Lecture 1_EMR, Photometry, Spectrometry..pptx

Biomedical Techniques CLSB-222 Dr. Ahmad Alamri, M.H.S, Ph.D. Assistant professor of clinical laboratory sciences [email protected]  AGENDA 01 Course Description and Main Objectives 02 Electromagnetic Radiations Absorption spectrosc...

Biomedical Techniques CLSB-222 Dr. Ahmad Alamri, M.H.S, Ph.D. Assistant professor of clinical laboratory sciences [email protected]  AGENDA 01 Course Description and Main Objectives 02 Electromagnetic Radiations Absorption spectroscopy and application of Beer’s law, Beer’s Lambert’s law 03 Spectrophotometer, components and applications 04  Course Description The course describes basic principle of analytical techniques and equipment used in clinical laboratory. Course Main Objectives OBJECTIVE 01 This course intends to know the basic principles of analytical techniques with an emphasis on the use of basic and advanced equipment routinely used in clinical laboratories. OBJECTIVE 02 The course provides the theoretical concepts of the techniques, demonstration of equipment and their use in clinical laboratory.  Required Textbook s 1. Practical Biochemistry: Principles and techniques: K. Wilson and J. Walker (7th edition) 2. McPherson, R.A. &Pincus, M.R. (2016). Henry’s Clinical Diagnosis and Management by Laboratory Methods (23rd ed.). Philadelphia: Elsevier 3. Rifai, N. (2017). Tietz Textbook of Clinical Chemistry and Molecular Diagnostics (6th ed.). Elsevier Health Sciences. 4. Davis, D. L. (2016). Laboratory Safety A Self- Assessment Workbook (2nd ed.). Chicago: ASCP Press Assignment = 10 Marks BB Discussions = 10 Marks MID Theory= 30 Marks Final MCQ = 30 Marks Grades and credits CREDIT HOURS 3 THEORY (+) 0 PRACTICAL Final Short questions = 20 Marks Total Marks = 100 What is Biomedical technique is the application of engineering principles and techniques to the Biomedic medical field. al It combines the understanding, design and problem-solving skills of any instrumentation or Techniqu technique with medical and biological sciences to e? improve healthcare diagnosis and treatment.  List of Broad Topics Covered in This Course Spectroscopy Bacterial and Cell culture Techniques Osmometry Emission spectroscopy Sedimentation Techniques Ion Selective Microscopy electrodes Flow - Electrophoresis cytometry Immunochemical Techniques Chromatography Molecular Biology techniques Gel filtration Techniques Radio isotopes Automated Techniques Procedures Electromagnetic Radiations Electromagnetic radiation (EMR) (also referred at ER or EMR) is defined as a type of radiation that has both magnetic fields and electric fields. Characterized by their wavelengths or frequencies or wave numbers The energy carried by an EMR is directly proportional to its frequency Electromagnetic waves require no medium for transmission and can rapidly propagates through the vacuum  Electromagnetic Waves Electromagnetic waves can be characterized by wavelength and frequency Wavelength (λ): Is described as the distance between identical points on a wave. It is typically measured using the distance between the crests/peaks of two waves. Wavelength is measured in Frequency (𝜈): Is described as the nanometers (nm) number of waves that pass a given point for a given amount of time. Frequency is measured in Hertz (Hz) Wavelength and Frequency are inversely related. This means that when the frequency of a wave is big, the wavelength will be small (and vice versa). This relationship is https://www.expii.com/t/electromagnetic-radiation-overview-  Smaller wavelengths and higher frequencies result in stronger electromagnetic waves. Bigger wavelengths and smaller frequencies result in weaker electromagnetic waves. https://www.expii.com/t/electromagnetic-radiation-overview- PHOTOMETRY-COLORIMETER AND SPECTROPHOTOMETER  PRINCIPLES of PHOTOMETRY: The specificity of a It is the study of compound to absorb light at a particular the phenomenon wavelength of light absorption (monochromatic by molecules in light) is used in the laboratory for solution. quantitative measurements. Photometry forms an important Clinical Biochemistry Laboratory tool for accurate estimation of different compounds in biological samples.  Nature of light: Light is a radiant energy and travels in the form of waves. The distance between two waves is the wavelength which is measured in a nanometer (nm). A nanometer is equivalent to 10-9 meter. The visible light ranges from 380 - 750 nm. The UV light ranges below 380 nm. The Infrared light ranges above 750 nm.  LAWS of PHOTOMETRY: The measurement of color by photometry is based on the principle of two laws known as BEER’S and LAMBERT’S laws. BEER’S LAW: states that the amount of light absorbed by a colored solution is proportional to the concentration of the solution. If A is the absorbance and C is the concentration of the color in the solution, then absorbance is directly proportional to the concentration of the solution. A α C LAMBERT’S LAW: States that the amount of light absorbed by a colored solution is proportional to the depth through which light passes in the solution. If L is the depth through which light passes in the solution, then A α L Combining the two laws that are: AαCx L A=K x C x L, where, K=Extinction Coefficient constant for color, Extinction Coefficient (k): is a measure of light lost due to scattering and absorption per unit volume  Suppose, A1=absorbance of the TEST solution, C1=concentration of the TEST solution A2=absorbance of STANDARD solution C2=concentration of STANDARD solution All photometric measurements are taken using CUVETTES having the same depth(L),therefore, we would get the following expression:  A1 K 1 x C1 x L C1 ---------- = ------------------------- = ------- A2 K 2 x C2 x L C2 Or A1 C1 = ---------- x C2 A2 That is, the concentration of TEST solution would be = Absorbance of TEST ------------------------------------ x Concentration of SPECTROPHOTOMETE R A spectrophotometer is an instrument that measures the number of photons (the intensity of light) absorbed after it passes through sample solution.  1. Source of light: It is an electric lamp. Most commonly is tungsten lamp which emits light from wavelength 320 – 700 nm. Other lamps used are deuterium lamp or hydrogen lamp for UV region(below320nm) Components 2. Entrance slit: It allows only a beam of light. It excludes unwanted of the light. Spectrophoto 3. Filter (Monochromator): meter It is made up of colored glass or a filter that selects one single light, i.e., one wavelength. The equipment that use filters for this purpose are referred to as filter Photometers whereas those that use prisms or gratings are called Spectrophotometers.  4. Cuvette holder: This is where the sample of the colored solution is inserted. The cuvette can be either in the round shape like a test tube or square form. For measuring absorbance in the visible region, the cuvette is usually Components made of glass, while in measuring absorbance at the of the low UV region cuvettes are made of quartz. Spectrophoto 5. Photocell (detector) : This cell converts light energy into electric energy. meter 6. Galvanometer (amplifier/readout): The potential difference (current) generated in photocell is measured by GALVANOMETER Source of Entrance Gratin Monochroma Detect light slit gs tor or Cuvette holder Definitio ns BLANK solution: It is a solution that contains all the reagent of the assay except the compound (sample) being measured in the solution. All light absorption measurements are made relative to blank solution. Because the absorbance of all regents is adjusted to zero or 100% Transmittance. MAXIMUM ABSORBANCE (λ Max): It is the WAVELENGTH at which ABSORBING COMPOUND (sample) absorbs MAXIMUM AMOUNT of MONOCHROMATIC LIGHT in a solution.  Spectroscop y In chemistry or physics is the learning of emission or absorption radiations from matter (atom, ion, and molecule) when dispersed through a suitable prism. Spectroscopy is the most important tool for determining the structure of an atom, ion, or molecule. The atom of an element always Atomic Absorption Spectroscopy Atomic absorption spectroscopy is a spectro analytical procedure useful in determining the chemical elements in a sample quantitatively. This procedure depends on the absorption of light by free metallic ions. Atomic absorption spectroscopy (AAS ) The principle is based upon the absorption and emission of light by atoms in the gaseous state. It was first observed by Fraunhofer while studying dark lines in the solar spectrum. In 1955 Alan Walsh from Australia applied the principle and instrumentation of atomic absorption spectroscopy for the analysis of chemical elements. Many difficult and time- consuming instruments were replaced after the discovery of AAS.  Atomization Any atomic absorption Hollow cathode lamp spectroscopy instrumentati Monochromator on has the following Detector types of components Recorder  Atomic absorption spectroscop y instrumentat ion ‫محذوف من المحاضرة‬ Application of atomic absorption spectroscopy Today, the atomic absorption spectroscopy technique is the most powerful tool in: Analytical chemistry Forensic sciences Environmental analysis Food industries The most It is possible to determine all elements at trace concentration. important advantage is the speed of It is not always essential to analysis. separate the element before analysis because AAS can be used It can to determine one element in presence of another. analyze various The atomic absorption samples spectroscopy principle or within a day. instrumentation can be used to analyze sixty-seven metals and several nonmetals such as phosphorus and boron.  Video Clip https://csiropedia.csiro.au/a-long-shot-that-paid-off-csiros-atomic- absorption-project-1970/