Spectrophotometry Instrumentation PDF

SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY INSTRUMENTATIO LECTURER: MARK ALEXIS LIM CATAPANG, RMT, MLS (ASCPi)CM N  1. Describe the general characteristics of the n...

SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY INSTRUMENTATIO LECTURER: MARK ALEXIS LIM CATAPANG, RMT, MLS (ASCPi)CM N  1. Describe the general characteristics of the nature of electromagnetic radiation 2. Define light transmittance and LEARNING absorbance and their relationship OBJECTIVE 3. Discuss the theory and practical application of Beer-Lambert Law S By the end of the lesson, the students are expected to: 4. Describe the principle of operation and component parts of the SPECTROPHOTOMETER. SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY  SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY TOPIC We will learn the following: OUTLINE Electromagnetic radiation (EMR) Spectrophotometry Components of a spectrophotometer Double beam spectrophotometry Flame emission spectrophotometry Atomic absorption spectrophotometry INTRODUCTION ANALYTIC Spectrophotometry Mass spectrometry TECHNIQUES SPECTROMETRY Atomic absorption Fluorescence Chemiluminescence LUMINESCENCE ELECTROANALYTIC Electrophoresis Amperometry METHODS Potentiometry CHROMATOGRAPH Gas chromatography Thin layer chromatography Y Liquid chromatography SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY  SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY SPECTROPHOTOMETR Y INTRODUCTION ELECTROMAGNETIC RADIATION ▪A.k.a. radiant energy ▪ a particle of electromagnetic radiation is known as a photon ▪ exhibits both wave-like and particle-like properties SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY INTRODUCTION ELECTROMAGNETIC RADIATION ▪A.k.a. radiant energy ▪ a particle of electromagnetic radiation is known as a photon ▪ exhibits both wave-like and particle-like properties SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY INTRODUCTION ELECTROMAGNETIC RADIATION ▪Have both electric and magnetic field components that travel at right angles to one another. ▪ These fields oscillate in phase, and are also perpendicular to the direction of the radiation's propagation. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY ELECTROMAGNETIC WAVES ▪Wavelength: distance between identical points on consecutive waves ▪ Amplitude: distance between origin and crest (or trough) ▪ Frequency: number of waves that pass a point per unit time ▪ Speed (c): wavelength (λ) x frequency (v) SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). CLINICAL CHEMISTRY ELECTROMAGNETIC WAVES SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). CLINICAL CHEMISTRY ELECTROMAGNETIC WAVES SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). CLINICAL CHEMISTRY ELECTROMAGNETIC WAVES ▪Wavelength is inversely proportional to the frequency of the light wave ▪ Energy is proportional to the frequency of the radiation ▪ Inversely proportional to the wavelength of light. ▪ Shorter wavelengths have higher energy and frequency, and vice versa. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). CLINICAL CHEMISTRY ELECTROMAGNETIC SPECTRUM ▪Light: is a wave of alternating electric and magnetic fields ▪ UV light has ↓ wavelengths and ↑ energy than visible light ▪ IR light has ↑ wavelengths and ↓ energy than visible light. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). CLINICAL CHEMISTRY ELECTROMAGNETIC SPECTRUM SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ https://stickmanphysics.com/stickman-physics-home/electromagnetic-waves/waves-of-the-electromagnetic-spectrum/ CLINICAL CHEMISTRY SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). CLINICAL CHEMISTRY SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). CLINICAL CHEMISTRY VISIBLE LIGHT Visible light is a form of electromagnetic radiation (EMR) that is detectable by the human eye The color of an object is determined by the wavelengths of light that are transmitted or reflected by it. The transmitted light and the absorbed light are complementary to each other. The visible color of a solution will be the complement of the wavelength being absorbed. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). CLINICAL CHEMISTRY VISIBLE LIGHT SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY VISIBLE LIGHT Wavelength (nm) Color (absorbed) Color emitted/ transmitted 350-400 Violet Yellow 400-450 Indigo Yellow 450-500 Blue Orange 500-550 Green Red 550-600 Yellow Indigo 600-650 Orange Blue 650-700 Red Green SAN PEDRO COLLEGE SCHOOL OF MEDICAL LABORATORY SCIENCE CLINICAL CHEMISTRY BEER LAMBERT’S LAW ▪ States that the concentration of a substance is: ▪ directly proportional to the amount of light absorbed ▪ inversely proportional to the logarithm of the light transmitted SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY BEER LAMBERT’S LAW ▪ States that the concentration of a substance is: ▪ directly proportional to the amount of light absorbed ▪ inversely proportional to the logarithm of the light transmitted SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY BEER LAMBERT’S LAW ▪ The relative amount of light passing through the sample is known as Transmittance (T) ▪ Percent transmittance (%T) ▪ the ratio of the radiant light transmitted divided by the radiant energy incident to the sample. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY BEER LAMBERT’S LAW ▪ The relative amount of light passing through the sample is known as Transmittance (T) ▪ Percent transmittance (%T) ▪ the ratio of the radiant light transmitted divided by the radiant energy incident to the sample. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY BEER LAMBERT’S LAW ▪ The difference in the amount of light transmitted by the blank and that transmitted by the sample is due only to the presence of the compound being measured. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY BEER LAMBERT’S LAW ▪ Absorbance (A) ▪ Is the relative amount of light absorbed by the sample and is related to transmittance (T). ▪ It cannot be measured directly by the spectrophotometer, but rather is mathematically derived from the percent transmittance. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY BEER LAMBERT’S LAW ▪ Absorbance (A) ▪ Directly proportional to concentration: A=ε×b×c ▪ Where: ▪ ε = molar absorptivity, the fraction of a specific wavelength of light absorbed by a given type of molecule; ▪ b = the length of light path through the solution; ▪ c = the concentration of absorbing molecules. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY BEER LAMBERT’S LAW SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY BEER LAMBERT’S LAW SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY DEFINITION SPECTROPHOTOMETRY ▪ is the measurement of the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY EXTERNAL COMPONENTS SPECTROPHOTOMETER SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY INTERNAL COMPONENTS SPECTROPHOTOMETER SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY INTERNAL COMPONENTS SPECTROPHOTOMETER SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). CLINICAL CHEMISTRY INTERNAL COMPONENTS OF A SPECTROPHOTOMETER LIGHT SOURCE ▪ Exciter lamp ▪ Provides the radiant energy needed to measure the analyte of interest ▪ A spectrophotometer generates polychromatic light and uses a monochromator to isolate a specific wavelength of interest. SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). CLINICAL CHEMISTRY INTERNAL COMPONENTS OF A SPECTROPHOTOMETER LIGHT SOURCE Type Description Incandescent Tungsten or Tungsten- Most common source for visible and near- iodide lamp infrared regions. Deuterium discharge lamp and Ultraviolet (UV) work Mercury arc lamp Mercury vapour/ Hydrogen lamp/ Fluorometry Xenon arc Hollow cathode lamp AAS (Atomic absorption spectrophotometry) Globar lamps, Nernst glower Provides infrared light SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Bishop, M. L., & Fody, E. P. (2022). Clinical Chemistry: Principles, Techniques, and Correlations. Jones & Bartlett Learning. CLINICAL CHEMISTRY ▪ McPherson, Richard A. and Pincus, Matthew R., "Henry's Clinical Diagnosis and Management by Laboratory Methods, 24th ed" (2021). INTERNAL COMPONENTS OF A SPECTROPHOTOMETER ENTRANCE SLIT ▪ Reduces stray light ▪ Stray light refers to any wavelengths outside the intended band that do not originate from the light source. ▪ If the stray light is allowed to pass through the analytical cell, it would cause a deviation from the readings SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Review Handbook in Clinical Chemistry by Maria Teresa Rodriguez (2023) CLINICAL CHEMISTRY INTERNAL COMPONENTS OF A SPECTROPHOTOMETER MONOCHROMATOR ▪ The primary function of the monochromator is to isolate a specific or individual wavelength of light from the polychromatic light source ▪ Types: ▪ Prisms ▪ Diffraction gratings ▪ Interference filters SAN PEDRO COLLEGE REFERENCES: SCHOOL OF MEDICAL LABORATORY SCIENCE ▪ Review Handbook in Clinical Chemistry by Maria Teresa Rodriguez (2023) CLINICAL CHEMISTRY INTERNAL COMPONENTS OF A SPECTROPHOTOMETER MONOCHROMATOR BANDPASS ▪ Total range of wavelengths that a monochromator allows to pass through ▪ Determines the efficiency of monochromator ▪ Generally, the narrow range of wavelength is the most desirable ▪ Prisms & gratings =