Introduction to Spectrophotometry PDF

College of Science Introduction to Spectrophotometry *Do NOT distribute, re-upload in other online platforms...

College of Science Introduction to Spectrophotometry *Do NOT distribute, re-upload in other online platforms © McGraw-Hill Education. All rights reserved. Authorized only for instructor use in the classroom. No reproduction or further *Intended for TUP online classes ONLY distribution permitted without the prior written consent of McGraw-Hill Education.  College of Science UV-Vis Spectroscopy UV-Vis spectroscopy is an analytical technique that measures the amount of discrete wavelengths of UV or visible light that are absorbed by or transmitted through a sample in comparison to a reference or blank sample. Applications: 1. DNA & RNA analysis 2. Pharmaceutical analysis 3. Bacterial culture 4. Beverage analysis UV-Vis Spectrophotometer 5. Quality assurance Have been in general use for the last 80 years and over this period have become the most important analytical instrument in the modern day laboratory. *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science UV-Vis Spectrophotometer *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science Cuvettes (Glass or Quartz) *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science Spectrophotometry Spectrophotometry is any technique that uses light to measure chemical concentrations. Light is both a particle and a wave. Light waves consist of perpendicular, oscillating electric and magnetic fields. *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science Spectrophotometry λ Wavelength ν Frequency is the number of complete oscillations that the wave makes each second. The unit of frequency is 1/second. *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science Electromagnetic Spectrum Electromagnetic spectrum showing representative molecular processes that occur when light in each region is absorbed. The visible spectrum spans the wavelength range 380 to 780 nanometers (1 nm = 10-9 m). *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science Electromagnetic Spectrum The part of a molecule responsible for light absorption is called a chromophore. Any substance that absorbs visible light appears colored when white light is transmitted through it or reflected from it. The substance absorbs certain wavelengths of the white light, and our eyes detect the wavelengths that are not absorbed. The observed color is called the complement of the absorbed *Do NOT distribute, re-upload in other online platforms color. *Intended for TUP online classes ONLY  College of Science Absorption of Light Absorption of light increases the energy of a molecule. Emission of light decreases its energy. Visible and ultraviolet radiation promote electrons to higher energy orbitals. *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science Absorption of Light Sample problem: By how many kilojoules per mole is the energy of O2 increased when it absorbs ultraviolet radiation with a wavelength of 147 nm? 1 m = 1x109 nm *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science How UV-Vis Spectrophotometer works *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science How UV-Vis Spectrophotometer works Monochromatic light consists of “one color” (one wavelength). The better the monochromator, the narrower is the range of Irradiance (I0 or P0) wavelengths in the or Intensity emerging beam. When light is absorbed by a sample, the irradiance of the beam of light is decreased. *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science How UV-Vis Spectrophotometer works Transmittance, T, is defined as the fraction of the original light that passes through the sample. Beer-Lambert’s Law c = concentration of the sample (M) (Absorbance) b = pathlength (cm) ε = molar absorptivity (M-1 cm-1) *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science Absorbance, Transmittance, Beer’s Law Sample problem: Find the absorbance and transmittance of a 0.00240 M solution of a substance with a molar absorptivity of 313 M-1 cm-1 in a cell with a 2.00-cm pathlength. *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science Limitations to Beer’s Law Results in non-linear calibration curve. Affected by high concentration of electrolytes/analyte. Only true for dilute solution ( 0.01 M). Scattering of light due to particulates in the sample. Stray light. Compartments must be tightly closed to avoid stray light, which leads to false readings. If too little light gets through the sample (high absorbance), intensity is hard to measure. If too much Dilute solutions that are dark colored. light gets through (low absorbance), it is hard to distinguish transmittance of the sample from that of the reference. It is desirable to adjust sample concentration so that absorbance falls in the intermediate range. *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science Standard Calibration Curve Calibration is the empirical determination of the relationship between a measured quantity and a known reference or standard value. It is used to establish analytical signal versus concentration relationships in a calibration or working curve. A calibration curve shows the response of an analytical method to known quantities of analyte. Solutions containing known concentrations of analyte are called standard solutions. Solutions containing all reagents and solvents used in the analysis, but no deliberately added analyte, are called blank solutions. Blanks measure the response of the analytical procedure to impurities or interfering species in the reagents. *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY  College of Science Reference Harris, D. (2016) Quantitative Chemical Analysis, 9th edition. New York: W.H. Freeman and Company. *Do NOT distribute, re-upload in other online platforms *Intended for TUP online classes ONLY