U2 Ch7 - Basic and Contemporary Techniques in Clinical Laboratory PDF

Chapter 7 (Pages 130-138) Basic and Contemporary Techniques in the Clinical Laboratory Preamble PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details neede...

Chapter 7 (Pages 130-138) Basic and Contemporary Techniques in the Clinical Laboratory Preamble PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES Unit Objectives are your study guide (not this PowerPoint) Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  Chapter 8 Objectives 1. Describe the principle of absorbance spectrophotometry and list the components. 2. Define Beer’s Law. 3. Compare observed colors of the visible spectrum and the corresponding wavelengths. 4. Describe the proper care for cuvettes. 5. Given the concentration and optical density reading prepare a Standard Curve. 6. Demonstrate how to use a spectrophotometer and prepare a Standard Curve.. 7. Demonstrate how to use a Standard Curve for reporting results. 8. Define the following words: a. meniscus f. blank b. absorbance g photoelectric cell c. %T h. monochromator d. reagent i. diffraction grating e. standard j. galvanometer Measurement Techniques Most measurement techniques fall into one of four categories: 1. Spectrometry- Focus of this course Others will be covered in Immunology 2. Luminescence 3. Electroanalytical methods 4. Chromatography 2 Absorbance Spectrophotometry The Nature of Light Light is a type of radiant energy, and it travels in the form of waves. The wavelength of light is the distance between waves. The term light is used to describe radiant energy with wavelengths visible to the human eye or with wavelengths bordering on those visible to the human eye. Spectrophotometer An instrument used to determine the concentration of colored solutions. Determined by passing a beam of light through the solution 3  Spectrophotometer Principle PRINCIPLE: The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. Observed Colors of Visible Spectrum 4  Spectrophotometer The portion of light which passes through the colored solution is the %TRANSMISSION (%T) Light that does NOT pass through the solutions is absorbed and is measured as ABSORBANCE Concentrated solutions allow less light to pass through than diluted solutions. For most colored solutions the > concentration the > absorbance Spectrophotometer  This follows Beer’s Law which states:  Absorbance of a colored substance is directly proportional to its concentration.  This is a linear relationship. -----------------------------------------------------------------------------------  For most colored solutions, the % transmittance decreases as the concentration increases  This is a geometric relationship or non-linear. 5 Spectrophotometer Parts of the Spectrophotometer 1. Light Source – Provides a beam of light 2. Monochromoter (diffraction grating) -disperses light into a spectrum 3. Monochromoter light - Contains a slit to isolate a beam of light (wavelength) Spectrophotometer 4. Cuvette – Holds the colored solution 5. Photo-Detector – Detects the light that was transmitted and converts it to electricity 6. Galvanometer – Measures and records the electrical current. 6 Spectrophotometer and Standard Curves Standard curves are used to determine the concentrations of colored solutions with follow Beer’s Law. Solutions are made with specific dilutions of a STANDARD or KNOWN concentration. The absorbance of the standards are read on the spectrophotometer. Spectrophotometer and Standard Curves  The readings are plotted versus the known concentrations of the dilutions  Absorbance is plotted using linear graph paper. (Absorbance is LINEAR)  Absorbance is plotted on the “Y” axis (Vertical alignment I__)  Concentration is plotted on the “X” axis (Horizontal alignment I__ )  See following chart 7  Standard Curve  Preparation and Use of a Standard Curve  Prepare on Graph Paper  Criteria for a good standard curve: 1. The line is straight. 2. The line connects all points. 3. The line goes through the origin, or intersect, of the two axes.  See Following Charts:  Plotting a Standard Curve  Using a Standard Curve Standard Curve Y X 8 Make a Hemoglobin Standard Curve Absorbance Hgb Concentration (Y axis) g/dl (X axis) 0 Blank 1.5 5 3.0 10 4.5 15 6.0 20 9 UNKNOWN (Patient result) Based on your hemoglobin standard curve, find the patient result with an absorbance result of 3.6 RESULT: 12 g/dl Spectrophotometer Instructions for operation: See handout https://youtu.be/-et7jDXOLB4 10 Postamble READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. USE THE UNIT OBJECTIVES AS A STUDY GUIDE All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 11