X?Ray Diffraction Studies 
    9. Scanning Electron Microscopy (SEM) 
    10. Quantitative Techniques using UV Spectroscopy 
    11. LC?NMR and Q?NMR 
    12. LC?MS/MS Analysis Applications
    LanguageEnglish
    PublisherBSP BOOKS
    Release dateMar 28, 2020
    ISBN9788194172772
    Application of Spectral Studies in Pharmaceutical Product development: (Basic Approach with Illustrated Examples) First Revised Edition

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      Book preview

      Application of Spectral Studies in Pharmaceutical Product development - Ramalingam Peraman

      Chapter 1

      UV-Visible Spectroscopy

      Introduction to Spectroscopy

      Spectroscopy and spectrography are the techniques developed based on the measurement of radiation or light intensity (absorbed or emitted or scattered by the sample or analyte) as a function of wavelength. Spectral measurement instruments are more commonly referred to as spectrometers, spectrophotometers, spectrographs, or spectral analyzers. These instruments measure the light intensity or resultant light from the sample, the light may be emitted or transmitted or scattered. Absorption is the measure of the difference between incident and transmitted light (Figure 1.1).

      Figure 1.1 Various interaction effects of light with matter (Analyte).

      In instrumentation, the output of the scan measurement of a sample from a range of wavelengths is represented as 2D and 3D UV-Spectrum. In general X axis (Input) will be wavelength; y-axis (output) will be light absorbed (eg. UV-Visible, IR, NMR, or any absorption spectroscopy) or light emitted (eg. fluorescence or any emission spectroscopy) or scattered (eg. Nephelometry, Raman spectroscopy). Three types of spectra can be produced by spectroscopy

      1. Continuous spectrum (UV-Visible Spectra, IR Spectra etc.)

      2. Emission lines (Atomic emission spectra)

      3. Absorption lines (Atomic absorption Spectra)

      Selection of Region based on Sample Nature

      The difference between UV and visible spectroscopy is the wavelength region and the sample used in the measurement. The UV region (190– 380 nm) is used for colorless samples (white substances that they do not show colour in solution form) and the visible region (380–790 nm) is used for coloured sample. Because of instrumental scanning of the sample using the UV-Visible region, the obtained spectrum is the characteristic of sample/molecule nature, it is highly depended on double bonds (pi electrons) and its configuration in the chemical structure.

      Absorption/Emission of Light by Chemical Molecules

      The mechanism behind the absorption of light by a molecule (analyte) or/and emission of light from the molecule (Analyte) depends on the phenomena of electronic excitation/transition (Figure 1.2). The amount (probability) and type of electrons (sigma, pi, n) involved in the excitation process will determine the characteristic photon energy absorbed and emitted by the molecule, thus it will determine the shape and pattern of UV-Visible Spectrum.

      Two different chemical molecules will never have the same number of electrons and double bond configuration. Thus, the UV spectrum will be different for different compounds.

      Figure 1.2 UV-Visible absorption and emission process of matter (Analyte).

      •Figure 1.2 shows the electronic excitation process and the relaxation process of electrons in molecules when UV-Visible light is irradiated on the sample.

      •Blue and red lights indicate to understand the wavelength comparison.

      •NOTE : sample should be at low concentration (<100 µg/ml for absorption spectroscopy and <5 µg/ml for emission spectroscopy), pure and highly transparent for any qualitative and quantitative analyses.

      Types of Electronic Transitions

      •Four different types of electronic transitions take place in the molecule when UV-Visible light is irradiated. Each molecule undergoes more than one electronic transition and requires different energies ( Figure 1.3 ), which depends on the chemical structure.

      •The four types of transitions are π–π*, n–π*, σ–σ*, and n–σ* and they can be ordered as follows based on the energy required σ–σ* > n–σ* > π–π* > n–π*.

      •Sigma transition requires higher energy that may occur at very low wavelengths of the UV region and in Vacuum UV region (< 190 nm).

      •The diagram below shows the comparison of different electronic transitions and energies in which n’ electron transition require less energy among all transitions (absorbs higher wavelength)

      Figure 1.3 Types of electronic transitions in organic molecules upon UV-Visible light exposure.

      From the above figure, we can observe that the energy required for the sigma transition (eg. Alkane) is the highest, which is not available in the UV region, so sigma transition may not be observed practically. Sigma transition may be possible at the vacuum UV region (<190 nm).

      How to predict the possible electronic transition for a given structure?

      To predict a possible transition, we should know the chemical structure and the type of electron available in the chemical structure Table 1.1.

      Table 1.1 Types of electronic transition and Chemical structures

      Schematic Procedure for Measuring Absorbance

      •In the instrumentation, a monochromatic light is always used to measure the absorbance (in quantitative analysis).

      •The monochromatic light to be used is selected from the UV spectrum (spectrum because of scan for entire UV-Visible range).

      •Preferably the lambda max of the compound is chosen as wavelength for quantitative analysis.

      •The sample cell needs to be a quartz cuvette in UV light measurement because glass can absorb UV light. For visible light measurement (colorimetric) glass cuvette can be used ( Figure 1.4 ).

      •The sample concentration should be very low (1–100 µg/ml) and transparent to avoid both scattering and Beer’s law deviations.

      Figure 1.4 Basic Instrumentation of UV-Visible Spectrophotometer.

      •Io is the intensity of incident light (monochromatic), It is the intensity of transmitted light.

      •If, Io = I t : The sample is inactive and indicates no absorption. This means that the molecule structure may not have n’ and pi electrons.

      •If Io is greater than It: This indicates the reduction of intensity of transmitted light due to absorption of photon energy by the molecular electrons by the process of electronic excitation.

      •Hence, Absorbance (A)

      A = log (Io/It) or

      A = –log T; (where T = It/Io; T = Transmittance)

      The UV Spectrum

      For example (2D- UV spectrum) the following UV spectrum shows the absorption in UV region (190 – 380 nm) that indicated the sample is colorless (Figure 1.5).

      The UV spectrum (Figure 1.5) is for Colorless sample shows a cut-off wavelength around 340 nm and lambda max of 260 nm. Thus, the spectrum shows no absorbance in the visible region.

      In the same way-coloured compound showed absorption maximum at 630 nm, but showed no absorbance in UV region (Figure 1.6).

      Figure 1.5 UV – Spectrum and UVmax (Lambda max) : Colourless Compound.

      Figure 1.6 UV – Spectrum and absorption maximum: Coloured Compound.

      Visible spectrum of the colored sample (Figure 1.6) indicated the absorption characteristics of the sample in the visible region (400–780 nm). Furthermore, the spectrum (Figure 1.7) showed that a spectrum characteristic (shape or pattern) are not affected by the sample concentration.

      Figure 1.7 UV – Spectrum Vs. Concentration (Shows that there is no change in spectrum characteristics).

      Beer-Lambert Law

      Absorption and concentration is related to Beer-Lambert law. In a simple sentence, it can be stated that the absorbance is directly proportional to the concentration of analyte (August Beer’s law) and path-length of the sample cell (Lambert’s law).

      The Beer-Lambert equation shall be expressed as following,

      A = A¹%cm. b. c

      Where

      •A = Absorbance, (log (Io/I)

      •b = Path length and c is concentration (g/100 ml),

      •A1%cm = The specific absorbance of the compound, which is constant (it is the absorbance of 1 % w/v solution in 1cm path length).

      •The above formula is recommended by Indian pharmacopoeia (2010, 2014) for the quantification of drug in pharmaceutical dosage form, when standard substance is not available.

      •The relationship between concentration and absorbance is expressed as a linearity curve and Beer-Lambert curve. This curve can be used for quantification if both standard and sample are available ( Figure 1.8 ).

      •In the curve, after a particular concentration, the linearity does not exist. That’s called Beer Law deviation; usually it may be either positive deviation or negative deviation.

      •To avoid the Beer’s law deviation, the instrument should be calibrated in the stray light should be within the limit, the concentration of sample should be low and the solution and cuvette should be transparent.

      Figure 1.8

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