Introduction to Clinical Chemistry I PDF

ARSI UNIVERSITY COLLEGE OF HEALTH SCIENCE DEPARTMENT OF MEDICAL LAB Introduction to Clinical Chemistry I. By Alazar T.(Msc in Clinical Chemistry) 10/9/2024  Course syllabus 10/9/2024 By Alazar T.(Msc in Clinica...

ARSI UNIVERSITY COLLEGE OF HEALTH SCIENCE DEPARTMENT OF MEDICAL LAB Introduction to Clinical Chemistry I. By Alazar T.(Msc in Clinical Chemistry) 10/9/2024  Course syllabus 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Content of Clinical Chemistry I 1. Introduction to ClinicalChemistry 2.Solutions 3. Introduction to Radiant Energy 3.1 The electromagnetic spectrum 3.1.1 Radiation sources, measurement of absorption of ultra violet and Visible light 3.1.2 Interaction of RE withmatter 3.2 Application of Fundamental Laws of absorption 3.2.1 Beer’s Law 3.2.2 Lambert’s Law 3.2.3 Combined Beer’s-Lambert’sLaw Analytical Procedures &Instrumentation 3.3. Principles, concepts and fundamentals of: Photometer, Colorimeter, Spectrophotometer, Refractometer, Fluorometer, Turbidimeter, Nephelometer and Electrophoresis 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Contt…… 3.4 Measurement Procedures & Calculation in Clinical Chemistry 3.4.1 Assay Techniques in Clinical Chemistry: End Point assay, Differential assay, rate assays (fixed and continuous) Laboratory Practice - Pipettingtechniques - Dilution of concentrated solutions - Preparation of working solution from stock solutions - Components of spectrophotometer - Wave length selection for solutions - Demonstration of Beer’slaw 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Contt……….. 4. Specimen types and collection, processing and preservation for Clinical Chemistry tests 4.1 Common factors affecting quality of specimen for Clinical Chemistrytests 4.2 Stability of analytes in biologicalspecimens 4.3 Preservation of specimen for Clinical Chemistrytests 4.4 Transportation of specimen for Clinical Chemistrytests 5. Carbohydrates 5.1 Introduction to CHOs chemistry 5.2 Metabolism of CHOs 5.3 Digestion & absorption of CHOs, Cellular metabolism of CHOs Laboratory practice on - Specimen collection and processing - Blood glucose measurement 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  The rest chapter for C/chemistry I Renal Function tests Protein Lipid & Lipoprotein 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  ARSI UNIVERSITY COLLEGE OF HEALTH SCIENCE DEPARTMENT OF MEDICAL LAB Introduction to Clinical Chemistry. By Alazar T.(Msc in Clinical Chemistry) 10/9/2024  Learning Objectives Upon completion of this chapter the students will be able to: 1. Define clinical chemistry 2. Explain the significance of clinical chemistry 3. Describe units of measurements 4. List common apparatus and equipments used in the clinical chemistry laboratory 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Outline D e f in e c l i n i c a l c h e m i st r y a n d o t h e r i m po r t a n t terminologies Significance of clinical chemistry Units of Measurement Apparatuses and equipment's used in the clinical chemistry laboratories 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Definitions Clinical chemistry is a science, a service, and an industry. As a science, clinical chemistry links the knowledge of general chemistry, organic chemistry, and biochemistry with an understanding of human physiology. As a service, the clinical chemistry laboratory produces objective evidence from which medical decisions may be made. As an industry, clinical laboratories are businesses. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Definitions Clinical Chemistry is def ined as an area in laboratory sciences that deals with chemical analysis of body f lu ids such as blood, urine, spinal f luid as well as feces, tissue, calculi and other materials. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Definitions Terminology Analyte Analysis Types of Analysis – Qualitative – Semi-quantitative – Quantitative 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Cont… Analyzer Reagent Quality assurance 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) The main purpose of Clinical Chemistry tests To assess the physiological function of our body systems or organs To diagnose and monitor diseases To follow up response to treatment 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Significance of Clinical Chemistry Provides biochemical testing of patient sample – Glucose – Protein – Bilirubin – Creatinine – Lipids – Enzymes – Electrolytes – Hormones – And Other biochemicals 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Clinical Chemistry Equipment and Supplies Beakers and Erlemineir Flask Graduated cylinders 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Analysis in Clinical Chemistry Tips in discard Micropipette bottle 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Clinical Chemistry Instrument Spectrophotometer( Semi automated) 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Typical Clinical Chemistry Analyzer Spectrophotometer (Automated) 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Making Reagents for Clinical Chemistry Balance, cylinder and Distilled water 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Clinical Chemistry Reagents Reagents in Refrigerator 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Reagents for Testing in Clinical Chemistry 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Specimens Tested in Clinical Chemistry 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Clinical Chemistry Testing 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Quality Control in Clinical Chemistry 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Recording Clinical Chemistry Results 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) 
UNIT OF MEASURMENT
Measurement Name Symbol Length Meter M Time Second S Quantity of Subs Mole mol Electric current Ampere A Temperature Kelvin K Luminous intensity Candela Cd 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont.. 10-1 Deci d 101 Deka da 102 Hecto h 103 Killo k 106 Mega M 109 Giga G 1012 Tera T 1015 Peta P 1018 Exa E 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Derived unit I: is related mathematically to the basic or supplemental units Quantity Name Symbol Volume Cubic meter m3 Density Kilogram/cubic Kg/m3 meter Substance Mol/cubic moll/m3 concentration meter Frequency Hertz Hz Force 10/9/2024 Newton By Alazar T.(Msc in Clinical Chemistry) N  Summary Def inition of clinical chemistry and other terms used in clinical chemistry Signif ic anc e of c linic al c hemistry for laboratory diagnostics Different apparatuses and equipment's used in the clinical chemistry laboratories 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Reference 2. Arneson, W and J Brickell: Clinical Chemistry: A Laboratory Perspective 1st ed. 2007 FA Davis 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  ARSI UNIVERSITY HEALTH SCIENCE COLLEGE DEPARTMENT OF MEDICAL LAB
. Chapter Two Solution preparation By Alazar T.(Msc in Clinical Chemistry) 10/9/2024  Objective At the end of the chapter, the student will be able to Def in e solution, types of solutions and expressing concentration of solutions Discuss purpose and how to prepare dilutions Explain standard, primary, and secondary solutions 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Outline Definition of solution Types of solutions Expressing concentration of solutions Chemical units: molarities, normality Dilution: Definition and preparation Standard, primary, and secondary solutions 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Solution Solution Properties In clinical chemistry, substances found in biologic f lu ids are measured (e.g., serum, plasma, urine, and spinal fluid). A substance that is dissolved in a liquid is called a solute; in laboratory science, these biologic solutes are also known as analytes. The liquid in which the solute is dissolved—in this instance, a biologic fluid—is the solvent. Together they represent a solution. Any chemical or biologic solution is described by its basic properties, including concentration, saturation, colligative properties, redox potential, conductivity, density, pH, and ionic strength. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Concentration Analyte concentration in solution can be expressed in many ways. Routinely, concentration is expressed as  percent solution,  molarity,  molality, or normality and , because these non-SI expressions are so widely used, they will be discussed here. No te that the SI e xpre ssio n fo r the amo unt o f a substance is the mole. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Percent solutions is expressed as equal parts per hundred or the amount of solute per 100 total units of solution. Three expressions of percent solutions are weight per weight (w/w), volume per volume (v/v), and, most commonly, weight per volume (w/v). For v/v solutions, it is recommended that grams per deciliter (g/dL) be used instead of percent or % (v/v). 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Molarity is expressed as the number of moles per 1 L of solution. One mole of a substance equals its gram molecular weight (gmw). The SI representation for the traditional molar concentration is moles of solute per volume of solution, with the volume of the solution given in liters. The SI expression for concentration should be represented as mo les per liter ( mo l/L), millimo les per liter ( mmo l/L), micromoles per liter (mol/L), and nanomoles per liter (nmol/L). The familiar concentration term molarity has not been adopted by the SI as an expression of concentration. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Molality Represents the amount of solute per 1 kg of solvent. Molality is sometimes confused with molarity; however, it can be easily distinguished from molarity because molality is always expressed in terms of weight per weight or moles per kilogram and describes moles per 1,000 g (1 kg) of solvent. The preferred expression for molality is moles per kilogram (mol/kg). 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Normality is the least likely of the four concentration expressions to be encountered in clinical laboratories, but it is often used in chemical titrations and chemical reagent classification. It is def ined as the number of gram equivalent weights per 1 L of solution. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Colligative Properties of solutions The behavior of particles in solution demonstrates four repeatable properties based only on the relative number of each kind of molecule present. The properties of osmotic pressure, vapor pressure, freezing point, and boiling point are called colligative properties. Vapor pressure is the pressure at which the liquid solvent is in equilibrium with the water vapor. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Colligative Properties….. Freezing point is the temperature at which the vapor pressures of the solid and liquid phases are the same. Boiling point is the temperature at which the vapor pressure of the solvent reaches one atmosphere. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Osmotic pressure is the pressure that opposes osmosis when a solvent f lows through a semipermeable membrane to establish e q uilibrium be twe e n c o mpar tme nts o f d iffe ring concentration. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Redox Potential Redox potential, or oxidation-reduction potential, is a measure of the ability of a solution to accept or donate electrons. Substances that donate electrons are called reducing agents; those that accept electrons are considered oxidizing agents. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Conductivity Conductivity is a measure of how well electricity passes through a solution. A solution’s conductivity quality depends principally on the number of respective charges of the ions present. Resistivity, the reciprocal of conductivity, is a measure of a substance’s resistance to the passage of electrical current. The primary application of resistivity in the clinical laboratory is for assessing the purity of water. Resistivity or resistance is expressed as ohms and conductivity is expressed as ohms or mho. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Solution Definition A solution is a homogeneous mixture of one or more substances dispersed molecularly in a sufficient quantity of dissolving medium. A solution is made up of solute and solvent Solute + solvent = Solution 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Types of solution Solid solution: Liquid solution: Gaseous solution: 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Expressing concentration of solutions Relative expression Dilute solution: Concentrated solution Saturated solution Super saturated solution 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Making dilution Introduction: D i l u t i o n i s d e f in e d a s a p r o c e s s b y w h i c h t h e concentration of a given solution is decreased by the addition of solvents to make a weaker solution It represents the ratio of concentrated material to the total volume of a solution Total volume consists of the volume or weight of the concentrated plus the volume of the diluents. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Dilution, continued… This ratio of concentrated or stock solution to the total volume equals the dilution factor Dilution factor (df) = volume of stock OR Total volume of solution Dilution factor (df) = Sample Sample + Diluent The df is inversely related to the concentration thus, the dilution factor increases as the concentration decreases. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Reasons for dilution To prepare a weaker solution from stronger one If the specimen at hand is less than a procedure calls for If the concentration of substances (analyte) is too high to be accurately measured. To remove undesirable substances from the specimen 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Methods of dilution o Dilution can be made singly or in series. o It usually expressed as a ratio rather than with units of concentration. o X/Y where X= sample volume o Y= is the sum of the sample volume and the diluents o E.g. 1:2, 1:5, 1:10 or fraction ½, 1/5, 1/10. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Simple/single dilution Is def ined as one part of the original solution to the total part of f inal solution, which include both solute & solvent. In making a simple dilution, the laboratory technician must decide on the total volume desired & amount of stock solution to use 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  In the performance of single dilution, the most commonly used equation is: a. Using Proportion It is used when reagents are prepared by adding a specific amount of one solution to a specific amount of an other solution. C V= A+B Where: C – total volume of final reagent A – total parts of solution A B – total parts of solution B V – volume of each part 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) b. Using C1V1 = C2V2 This formula is useful only if the units for concentration & volume are the same & if three of the four variables are known. What volume is needed to make 500ml of 0.1M solution of tris-buffer from a solution of 2 M tris-buffer? Answer: 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Serial dilution Large dilutions may be dif fic ult to make because of the amount of diluent that needs to be added. A series of dilutions, also called serial dilutions, may be a better way to make the dilutions Generally, it is a multiple progressive dilution process in which the original solution is further diluted. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Serial dilution, continued… It is required for certain quantitative tests Serial dilution is extremely useful when the volume of the concentrate &/or diluents is in short supply 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  1 mL 1 mL 1 mL 1 mL -discard 1m 1m 1m 1m l l l l Sample: serum tube 1 tube 2 tube 3 Dilution: 1:2 1:4 1:8 Concentration: 1 0.5 0.25 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  The dilution fold of a system can be determined by the formula: 1 = volume transferred dilution fold total volume Volume transferred = is equal to the constant volume transferred to each successive tubes in the serial dilution system. Total volume = is equal to the volume being transferred plus the volume of diluents already in the tube. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Standard Solution is a solution whose concentration is exactly known. There are two types of standard solutions: – primary standard – secondary standard solution. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Primary standard solution a highly purif ie d chemical that can be measured d irectly to prod uce a substance of known concentration in a given solution. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  The solution must satisfy the following criteria: Must be essentially free of impurities Should be stable in both solid & solution form The concentration should be accurate. Should not be hygroscopic or vaporize at 20oc 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Example o Sodium carbonate(NaCO3) o Sodium oxalate o Sodium chloride, o Potassium dichromate 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Secondary standard solution is def in ed as a substance of lower purity & whose concentration is determined by comparison to a primary standard. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  To prepare secondary standard o We i gh so m e a m o unt o f t he subst a nc e usi ng analytical balance. o Dissolve in a given volume of solvent. o Using primary standard solution, determine the exact concentration of the substance. o I f nec essary d ilute to some volume to get the concentration required. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Example secondary standard o Oxalic acid o Nitric acid o Hydrochloric acid o Sulfuric acid. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  pH Buffers are weak acids or bases and their related salts that, as a result of their dissociation characteristics, minimize changes in the hydrogen ion concentration. Hydrogen ion concentration is often expressed as pH. A lowercase p in front of certain letters or abbreviations operationally means the “negative logarithm of” or “inverse log of” that substance. In keeping with this convention, the term pH represents t he ne gat i v e o r i nv e rse l o g o f t he hyd ro ge n i o n concentration. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Concept of acids and bases Acids & Bases According to Bronsted Lowry theory and Arrhenus theory respectively Acids – are proton donors - dissociate to furnish hydrogen ions Bases – are proton acceptors -dissociate to furnish hydroxyl ions in queous solution 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Dissociation of strong acids or bases o Strong acids are those which dissociate completely into their respective cations (H + ) &anions in aqueous (A -) solution.  HA H+ + A-  HNO3 H+ + NO3-  H2SO4 2H+ + SO4= By Alazar T.(Msc in Clinical Chemistry) 10/9/2024  Strong bases are those which dissociate completely into their respective hydroxyl ions (OH+) & respective cations (B-) in aqueous solution. o BOH B+ + OH- o NaOH Na+ + OH- 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Dissociation of weak acids or bases o Weak acids (HA) are those which partially dissociate when they are in aqueous solution o HA H+ + A- o Examples: Acetic acid, Carbonic acid, phosphoric acids, lactic acids, benzoic acids etc. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Weak bases are those which dissociate partially when dissolved in aqueous solution.  BOH B+ + OH-  Eg NH3, Ca (OH)2, Al2(OH)3 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Hydrogen ion concentration (pH) To express hydrogen ion concentration, the symbol pH was introduced by Sorensen in 1909. pH is defined as the negative logarithm of the Hydrogen ion concentration. pH = - log [H+] in mol/L pH of pure water = 7 pH of 1 M solution of strong acid = 0 pH of 1 M solution of strong base = 14 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Buffer solution & its action Buffers are chemical system that prevents change in the concentration of another chemical substance. For example, a proton donor & acceptor system of solutions are used as buffers to prevent change in hydrogen ion concentration. It consists of a mixture of weak acid (or base) & its conjugate base (or acid). E.g. CH3COOH(ethanoic acid) & CH3COONa (its salt) 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Buffers resists changes in pH when small quantities of an acid or a base is added to it. A buffer act as like an base if acid is added & like an acid if base is added. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Types of buffers 1. Neutral buffer – phosphate buffer 2. Acidic buffer – citrate buffer 3. Alkaline buffer – borate buffer 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  ARSI UNIVERSITY HEALTH SCIENCE COLLEGE DEPARTMENT OF MEDICAL LAB
Chapter Three. Introduction to radiant energy

By Alazar T.(Msc in Clinical Chemistry) 10/9/2024  Objective At the end of this chapter the student will be able to: Definition of terms Discuss radiant energy Describe properties of EMR Explain about interaction of EMR with matter Discuss basic law of absorption: Beer-Lambert’s law 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Outline Introduction to radiant energy Properties of EMR Interaction of EMR with matter Electromagnetic spectrum Absorption measurements: Beer-Lambert’s law, stray light 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  3.Introduction to radiant energy

Electromagnetic radiation Radiation showing electric & magnetic characterstics in the form of waves or photons is termed as electromagnetic radiation It travels at approx. 3 x 105 km/s in the vacuum of space. In materials which are transparent to electromagnetic radiation, the velocity is slightly less than the velocity in a vacuum. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  10/9/2024 By Alazar T.(Msc in Clinical Chemistry) EMR….. Dual Nature of EMR Energy Energy transfers in the physical world either by waves or particles In general, electromagnetic radiation behaves: as a wave when moving through space, as a particle when it interacts with matter 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  EMR….. Wave Properties Wave is the way of transferring an energy from one place to another Consists of discrete packets of energy or quanta called photons It Can be described by: 1. Velocity (c ) 2. Amplitude 3. Wave length (λ) 4. Frequency (ν) 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont.……. 1. Amplitude  The height the wave crest or troughs from the baseline  Governs brightness of light 2. Wavelength  distance between two wave crests or troughs  One full wave cycle (crest top to next crest top). 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont…. 3. Frequency how fast it oscillates (goes up & down) measured in cycles (remember crest to crest) per second. The number of wave crests per second, that is the number of wave crests that passes by a given point in one second. 1cycle/second = 1 Hz (hertz) = 1s-1. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont…. 4. Speed of Light (velocity)  Speed of the wave  For example – water - few meters per second – Sound wave – 340 m/sec – Light - 3 x 108 meters/second 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Cont…… Relationship between c, λ & ν.  The longer the wavelength the lower the frequency, or the shorter the wavelength the higher the frequency.  This relationship is expressed in the formula ν = c/λ where: - ν - frequency of light in cycle/sec. - c - speed of EM wave in vacuum - λ - wave length in cm 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont….  E = hv  Where h = planck’s constant (6.62x10-27 erg.sec) ν = frequency E = energy  E = h c /λ 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Interaction of EMR with matter In order to use photometric instruments correctly & to be able to develop & modify spectroscopic techniques it is ne c e ssary to und e rstand the princ iple o f interaction of radiation with matter. The only way to observe electromagnetic radiation is by its interaction with matter. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Interaction of EMR with matter It involves:  Diffraction  Reflection  Refraction  Dispersion  Absorption & transmission 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) 1. Diffraction
Is the change of direction of the EM beam when it strikes the edge of an opaque body or it passes through a small hole
10/9/2024 By Alazar T.(Msc in Clinical Chemistry) 2. Refraction bending of light as it passes through materials of different optical density 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) 3. Reflection  When radiation falls on silver coated glasses, the beam of the radiation returns towards the source of radiation 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) 
4. Dispersion

Is the change in refractive index with a change in wavelength The velocity of light in a materials & its refractive index depends on the wavelength of the light. This causes the light to be refracted by different amounts according to the wavelength (or color). This gives rise to Bythe colors10/9/2024 Alazar T.(Msc seen through a prism. in Clinical Chemistry) The following is a chart giving the index of refraction for various wavelengths of light in glass 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) The electromagnetic Spectrum Spectrum is an ordered arrangement of radiant energy according to the wavelength. A. Continuous spectrum A spectrum which is composed of visible lights of all wavelengths are called Continuous spectrum It is a continuous spectrum because one color fades into another. E.g. sun light or light from ordinary incandescent bulb 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Component energies of the electromagnetic spectrum a. Radio waves  The longest- from a few meters to longer than the size of the earth.  They can travel long distance in the atmosphere b. Microwaves  The wavelength is from about 1 millimeter to 1 meter.  Used in communication, radar & cooking 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) 
C. Infra red
 Wavelength from 10-3 to 10-6 (micron)  Ranges from approx 12,500 – 50 cm-1 U se d in toxic o lo gy and mo le c ular struc ture determination 4000 to 1000 cm -1 – used for the analysis of organic compounds 1000 to 400 cm -1 - is used for the analysis of inorganic compounds 12,500 to 4000 cm -1 is not helpful for such analysis. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  d. Visible It is a very small portion of the total EM spectrum visible to human eye It ranges from 700nm (at red light) to 400nm (violet) the visible color of a solution corresponds to the wavelength of the light that are transmitted , not absorbed, by the solution. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont… The d ifferent c olors have d ifferent wavelengths & frequencies. The rest of the EM spectrum is not visible to the human eye Source of visible light: o tungsten lamp. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  wavelength Color of Color of light /solution in nm light reflected/complementar absorbed y 350-430 Violet Yellow 430-475 Blue Orange 475-495 Blue-green Red -Orange 495-505 Blue-green Orange - Red 505-555 Green red 555-575 Yellow- Violet red green 575-600 Yellow Violet 600-650 Orange Blue 650-700 Red green 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Note: A substance that absorbs green light at 500 nm ref lects or transmits all other lights or wavelengths & appears as purple. To measure the concentration of a blue solution, light at about 590 nm is passed through the solution The amount of yellow light absorbed varies directly as the concentration of the absorbing substance in solution 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont…  The absorbed c olor is the c omplementary of the transmitted color.  Thus to make absorption measurement, one must use the wavelength at which a colored solution absorbs light.  For example, a red solution absorbs green light & transmitted red light. Therefore, a red solution should be measured at 490 to 550nm 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont… In photometer using f ilter used as a monochromator, the filter chosen is usually complementary to the color of the solution to be measured.  blue solution – yellow filter  Yellow solution –blue filter  Red solution – blue green filter  Blue green solution – red filter 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) E. UV Light
It ranges from 400 to 100 nm It is dangerous to tissue & cells (common sun burns) Wid ely used in the quantitative & qualitative determination of clinical chemistry tests. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont.. Source: – Discharge tube containing hydrogen or deuterium at reduced pressure. – High pressure mercury & xenon arc lamps. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) f. X- ray It ranges from 100 to 0.1 nm High frequency, high energy waves that can penetrate several centimeters into most solid matter. Used in radiological diagnosis 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) g. Gamma rays  it ranges from 0.1 to less than 10-16  It is the highest energy ray in the EM spectrum  It is generally produced in nuclear reactions & not as common in nature, 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  10/9/2024 By Alazar T.(Msc in Clinical Chemistry) B. Line/atomic emission spectrum a spectrum with only certain colors. (NOT continuous like sunlight) Samples of elements emit light when they are vaporized (heated) or electricity passes through them. Every element has a unique line 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Cont… The wavelength of the line are characteristics of a particular element It can be used for qualitative identification & quantitative determination of elements in an unknown mixture For example, f la me photometer, atomic absorption spectrophotometer. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  5. Absorption & transmission when some radiant energy passing through a solution, transparent glass, or semitransparent substances – Some amount of light is transmitted & – Some is absorbed or trapped by the medium. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Absorption measurement Many determinations in clinical chemistry are based on the measurement of the radiant energy  Emitted (e.g Fluorometer)  Transmitted  Absorbed (absorption spectrophotometer)  Reflected (reflectance photometer)  Refracted (refractometer) 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont.. Light transmittance Is def ined as the proportion of the incident light that is transmitted. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont… Transmittance (T) = I/Io where I = transmitted light Io = original incident Usually this ratio is described as a percentage: %T = I/ Io x 100% As the concentration of a compound in solution increases, more light is absorbed by the solution & less light is transmitted. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Light absorbance The relationship between %T & concentration is not linear but varies inversely & logarithmically. As a result it is more convenient to use the concept of absorbance to avoid the use of logarithmic units. However the concept of transmittance is important because only transmitted light can be measured. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Relationship between absorbance & transmittance 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  1. Absorption spectrophotometry Fundamental law of absorption a. Introduction When a radiant energy, Io, passes through a solution to be analyzed, – some of the radiant energy will be absorbed – some of it will be transmitted 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont…….. The transmitted light, I, is affected by factor such as: o Incident light o Optical path length o Concentration of solution 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  2. Beers- Lambert's law It is commonly referred to as Beer’s Law 1. Beer’s law It states that concentration of a substance is: o d ire c tly pro po r tio nal to the amo unt o f light absorbed by the solution & o i n v e r s e l y pr o po r t i o n a l t o t h e l o ga r i t h m o f transmittance o A=C o A = a. c 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  2. Lambert's law It states that the amount of radiant energy absorbed is directly proportional to the thickness of the medium through which the light pass. A=b A = a.b By Alazar T.(Msc in Clinical Chemistry) 10/9/2024  10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  3. Beers-Lambert’s law  A = a.b.c  It is the combination of the two laws. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Beer-Lambert's law indicates a direct proportionality between A and c only if:
– incident radiation is monochromatic – each molecule in solution acts as an independent absorbing species in solution – Absorption takes place in a solution of uniform cross- section (a well mixed solution) 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Limitations of Beer’s law-cause for deviation from the law  non-monochromatic light  Elevated concentration  Solvent absorption  Transmitted light by other mechanisms  Non-parallel sides of cuvets 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Deviations from beer’s law a. Spectral interference The beers-lambert’s law express the linear relationship b/n the concentration of the sample & the absorbance value recorded. However, the relationship is only an experimental one & not a fundamental law of nature. As a result, the linearity is only true under certain limiting conditions 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Some amount of radiation will be – Reflected from the surface of the sample holder, – Absorbed by the material of which the cell is composed or – The solvent may also absorb or reflect radiation. Io = absorbed + transmitted + others 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Cont… To foc us attention on the c ompound of interest, elimination of these factors is necessary. This is done through the use of blank or reference solution This blank should be identical to the test sample in all aspects except the presence of the test substance Blank reading = Io - other loses 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont.. Hence: Absorbed = blank – transmitted. Types of blank solution 1. reagent blank 2. Sample blank 3. Water saline or air blank 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont… 1. Reagent blank reagent + solvent or A solution of reagents with out sample Used to correct high absorbance of the reagent 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont… 2. Sample blank Sample + diluent A solution of sample & reagents missing a key reagents that initiate the rxn or cause formation of f in al rxn product. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  b. Stray light Quantitative radiation rely on radiation that reach the detector passing through the sample But it is impractical, because it is difficult to design instrument which are capable of effectively eliminating all extraneous radiation Much of these unwanted radiation arises from the scattering of the incident radiation by irregularities in surfaces (by faults in manufacturers) or scratches 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Contt… Such light, stray light, results in a deviation from Beer’s law & The effect is that absorbance measurements are lower than they should be. It is possible to asses the proportion of stray light by measuring the amount of radiation transmitted by samples which are optically opaque at the wavelength to be assessed but which transmit radiation of other wavelengths 10/9/2024 By Alazar T.(Msc in Clinical Chemistry) Cont… The instrument is set to zero %T with blocking light path & 100% transmittance with a reagent blank in the normal way & opaque substance introduced into the sample compartment. The amo unt o f l i ght t ransmi t t e d by t he sampl e , measured in percentage transmittance is quoted as the stray light at a specified wavelength. 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)  Quiz 5% 1. Write the relationship b/n absorbance and Transmittance of light? 2. Write the difference and similarity of beers and lamberts low? 3. List continues spectrum of radiant energy in increasing order? 10/9/2024 By Alazar T.(Msc in Clinical Chemistry)