ACHARYA NAGARJUNA UNIVERSITY COLLEGE OF PHARMACEUTICAL SCIENCES

SEMINAR PRESENTED BY O.SASIVARDHAN

Roll.no:Y15MPH326 IN PHARMACEUTICAL ANALYSIS

UV-VIS REFLECTANCE SPECTROSCOPY

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TS • Introduction

• Types of reflection• Principle• Instrumentation• Features of uv –visible reflection

spectroscopy spectra• Factors affecting the reflectance• Applications• References

INTRODUCTION

• Reflectance, which is the base quantity that characterizes the process of reflection, is defined as the ratio of the reflected radiant flux to the incident radiant flux (or power)

• Reflectance is introduced by Kubelka–Munk scientist’s.

• Reflectance can be measured wide wavelength range from 250-950 nm.

• Reflectance is characteristic for each of drug molecules, solid samples and paints

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1.Specular reflectance Mirror like reflectance from a surface with well defined angle of reflection

2.Diffuse reflectance Reflected radiant energy that has been partially absorbed, transmitted and partially scattered by a surface, with no defined angle of reflection

SPECULAR REFLECTANCEMirror like reflectance from a surface with well

defined angle of reflection.Use:-Analysis of films or coatings on reflective surfaces

e.g. polymer coatings on food containers

DIFFUSE REFLECTNCEReflected radiant energy that has been partially

absorbed, transmitted and partially scattered by a surface, with no defined angle of reflection.

Example :-Strongly absorbing samples like coal,

pharmaceuticals, plastics...

PRINCIPLE

SPECTRA

MODES OF REFLECTANCE MEASUREMENT

• This is the ratio of the diffusely reflected part of the (whole) reflected flux to the incident flux. The measurement of diffuse reflectance is made in comparison to a reflection standard (reflectance N) with a collimated or conical radiation beam. The signals of the detector are calculated as follows:

• ρd =   I (X) - I (stray) - ρ [ I (mi) - I (stray) ]   I (N) - I (stray) - ρN [ I (mi) - I (stray) ]

WHERE,I (X): signal with sample irradiationI (N): signal with standard irradiationI (stray): signal with open measurement portI (mi): signal with irradiance of a mirror

INSTRUMENTATION

• UV-Vis diffuse reflectance spectrophotometer the consist mainly following components.1.light source2.Interating sphere3.Sample holders4.Detectors

INTEGRATING SPHERE• An integrating sphere is an optical device used to

collect and measure electromagnetic radiation• An integrating sphere (also known as an Ulbricht

sphere) is an optical component consisting of a hollow spherical cavity with its interior covered with a diffuse white reflective coating, with small holes for entrance and exit ports

• Various coating material for integrating sphere are MgO, BaSO4 and PTFE (polytetrefluoroetylene) compounds .

WORKING OF I.S Upon entering the sphere, the light strikes the sample surface

and the highly reflective walls of the cavity, undergoing many diffuse reflections.

Radiation trapped inside the sphere cavity can dissipate by a combination of three ways. 1. Most of the light eventually is absorbed into the sphere walls.

2.Some of the light exits the cavity through one of the empty ports, and

3.A small portion of the trapped radiation strikes the active surface of the sphere detector

A steady state radiant flux distribution is established almost instantaneously within the sphere and remains as long as the beam source is active.

SAMPLE HOLDERS

Various types of sample holders used in DR UV-VIS Spectroscopy,

1.Centre-Mount Cuvette-style sample holder2.Clip and Jaw style centre mount sample

holders

Centre-Mount Cuvette-style sample holder The device is ideal for measuring turbid samples

such as sea water, proteins, and other biological solution.

Measurements performed with this device are in the 0°/d geometry. Since the cuvette is fixed at 0° angle of incidence, the specular component of reflectionis directed out of the sphere through the transmission port.

Clip and Jaw style centre mount sample holder

The clip style sample holder, uses a spring-loaded clip to hold the sample.

The sample must be in a sheet configuration and large enough to accommodate the entire sample beam

The jaw style sample holder is better for holding large, bulky samples. The sample must be opaque or an absorbent backing must be applied. The sample holder is designed for reflectance measurements of samples that do not transmit light

DETECTORS

The most commonly used detector in DR UV-VIS Spectrophotometer is photomultiplier tube.

• Principle of PMT is Photoelectric effect, Secondary emission of electrons

Features of Reflection uv –visible spectroscopy spectra

The R UV-VIS Spectra plot of percentage of reflectance against wavelength.

The 100% R implies sampleeffectively reflects 100% of all uv visible wavelengths of light that interacts with it.

Different types of samples will show different reflectance.

Factors affecting the reflectance 1.Particle Size – reducing the size of the sample particles

reduces the contribution of reflection from the surface. Smaller particles improve the quality of spectra (narrow bandwidths and better relative intensity). The recommended size of the sample/matrix particles is 50 micrometers or less. This fine powder is easily achieved by using a ShakIR mixer.

2.Refractive Index – effects result in specular reflectance contributions (spectra of highly reflecting samples will be more distorted by the specular reflectance component). This effect can be significantly reduced by sample dilution.

• 3.Homogeneity – samples prepared for diffuse reflectance measurements should be uniformly and well mixed. Non-homogenous samples will lack reproducibility and will be difficult to quantify. An ideal way to mix samples for diffuse reflectance is by using a ShakIR.

• 4.Packing – the required sample depth is governed by the amount of sample scattering. The minimum necessary depth is about 1.5 mm. The sample should be loosely but evenly packed in the cup to maximize IR beam penetration and minimize spectral distortions.

SOFTWARES USED FOR RFLECTANCE MEASUREMENTS

Win UV Software UV WinLab software ASSP software

APPLICATIONS OF D R UV-VIS SPECTROSCOPY

• The D R UV-VIS Spectroscopy have applications widely in pharmaceutical and food and chemical industry.

• The D R UV-VIS Spectroscopy used for estimation of furosemide in bulk and dosage form

• D R UV-VIS Spectroscopy used for measurement of enzyme activity of –ALP

• D R UV-VIS Spectroscopy used for quantitative estimation of proteins

• D R UV-VIS Spectroscopy used for non-invasive monitoring of tissue haemoglobin.

• D R UV-VIS Spectroscopy used for measurements of small quantities of DNA samples and etc.

CONCLUSION

• D R UV-VIS Spectroscopy Is a novel analytical technique used as qualitative and quantitative purpose in pharmaceutical, food and chemical industry.

REFERENCES

• Taylor, A.H., J. Opt. Soc. Am. 4, 9 (1919); Benford, F., Gen. Elec.Rev. 23, 72 (1920).

• Hardy A.C., J. Opt. Soc. Am. 18,96 (1929).• Hardy; A.C., Handbook of Colorimetry (M.

LT.Press Cambridge, Mass. 1936).• Evans, R.M., An Introduction to Colour (John Wiley

and Sons, London, 1948).• Le Grand, Y., Light, Colour and Vision (Chapman

and H all, London, 1957).

• Judd, D.B., and Wyszecki, G., Color in Business, Science and Industry (John Wiley and Sons, London, 1963), 2d. edition.

• Billmeyer, F. W., and Saltzmann, M., Principles of Color Technology (Intersciencc, New York, 1966).

• Wyszecki, G., and Stiles, W.S., Color Science, Concepts and Methods, Quantitative Data and Fon~~llas (John Wiley and Sons, New York, 19.67), 2d. edltlon.