AUTOMATION Instrumentation 4 types of analyzer 1. Continues flow - more than 1 test in one sample 2. Centrifugal analysis - multiple sample in one test 3. Discrete analyzer - random access testing 4. Dry slide - uses micro moles and reagents on slide of dry chemistry analysis (kodak electron) BASIC APPROACHES Continues Flow Analysis • Reagents, diluents, and samples are pumped through a system of continuous tubing • Air bubbles ( to minimize diffusion of reagents and mixing of samples) serve as separating and cleaning • All tests are performed in parallel testing. (more than one test is analyzed concurrently on organism specimen) Disadvantage 1. Carry over effect 2. Wasteful use of reagents Centrifugal Analyzer • It uses the force generated by centrifugationto transfer specimen and reagents • Tests are performed as Batch analysis. (all samples are loaded atthe same time but one test only) HbA1C = whole blood (long term monitoring of DM) Discrete Analyzer • Most Popular & Versatile Analyzer (it only measures the test that is/are requested on a sample) • It employs a variety of syringe pipets to aspirate & dispense samples & reagents • Uses random access capability / testing (any test can be performed on any sample, in any sequence) Allows STAT samples to be easily repeated Sequential testing - multiple test analyse on one sample after another on a given specimen LIGHT ENERGY, WAVELENGTH, RADIANT ENERGY Radiant Energy • characterized as a spectrum from short wavelength to long wavelength: cosmic, gamma rays, X-rays, ultraviolet, visible,infrared, microwaves, radiowaves. Wavelength • Distance between 2 successive peaks • Expressed in nanometers (nm) • Ultraviolet Light : < 400 nm • Visible Light : 400-700 nm • Infrared Light :>750 nm/>700 nm X-rays = have long wavelength Microwave = shortest wavelength Very long wavelength = infrared Very short = ultraviolet Frequency • number of vibration of wave per second Wavelength is inversely related to Frequency & Energy SHORTER wavelength :High Frequency, High Energy LONGER Wavelength : Low Frequency, Low Energy Wavelength <400 400-430 430-480 480-560 560-590 590-630 630-750 >750 Color Absorbed Not visible Violet Blue Green Yellow Orange Red Not visible Spectrophotometry • Instrument that measures light energy () • Involves measurement of the light Transmittance by a solution to determine the concentration of the light absorbing substances in the solution. SPECTROPHOTOMETRY • PRINCIPLE:Beer Tambert’s Law • States that Concentration of Unknown Substance is directly proportional to Absorbed Light (Absorbance) Kung gaano kconcentrated ang specimen ganun din kadami ang maabsorb na light • Concentration of Unknown Substance is inversely proportion to the Transmitted Light (Transmittance) dipende ang concentration sa na-absorb na light. Kung gaano kaconcentrated ang specimen, ganun naman ang kinakonti ng matratransmit na light Absorbance Light absorbed by the solution to the concentration of solution Transmittance Amount of light that passes through colored solution Indirectly proportional to the concentration of solution calculate analytic concentration through 1. Molar absorptivity value A= abc a = molar absorptivity b = length of light c = concentration molecules A= 2-log %T A= log (100/%T) 2. Ratio of standard to the unknown Cu= Au / As x Cs 3. Standard curve (graph paper) A. Semilogarithmic paper X- concentration of the standard solution Y - transmittance or the absorbance 1. Line is straight 2. Line connects all post 3. Line goes to the origin or intersect the axes B. Linear graph paper SPECTROPHOTOMETRY Parts of Spectrophotometer: 1.Light Source • Provide Incident Light/ Radiant Energy Types of Light Source: • Continuum source • Emits Radiation that changes in intensity (Widely Used:Tungsten; Deuterium, Xenon) • Line source • Emits Radiation & Wavelength (Hg; Hollow-Cathode;LASER light amplification by stimulated emission of radiation) 1. LIGHT SOURCE Types Incandescent lamp/ tungsten iodine lamp Deuterium discharge lamp Mercury lamp Silicon carbide/ quartz halogen Xenon lamp Description Typical source in most spectrophotometers For Visible & Near Infrared Region (320-700 nm) For UV spectrum Used in UV & Fluorescent & HPLC For Infrared Region Produce a continuous spectrum of fairly uniform intensity from 300-2000 nm Useful for Visible, & UV applications Parts of Spectrophotometer: 2. Entrance Slit • Minimizes unwanted/ Stray Light and prevents entrance of scattered light into the monochromator system. •Stray light : Presence of any light other than the wavelength of measurement reaching the photodetector. • Most common cause of loss of linearity 3. Monochromator • Isolates specific or individual wavelength of light. Types: • Diffraction gratings • most commonly used because it has a better solution than prism • Glass with grooves and slit used for UV, Visible & Infrared • Diffraction, the separation of light into component wavelengths, is based on the principle that wavelengths bend as they pass a sharp corner. 3. MONOCHROMATOR Types: • Prism glass, quartz, sodium chloride • Produces the purest monochromatic light in UV & Separates white (Visible) Light into a continuous spectrum • Interference filter semi transparent silver films • Produce monochromatic light based on the principle of constructive interference of waves Parts of Spectrophotometer: 4. Exit Slit • Controls the width of light beam (Bandpass) • Bandpass : Total Range of wavelengths transmitted • Exit slit selects the bandpass of the monochromator that allows light of the selected wavelength to pass through the cuvet onto the detector. Parts of Spectrophotometer: 5.Cuvet/ Cuvette/ sample cell • Analytical cell / absorption cell / photometer tubes • It holds the solution whose concentration is to be measured TYPES DESCRIPTION Alumina silica glass Quartz/plastic/fused silica Borosilicate glass Soft glass Most Commonly Used in Visible Region Measurements below 340 nm (UV & Visible) For Alkaline solution For Acidic solution Parts of Spectrophotometer: 6. Photodetector • Detects and converts transmitted light into photoelectric energy • The more light transmitted, the more energy, and the greater the electrical signal that is measured TYPES Photocell Phototube Photomultiplier tube Photodiode DESCRIPTION Aka “Barrier Layer Cell/ Photovoltaic cell” Simplest Detector; least expensive; TemperatureSensitive Contains cathode & anode enclosed in glass case Most Commonly Used (Visible & UV)Detects Very Low Levels of Light (↑ Sensitivity; Rapid response) Excellent linearity Parts of Spectrophotometer: 7. Meter/Read-out Device • Displays the output of the detection system TYPES OF SPECTOPHOTOMETERS: • Single Beam • Simplest Type of Absorption Spectrometer and designed to make one measurement at a time at one specifies wavelength. •Double Beam • An Instrument that splits the monochromatic light into 2 components- one beam passes through the sample and through a reference solution or blank. Reagent blank (compensate reading), water blank (set spectro reading to 0), sample blank(compensate for unsafe absorbance (hemolysis, steric sample,lypinic sample)) • Double-Beam in Space : 2 photodetector. 1 for sample, 1 for reference beam • Double-Beam in Time : 1 photodetector Wavelength accuracy ISSUES IN QUALITY CONTROL OF SPECTROPHOTOMETER Means the wavelength indicated on the control dial is actual wavelength of light passed by the monochromator Stray light (entrance slit) Determined by use of Holmium Oxide & Didynium Filter Presence of any light other than the wavelength of measurement reaching the photodetector Most Common Cause: Reflections of light from scratches on optical surfaces Linearity Remedy:use a sharp cut of filter Change in the concentration results to a straight line Atomic Absorption Spectrophotometer Principle: Ground state atoms absorb light at defined wavelength • Measures the light absorbed by Ground State Atoms (Unexcited atoms) hollow cathode lamp • Measures concentration by detecting the absorption of electromagnetic radiation by atoms rather than by molecules •More sensitive that FES • Reference Method for Calcium, magnesium • Lanthanum/ Strontium Chloride: Form stable complexes with PO4 (PO4 causes interference to Ca) • Components • hollow cathode lamp - chopper - flame- monochromator Flame Emission Spectrophotometry • Measures the light emitted by single atom burned in a flame • Measures the light absorbed by Excited State Atoms • Lithium / cesium - internal standard correct variation in flame and atomizer characteristics • Measurement of Na; K; Li (Excited atoms) Turbidimetry • Measures the amount of Light Blocked by suspension of particles at 180 Angle • Measures abundant large particles (proteins) & bacterial suspension. • Ex: Protein Measurement; Clot formation Detection; Bacterial growth Nephelometry • Measures the amount of Light Scattered by small particles at 90 degree angle to the incident light • The amount of scatter is Dependent to the number and size of particles present in the solution • Light Source : Merciry arc, tungsten, light emitting diode • Measurement of CRP; Antigen-Antibody complexes, proteins. Rayleigh’s law __________________ • Measurement of light emitted by a substance that has been previously excited by a source of UV light • Measures the amount of light present over a zero background • Emitting Light energy of a ______________________ • Light Source : _____________________ • ______________________ • Primary Filters : _________________________ • Secondary Filters : _______________________ __________________ • Advantages: • Fluorometry is about 1000 times more sensitive than absorption techniques and has increased specificity because optimal wavelengths are chosen both for absorption (excitation) and for monitoring emitted fluorescence. • Limitations: • Changes from the established protocol that affect pH, temperature, and solvent quality; self-absorption; quenching • _______________ : Process by which fluorescence of an analytes is reduced due to excited molecule losing some of its energy by interacting with other substances in a solution FLUOROMETRY • The emission of light produced by certain substances after they absorb energy. • It is similar to fluorescence except that the time delay is ______________________ between absorption of radiant energy and release of energy as photons of light. __________________ • Diff. from Fluorometry since it does not require Excitation Energy (Light source) & Monochromators • The process where the chemical energy of a reaction produces excited atoms, and upon electron return to ground state, photons of light are emitted • Reactions are oxidation reactions of _________________ __________________ • A technique where solutes in a sample are separated for identification based on physical differences that allow their differential distribution between a mobile phase and a stationary phase. • Components: • __________________ : carries the sample • __________________ : Through w/c the mobile phase flows. • __________________ : Holding the stationary phase • __________________ : Separated Components __________________ CHROMATOGRAPHY TYPES OF CHROMATOGRAPHY PLANAR CHROMATOGRAPHY 1. ____________________ • Used for the fractionation of sugar and Amino Acid 2. ____________________ • ____________________________________ TYPES OF CHROMATOGRAPHY COLUMN CHROMATOGRAPHY 1. ____________________ • Separate mixtures of compounds that are ____________ or can be converted to volatile form • It is used for separation of steroids, Barbiturates, Blood, Alcohol, Lipids • An inert carrier gas (mobile phase) carries the vaporized sample into the column. Carrier gases commonly used include hydrogen, helium, nitrogen, and argon CHROMATOGRAPHY TYPES OF CHROMATOGRAPHY COLUMN CHROMATOGRAPHY 1. Gas Chromatography • ____________________ : separation occurs in absorption at the solid phase • ____________________ : Occurs by diff. in solute between the gas mobile & Liquid stationary CHROMATOGRAPHY TYPES OF CHROMATOGRAPHY COLUMN CHROMATOGRAPHY 2. _______________________ • ________________________________ • ___________________ : Isolate drug • ___________________ : Based on the fragmentation & ionization of molecules using energy CHROMATOGRAPHY TYPES OF CHROMATOGRAPHY COLUMN CHROMATOGRAPHY 2. _______________________ • ________________________________ • ___________________ : Isolate drug • ___________________ : Based on the fragmentation & ionization of molecules using energy CHROMATOGRAPHY TYPES OF CHROMATOGRAPHY COLUMN CHROMATOGRAPHY 3. ______________________ • Based on the distribution of solutes between a mobile Liquid phase & Liquid Stationary Phase • _____________________________ • ______________________________________ • Uses pressure for fast separations, controlled temp, in-line detectors • Used for Fractionation of Drugs, Hormones, Lipids, CHO, CHON • Separation of Hemoglobins (Thalassemia) & for HbA1C Test CHROMATOGRAPHY TYPES OF CHROMATOGRAPHY COLUMN CHROMATOGRAPHY 3. LIQUID CHROMATOGRAPHY • _____________________________ • Used for detecting Non-Volatile Subs • Confirm positive drug screening tests; • Complementary to GC-MS CHROMATOGRAPHY CHROMATOGRAPHY Separation Mechanisms Used In Liquid Chromatography • Exchange of sample ions and mobile-phase ions w/ the charged group of stationary phase • Separation of Amino Acids, proteins & Nucleic Acids • Liquid-Liquid Chromatography • Based on solubility (Polar/Non-Polar) • Separation of lipoproteins, CHO, HBA1C, ab • Liquid-Solid Chromatography (Separation is based on the differences bet. absorption and desorption of solutes at the surface of a solid particle The measurement of current or voltage generated by the activity of specific ion. ___________________ • Determine the concentration of a substance in solution employing an electrochemical cell that consists of two halfcells, where the potential difference between an indicator electrode and a reference electrode is measured. • Measurement of electrical potential (Voltage) between 2 electrodes • Use : ________________________ ELECTROCHEMISTRY ________________________ • Electrochemical Transducer capable of responding to one given ion • Direct ISE (Without Dilution): • _______________________________ • Indirect ISE (With Dillution): • _______________________________ POTENTIOMETRY ISE MEMBRANES: • Sodium : ______________________ • Potassium : ______________________ • Calcium & Lithium : ______________________ • Hydrogen : ______________________ POTENTIOMETRY ________________________ • Glass Electrode • Silver-Silver Chloride Electrode • Calomel Electrode (Hg2Cl2) ________________________ • Severinghaus Electrode POTENTIOMETRY • Amount of increase in current (i.e., the wave height) proportional to the concentration of analyte. • Current measured; voltage change versus current plotted to produce a polarogram. • Voltage at which sharp rise in current occurs characteristic of the electrochemical reaction involved ___________________ • Measurement of Current Flow produced by oxidation-reduction reaction • Use : _______________________ • Also used in Glucose (Polarography); Chloride; Peroxidase ___________________ • Measures the quantity of Electricity (Coulombs) needed to convert an analyte to a different oxidation state • Use : _____________________ • Interferences : ____________________ ___________________ • Method in which a potential is applied to an electrochemical cell and the resulting current is measured • Use : ____________________________ ___________________ • The Process of separating the charged constituents of a sample by means of electrical current • Electrophoresis is defined as the movement of charged molecules in a liquid medium when an electric field is applied ___________________ • Components: • _________________________ • _________________________ • _________________________ • _________________________ • _________________________ ELECTROPHORESIS ELECTROPHORESIS SUPPORTING MEDIA molecular size charge Molecular Size surface charge & Molecular Size mon (Routine) (Routine) isozenzymes used) ELECTROPHORESIS • Detecting System : ___________________ • Colorimetric Stains Or Fluorescent Chemicals. • Serum proteins : _____________________________ • CSF proteins : _____________________________ • Lipoproteins : _____________________________ • Lactate dehydrogenase isoenzymes : __________ ELECTROPHORESIS Commonly Encountered Problems In Electrophoresis • Holes in staining pattern • _______________________________ • Very slow migration • _______________________________ • Sample precipitates in support • _______________________________ ELECTROPHORESIS ELECTROPHORESIS TYPES OF ELECTROPHORESIS • Migration of Small Ions • Migration of Charged Macromolecule • The movement of buffer ions and solvent based fixed support • Migration through pH gradient (Isoenzymes, CSF oligoclonal) • It is ideal for separating proteins of identical sizes with different net charges • Molecules are separated by electro-osmotic flow (EOF) • Most Important Application: _______________________ • Hemoglobin Electrophoresis • _______________ : Tetramer composed of four globin chains, four heme groups, and four iron atoms a. Hemoglobin A1 : __________________________________ b. Hemoglobin A2 : __________________________________ c. Hemoglobin F : __________________________________ ELECTROPHORESIS • __________________________ A number of exist where a substitution of one amino acid on either the alpha chain or the beta chain causes the formation of an abnormal hemoglobin molecule ELECTROPHORESIS Hemoglobinopathies Substitution for glutamic acid in position 6 of the beta chain chain chain THANK YOU!!! JBCRMT DO NOT PHOTOCOPY / REPRODUCE WITHOUT PERMISSION! WASH YOUR HANDS AND WEAR YOUR
