Clinical Chemistry instrumentation PDF

CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA...

CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA INSTRUMENTATION: PART 1 IMPORTANT TOPIC TODAY: (SPECTROSCOPY) 2.) TRANSMISSION ▪ This deals with the interaction between light and matter. ELECTROMAGNETIC WAVES ▪ The light that we see is composed of electromagnetic waves. The following picture shows the different parts of a wave: - When light-waves enter matter and passes through matter in a straight-line. 3.) REFRACTION ▪ This wave will move from here to there, wherever you might have your light source, trending to wherever you want it. - When light-waves are bent when it enters matter and  PARTS OF AN ELECTROMAGNETIC WAVE passes through matter. 1. Crest – the highest point of the wave. 4.) DIFFRACTION 2. Trough – the bottom/lowest-point of the wave. 3. Amplitude – the distance between the middle- part/center-point of the wave to the highest point and the distance between the middle part/ center-point of the wave to the lowest point. 4. Wavelength – The distance between one crest to another crest, one trough to another trough, or any part of the wave that corresponds to the next point in that wave (when the wave reaches that same point). 5. Wave number – the reciprocal of the wavelength. 1 - When light-waves enters or comes through some sort of divided by Wavelength. slit in your material and electromagnetic waves, which DIFFERENT INTERACTIONS OF ELECTROMAGNETIC WAVES are composed of many wavelengths, they are bent. 1.) REFLECTION - Each wavelength component of the light is bent towards different directions. - This is when you stand in front of a mirror, then the light approaches a mirror and bounces back/ reflects. FRANCISCO, RAPHAEL CARLOS Y. 1  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA 5.) ABSORPTION 8.) LUMINESCENCE, FLUORESCENCE, & PHOSPHORESCENCE - When light comes through matter and some part of the wave is absorbed and only a small portion goes through it or sometimes none-at-all. - When electromagnetic waves react with matter those waves give out energy to the electrons of your matter. - When you have your light absorbed into the material, from relaxed state it goes to your excited state. 6.) SCATTERING - When light comes in-contact with matter and instead of bouncing back or going through in one direction, it scatters all-around the 3D volume of that material. - There is an absorbance of electromagnetic waves from 7.) SPONTANEOUS EMISSION the ground or from the relaxed state then the electrons they go to your excited state. Then you take away the source of the electromagnetic waves and the electrons start to relax, when your electrons relax fast enough, they give off luminescence. - Fluorescence only happens when the electromagnetic waves are still reacting towards your matter. - Phosphorescence, on the other hand, happens when you completely take off the light source/electromagnetic wave and the object is still keeps on giving off light energy. - This is for example when you burn your charcoal, and when you add more thermal energy to your charcoal and the charcoal is already ignited when you blow more air into your charcoal, the glow of your charcoal intensifies. FRANCISCO, RAPHAEL CARLOS Y. 2  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA ▪ Frequency is directly proportional to energy. RELATIONSHIP BETWEEN WAVELENGTH AND ENERGY - Meaning to say the higher the frequency, the higher the energy yield, the lower the frequency the lower the PLANCK’S FORMULA ▪ E = hv energy yield. ▪ h is a constant at (6.63 x 10-34 J sec), this is known as ▪ Wavelength is inversely proportional to energy. Planck’s constant. - Meaning to say the longer the wavelength the lower the ▪ v is frequency. energy yield, the shorter the wavelength the higher the energy yield. FREQUENCY  is the number of vibrations of wave motion per second. VISIBLE LIGHT SPECTRUM  Frequency of a wave is inversely proportional to the wavelength; it follows that the energy of electromagnetic radiation is inversely proportional to wavelength. THE ELECTROMAGNETIC SPECTRUM ▪ Has different energies and that energy is based on the frequency and that frequency when computed for mathematically is dependent on the wavelength.  Spectroscopy was first noticed during the time of Newton, ▪ Electromagnetic waves according to size of wavelength: wherein he had this prism and he allowed white light from 1.) Radio wave the sun to go through the prism and this prism allowed 2.) Micro wave the light to separate into different colors on his dark wall. 3.) Infrared  It was later found out that what we see directly as color, 4.) Visible Light (700nm to 200nm); (700nm to 400nm in is what is reflected to us by the object. When we see reality) shining light on a material, for example when it absorbs 5.) Ultra-Violet (UV) violet, all the rest is reflected to us and what we see is 6.) X-ray not the color violet. 7.) Gamma Ray ▪ Electromagnetic waves according to frequency: 1.) Gamma Ray (Highest Energy Yield) 2.) X-ray 3.) Ultra-violet (UV) 4.) Visible Light 5.) Infrared 6.) Micro wave 7.) Radio wave (Lowest Energy Yield) FRANCISCO, RAPHAEL CARLOS Y. 3  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA COLORIMETRY OPERATION OF SPECTROPHOTOMETER ▪ The solution of many compounds has characteristic colors. ▪ The intensity of color is proportional to the concentration of the compound. ▪ Example: when we have a particular solution of copper sulfate, what is the color of copper sulfate? It is the color blue, which is the color that is reflected back to us. PHOTOELECTRIC COLORIMETRY  The primary analytical utility of spectrophotometry or filter photometry is the isolation of discreet portions of the spectrum for purposes of measurement. SPECTROPHOTOMETRIC MEASUREMENT  Is a measurement of light intensity in a narrow wavelength. PHOTOMETRIC MEASUREMENT  Is a measurement of light intensity. SPECTROPHOTOMETRY ▪ The figure above shows the basic set-up of a spectrophotometer. ▪ Measures amount of light transmitted by a solution to 1.) Light source – gives us our light/ sets of wavelengths. determine the concentration of the light absorbing This gives us our electromagnetic spectrum. substance in a solution. It can be: ▪ The more light absorbed the higher the concentration. a.) White light – all colors, polychromatic light ▪ Measures the amount of light reflected by that particular b.) Monochromatic light – light of one color. material or solution and it is basically compared to how 2.) Wavelength selector or Monochromator – it allows us light might be transmitted by a particular concentration. to select which wavelength of light passes through our BEER-LAMBERT LAW sample. It is composed of:  Measuring the amount of light absorbed by a solution a.) Entrance Slit – it should be narrow, so that the relating that absorption to the solution’s concentration. color that we really want is the one that is passing  It states that the concentration of the unknown through to the dispersing device. substance is directly proportional to the absorbed light. b.) Dispersing Device (absorbance or optical density) and inversely c.) Exit Slit proportional to the amount of transmitted light 3.) Sample Holder (%transmittance). 4.) Detector  Mathematically establishes the relationship between 5.) Data readout concentration and absorbance. INTENSITY – How much light or power of the light that  Allows us to correlate the absorbance of light with the comes through. concentration of a particular solution. (This is the basic TRANSMITTANCE – Defined as how much light came out idea about spectrophotometry/spectroscopy). over how much light initially came in. A = abC Where: A is absorbance b is path length a is absorptivity C is concentration ▪ Since a (absorptivity) and b (path length) are constant, there is a linear relationship between absorbance (A) of material and concentration (C) of that material in that solution. FRANCISCO, RAPHAEL CARLOS Y. 4  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA PERCENT TRANSMITTANCE ABSORBANCE (A) ▪ It is the ratio of the radiant energy transmitted (I t) divided ▪ It is the amount of light absorbed. by the radiant energy incident on the sample (I0) ▪ Proportional to the inverse log of transmittance ▪ Mathematically derived from %T FORMULA: FORMULA: ▪ The %T measured by the commercial spectrophotometers is the ratio of the sample Where: transmitted beam divided by the blank transmitted A is Absorbance beam. a is coefficient of absorptivity (specific for each compound) ▪ In actual practice, the light transmitted by the blank is b is path of cuvette (container) submitted for I0. c is concentration of solution FORMULA: LIGHT SOURCE or (WAVELENGTH SOURCE) ▪ Provides polychromatic light and must generate sufficient radiant energy or power to measure the analyte of interest. ▪ An intense beam of light is directed through the monochromator and the sample. ▪ To give accurate absorbance measurements throughout its GRAPHICAL RELATIONSHIP absorbance range, its response to change in the light ▪ %transmittance and %absorption is not linearly related intensity must be linear. to concentration. ▪ Wavelength source for our instruments. ▪ For a graph to be useful, straight line is needed. ▪ When we talk about UV visible spectrophotometry, we have ▪ Gives us the idea that we have an absorbance, and we here the bulbs or the visible arrange. can equate it with Beer-Lambert’s Law.  LINEAR AND DYNAMIC RANGE is a certain useful region of the graph between the relationship of absorbance or instrument response and the analyte concentration.  Absorbance should be useful in getting the concentration of a sample. FORMULA: Tungsten Lamp Deuterium lamp with UV glass envelope  Deuterium lamps gives us the UV range of light. FRANCISCO, RAPHAEL CARLOS Y. 5  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA TWO TYPES OF LIGHT SOURCE: 1.) Line Source 2.) Continuum Source  Emits limited radiation and wavelength.  Emits radiation that changes in intensity; widely  Very specific lamp for very specific uses. used in the laboratory. Examples:  Emits a wide range of wavelengths. a.) Mercury and sodium vapor lamps in  They find widespread use in absorption and spectrophotometers (UV and visible regions) – when fluorescence spectroscopy. you light them up, all the wavelengths given by mercury  For the UV region, the most common sources is and sodium vapor those are the ones you can only get. the deuterium lamp. b.) Hollow cathode lamp (AAS: atomic absorption  Monochromator is needed for this to be used. spectroscopy) Examples: – Line source that emit a few discrete lines find wide a.) Tungsten light bulb – commonly used light source use in atomic absorption, molecular and fluorescent for visible and infrared region. spectroscopy. – whatever material you are analyzing is what composes your filament. For example you are analyzing for gold, you only have gold in your filament. And if you have nickel, that is you filament, and so on. c.) LASER (Light Amplification by Stimulated Emission of Radiation) – is also used as a light source in spectrophotometry. b.) Deuterium lamp – routinely used to provide UV radiation in analytical processes. FACTORS OF CHOOSING LIGHT SOURCE Range – like if you need a lamp for Visible light and UV rays, it should give you wavelengths for it. Spectral distribution with the range Source of radiant production Stability of radiant energy and temperature Cheap and easily assessable c.) Xenon discharge – produces a continuous source ALTERNATIVES OF TUNGSTEN LAMP of radiation which converts both the UV and visible 1.) Mercury arc (Visible & UV) region. (Expensive One) 2.) Deuterium Lamp 3.) Hydrogen Lamp (UV) 4.) Xenon Lamp 5.) Merst Glower (IR) 6.) Globar (Silicone Carbide for IR) ENTRANCE SLIT ▪ Minimizes unwanted or stray light and prevents the entrance of scattered light into the monochromator system. d.) Argon Lamp Stray Light  Refers to any wavelengths outside the band transmitted by the monochromator; it does not originate from polychromatic light source; it causes absorbance error.  Limits the maximum absorbance that a spectrophotometer can achieve.  Is the most common cause of loss of linearity at high- analyte concentration. FRANCISCO, RAPHAEL CARLOS Y. 6  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA MONOCHROMATOR PHOTODETECTOR ▪ It isolates specific or individual wavelength of light. ▪ It detects and converts transmitted light into photoelectric ▪ In the olden days, it was just a prism. energy. ▪ Detects the amount of light that passes through the sample Kinds of Monochromator: in the cuvette. a. Prisms b. Diffraction gratings – you get this when you put lots of prisms side-by-side. c. Filters – this is used if you want a specific type of light to pass through. Such as if you want only red light to pass through you have your red filter. d. Holographic gratings EXIT SLIT ▪ Controls the width of beam of light (bandpass). Photomultiplier Tubes Light Detecting Diode ▪ Allows only a narrow fraction of the spectrum to reach Kinds of Photodetector the sample cuvette. a. Barrier Layer ▪ Bandpass – is the total range of wavelengths  Simplest detector, least expensive, temperature sensitive transmitted.  Used in filter photometers with a wide bandpass. ▪ Accurate absorbance measurement requires a bandpass less than 1/5 the natural bandpass of the  Is a basic phototransducer that is used for detecting and spectrophotometer. measuring radiation in the visible region. ▪ The degree of wavelength isolation is a function of the  Composed of selenium on a plate of iron covered with a type of device used and the width of entrance and exit transparent layer of silver. slits.  Requires external voltage source but utilized internal ▪ Special purity of the spectrophotometer is reflected by electron transfer for current production-low internal the bandpass, that is, the narrower the bandpass, the resistance. greater the resolution.  Typically has a maximum sensitivity at about 550nm and the response falls off to about 10% of the maximum at 350 CUVET & 750. ▪ It is also called as absorption cell/analytical cell/sample cell. b. Phototube ▪ It holds the solution whose concentration is to be  Contains cathode and anode enclosed in a glass case. measured.  It has photosensitive material that gives off electron when ▪ Designed to pass most of the incident light through light energy strikes it. without absorbing it.  It requires an external voltage for operation. KINDS OF CUVETS: 1.) Alumina silica glass – most commonly used (can be used in 350-2000nm) 2.) Quartz/Plastic – used for measurement of solution requiring visible and ultraviolet spectra. 3.) Borosilicate Glass 4.) Soft Glass FRANCISCO, RAPHAEL CARLOS Y. 7  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA c. Photomultiplier Tube d. Photodiode  Most commonly used detector to measure visible and  Not as sensitive as Photomultiplier Tubes (PMT) but UV regions. excellent linearity.  Excellent sensitivity and has a rapid response detects  Measure light at a multitude of wavelengths-detects less very low levels of light. amount of light.  Response of the PMT begins when incoming photons  Has a lower dynamic range and higher noise compared to strike a photocathode. PMT.  These tubes are limited to measuring low power  Most useful as a simultaneous multichannel detector. radiation because intense light causes irreversible  Preferred because it is cheap. damage to the photoelectric surface.  Should never be exposed to room light because it will burn out.  This instrument is simply when you put many phototube detector inside. Photodiode METER OR READ OUT DEVICE ▪ Displays output of the detection system Example/s: Galvanometer or Ammeter or Light-Emitting Diode (LED) Inside a Photomultiplier Tube Display When incident light comes in and for example ejects one electron it goes to where photoelectrons gets ejected and then it moves along and touches dynode number 1 it emits two or three electrons and it multiplies each time it goes to another dynode. Galvanometer Computers/LED Display Photomultiplier Tubes Phone used as Ammeter FRANCISCO, RAPHAEL CARLOS Y. 8  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA TWO TYPES OF UV-VIS SPECTROPHOTOMETER B. Double Beam in Time – with one photodetector and alternatively passes the monochromatic light through the 1.) SINGLE BEAM SPECTROPHOTOMETER sample cuvet and then reference cuvet using a chopper or ▪ It is the simplest type of absorption spectrometer. rotating mirror. ▪ Designed to make one measurement at a time at one specified wavelength. ▪ The absorption maximum of the analyte must be known in advance when a single beam instrument is used. ▪ The cheapest one and simplest one to manufacture, yet the most tiresome spectrophotometer to work with. ABSORBANCE MEASUREMENT ▪ Ascertain the wavelength of maximum absorbance for the compound of interest! 2.) DOUBLE BEAM SPECTROPHOTOMETER ▪ Each chemical has a region where light is absorbed, and no ▪ It is an instrument that splits the monochromatic light light is absorbed. into two components- one beam passes through the ▪ Allows only a narrow fraction of the spectrum to reach the sample, and the other is through a reference solution or sample cuvette. blank. BLANKING TECHNIQUE ▪ The additional beam corrects for variation in light source intensity. ▪ To compensate unwanted light absorption, we measure a ▪ The absorbance of the sample can be recorded directly reagent blank. as the electrical output of the sample beam. ▪ The light absorbed by the reagent blank can be subtracted from the measurement using the sample, and a “true” absorbance reading can be obtained! ▪ As long as the absorbance value of reagent blank is relatively constant, the reagents may be functioning properly. ▪ If the blank changed markedly: reagent may be deteriorated. ▪ Can at least compensate unwanted absorbance. CALCULATION OF ABSORBANCE MEASUREMENT KINDS OF DOUBLE BEAM SPECTROPHOTOMETER a. Ratio of Standard to Unknown b. Standard Curve A. Double Beam in Space – with 2 photodetectors, one c. Use of Absorptivity Value each for the sample beam and the reference beam. FRANCISCO, RAPHAEL CARLOS Y. 9  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA 1.) RATIO OF STANDARD TO UNKNOWN STEP#2: MEASURE THE ABSORBANCE OF THOSE STANDARDS IN THE SPECTROPHOTOMETER ▪ Simplest type of concentration measurement involves the determination of the absorbance value for a known concentration of the compound of interest. ▪ The absorption maximum of the analyte must be known in advance when a single beam instrument is used. ▪ Seldomly used in chemical analytical laboratories. Example: We wish to determine the bilirubin concentration in a sample. The absorbance of our unknown at 450nm is 0.428, & the absorbance of our 5.0 mg/dL standard is 0.372. What is the concentration of Bilirubin? STEP#3: GRAPH CONCENTRATION VS. ABSORBANCE – THIS IS THE ‘STANDARD CURVE’  THE STANDARD CURVE MUST BE LINEAR! STEP#4: GRAPH THE UNKNOWN ABSORBANCE ON YOUR 2.) USE OF STANDARD/CALIBRATION CURVE STANDARD CURVE STEP#1: MAKE YOUR STANDARD SOLUTIONS We measure the unknown’s absorbance for example the absorbance is 0.70, now we graph the unknown absorbance vs protein concentration and then we graph the line of best fit. This  LEAVE ONE CUVETTE BLANK/EMPTY is what you call a linear regression. After we get the line of best fit/linear regression, we will get our line equation. Now we have to check our r2. If we have and r2 = 1, it is perfectly linear. In sir’s chemistry lab I want it at 0.999, if it is not I redo the analysis. But if the accrediting body 0.9 is acceptable then its ok. Then solve. y is now the absorbance and x is now the concentration. FRANCISCO, RAPHAEL CARLOS Y. 10  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA 3.) USE OF MOLAR ABSORPTIVITY REMEMBER: ▪ Beer's Law may not last forever. As concentrations continue to increase, the linear relationship between absorbance and concentration may break down. For example: The reaction used to quantitate ethanol in blood involves enzymatic reaction: conversion of ethanol to acetaldehyde with reduction of NAD to NADH. For every molecule ethanol oxidized, one molecule of NAD is ▪ When this happens, we say that the methodology is "out of reduced to form NADH. linearity" or "out of range". Instruments will usually report Given: Required: C (Concentration) = ? an "error code" or "error message" when this occurs. Change in A340 = 0.421 Cuvet Path Length = 1 cm ▪ Specimens with extremely high concentrations will need to Sample Volume = 3mL be diluted with appropriate solutions and retested. We plug in molar absorption coefficient into “a” and “b” ▪ The final concentration from diluted specimens must be is the standard 1cm length. And usually A for multiplied by the dilution factor to give the correct absorbance is given, with all those given we can now concentration in the undiluted specimen (sometimes solve for concentration. analyzers will do this for you automatically). FLUOROMETRY ▪ When light strikes a compound, it may be absorbed. ▪ Part of the energy provided by the light is used to excite one or more electrons, moving them out in their normal pathway. ▪ FLUORESCENCE occurs when this electrons give off light as they pass from excited state back to their ground state level within the molecule. ▪ FLUORESCENCE SPECTRA ▪ EXCITATION SPECTRUM gives information proper wavelength of light in order to produce the maximum number of electrons raised to the excited state. FRANCISCO, RAPHAEL CARLOS Y. 11  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA TWO TYPES OF FLUORESCENCE SPECTRA FLAME EMISSION PHOTOMETRY 3.) EXCITATION SPECTRUM ▪ Measure the light emitted by a single atom burned in a flame.  Gives information proper wavelength of light in order ▪ PRINCIPLE: Excitation of electrons from lower to higher to produce the maximum number of electrons raised energy state. to the excited state. ▪ LIGHT SOURCE: Flame (also serves as cuvette)  Light of different wavelength passes through the ▪ METHOD: Indirect internal standard method sample, & the amount of fluorescence is ▪ Internal Standard: Lithium/ Cesium – corrects variations determined. in flame and atomizer characteristics.  The wavelength of excitation light that produces the ▪ Used for the measurement of excited ions (Na & K). maximum fluorescence is then used as excitation ▪ Flickering light indicates changes in the fuel reading of the wavelength. instrument. 4.) EMISSION SPECTRUM ▪ What happens here is that you have electrons and you put  Using the wavelength that stimulates the maximum it inside a flame and it becomes atomized and it passes no. of electrons, the intensity of the fluorescent through the flame, the flame gives the sample energy/ the produced is determined at different wavelengths. power to emit light.  The wavelength where the fluorescence is seen at ▪ This is like when you have your lighter and alcohol and you its highest is called emission wavelength. let your alcohol spray and lighter on and it’s a party-party. ▪ For example: if you have sodium in a sample and you let it blaze it becomes yellow in the flame, and if you have copper FLUORESCENCE INSTRUMENTATION in a sample and you let it blaze it becomes blue. ▪ This is similar to our UV-Vis Spectrophotometry, but ▪ Different atoms give different colored flames. there is a change in the set-up, there is now an addition to the excitation filter, which makes our sample “excited” and then fluoresce. ▪ You also place the detector instead of linear, it has to be 90 degrees from the path of the first excitation wavelength. And then when it starts to fluoresce 90 degrees from the first path then you have the second path and then you select the wavelength of excitation in the emission filter and then that is what you allow to the detector.  LIGHT SOURCE  WAVELENGTH SELECTOR  SAMPLE HOLDER MICROWAVE PLASMA-ATOMIC EMISSION SPECTROSCOPY  DETECTOR SYSTEM  DATA READOUT FRANCISCO, RAPHAEL CARLOS Y. 12  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA ATOMIC ABSORPTION SPECTROPHOTOMETRY ▪ Measure concentration by detecting the absorption of electromagnetic radiation by atoms rather than by molecules. ▪ It measures the light absorbed by atoms dissociated by heat. ▪ PRINCIPLE: Element is not excited by merely dissociated from its chemical bond and place in an unionized, unexcited, ground state. ▪ LIGHT SOURCE: HOLLOW CATHODE LAMP - (consists of an evacuated gas-tight chamber containing an anode, a cylindrical cathode, and an ▪ You have your hollow cathode lamp and then you have inert gas, such as helium or argon.) your sample then you have the fuel and the oxidizer mix the sample in the mixing chamber, and passes through the ▪ INTERFERENCES: Chemical, Matrix (differences in stove and then you atomized/vaporized your sample and viscosity) and ionization the light passes through and some of the light is absorbed ▪ It is more sensitive than FEPI (Flame Emission by specific elements that composes your hollow cathode Photometry) it is accurate, precise, and very specific. lamp, if it is a gold analyzer the cathode should be made ▪ Internal standard is not needed-changes in aspiration up of gold. It will analyze gold in the flame and you will have have little effect on the number of ground state atoms. atomic emission and pass through the slit and pass the ▪ An atomizer (nebulizer/graphite furnace) is used to monochromator then it will go through the photodetector. convert ions to atoms; a chopper is used to nodulate the The photodetector will now send the Absorption values to light-source. the computer. ▪ Lanthanum or strontium chloride is added to samples to form stable complexes with phosphate. ATOMIC ABSORPTION ◦ A cathode tube containing one given metal emits a light wavelength unique for that metal. Common metals are lead and mercury. ◦ The patient specimen is vaporized (literally !!!) into an acetylene flame and is dispersed into the light path. ◦ Metal atoms in the ground state from patient specimen absorb the light energy from the cathode tube. ◦ Because light is emitted from the metals in the cathode tube and the patient metals in the flame, the chopper breaks up the signal from the cathode tube. Atomic Absorption Machine ◦ The photo detector measures the difference in the signals to measure the light emitted from the flame only. ◦ Atomic Absorption is only suited for metallic substances that are not destroyed by the flame - Everything else gets "fried". FRANCISCO, RAPHAEL CARLOS Y. 13  CLINICAL CHEMISTRY-1 LECTURER: WILFREN A. CLUTARIO, RCH, MSC LEC. 7A BACHELOR IN MEDICAL LABORATORY SCIENCE : BATCH DESULFURELLA FRANCISCO, RAPHAEL CARLOS Y. 14