Unit I- Student Notes(2) 2.docx

Unit I Optical Techniques Many laboratory determinations are made by measuring light that has been: Transmitted Emitted Absorbed Reflected Scattered Photometry: The measurement of the luminous intensity of light or the amount of luminous light falling on a surface from such a source There are two...

Unit I Optical Techniques Many laboratory determinations are made by measuring light that has been: Transmitted Emitted Absorbed Reflected Scattered Photometry: The measurement of the luminous intensity of light or the amount of luminous light falling on a surface from such a source There are two types of photometers: Filter Photometers Spectrophotometers Filter Photometers: optical filters are used to isolate a narrow wavelength range of spectrum (to provide monochromatic light) Spectrophotometers: a monochromator (reducing light to one colour) with prisms or gratings is used to isolate specific wavelengths of light Introduction to Spectrophotometry: Involves interaction of electromagnetic radiation with matter Spectrophotometer is an instrument used to indirectly determine the amount of a compound present in a solution by shining a light of a specific wavelength through the solution and measuring how much was absorbed Each compound will absorb, transmit and reflect a certain wavelength For laboratory application, typically involves light in the ultraviolet and visible regions of the spectrum (290-800nm) More molecules that are present the more absorption there is. Like kool aid- the higher the concentration the darker it is going to be Wavelength, Frequency and Energy: Electromagnetic radiation is described as photons of energy traveling in waves The relationship between wavelength (λ) and energy (E) is described by Planck’s formula This equation demonstrates that the energy of light is inversely proportional to the wavelength Wavelength is peak to peak as the wavelengths get bigger they have less energy, smaller wavelengths is higher energy Shorter frequency higher energy longer frequency low energy Absorption of EM (Electromagnetic) Radiation Consider a beam of radiant energy with an original intensity, Io, impinging on and passing through a square cell (whose sides are perpendicular to the beam) containing a solution of a compound that absorbs radiant energy of a certain wavelength. The intensity of the transmitted radiant energy, Is, will be less than Io. Some of the incident radiant energy may be reflected by the surface of the cell wall or solvent, therefore, these factors must be eliminated if one is to consider only the absorption of the compound of interest. This is done by using a blank or reference solution containing everything but the compound to be measured. Derivation of Beer’s Law: T (transmittance) = IS/I0 %T = IS/I0 x 100 A = - log (IS/I0) = log (100%) – log % T OR … A = 2 – log % T The Beer-Lambert Law The Beer-Lambert law (most commonly referred to simply as Beer’s law) states that the concentration of a substance is directly proportional to the amount of light absorbed or onversely proportional to the logarithm of the transmitted light. If the concentration of a solution is constant and the path length through the solution that the light must traverse is doubled, the effect on the absorbance is the same as doubling the concentration, since twice as many absorbing molecules are now present in the radiant energy path. Thus, the absorbance is also directly proportional to the path length of the radiant energy through the cell. More light that gets through means the less amount of concentration The longer the light path the higher the concentration (if you have two cuvets side bvy side the concentration is doubled) The mathematical relationship that connects absorbance of radiant energy, concentration of a solution, and path length is shown by Beer’s Law. A = εbc A = absorbance = (epsilon) molar absorptivity (L. mole-1 cm-1), this is a constant for a given compound at a given wavelength b = light path (cell path length in cm), usually 1 cm c = concentration of the substance of interest (mole/L) This equation forms the basis of quantitative analysis by absorption photometry or absorption spectroscopy. Absorbance values have no units The absorptivity is a proportionality constant related to the chemical nature of the solute and has units that are reciprocal of those for b and c. When c is expressed in moles/L and b is expressed in centimeters, the symbol ε, called the molar absorptivity, is used in place of a { (A = abc) and is a constant for a given compound at a given wavelength under specified conditions of a solvent, pH, temperature. To review: Beer’s Law – The amount of light absorbed by a solution is directly proportional to the concentration of a solution … AND CONVERSELY … the log of the transmitted light of a substance is inversely proportional to its concentration As the concentration or cell path increases, the absorbance increases, and transmittance decreases Relationship of molar absorptivity to absorbance: A higher molar absorptivity will impart a greater sensitivity to the measurement Because of the linear relationship between absorbance and concentration, it is possible to relate unknown concentrations to a single standard by a simple proportional equation. Therefore: As / Au = Cs /Cu and Cu = Au Cs Where: As Cu = concentration of the unknown Cs = concentration of the standard Au = absorbance of the unknown As = absorbance of the standard The previous equation is valid only if the sample obeys beers law and both standard and unknown are measured in the same cell (only linear relationship between absorbance and concentration) Usually a linear relationship exists for a certain concentration and absorbance, when calculating an unknown, make sure that your data falls within the standard calibration curve (line of best fit) Limitations of Beer’s Law: Deviation from Beer’s Law, that is, variations from linearity of the absorbance versus concentration curve, occur in the following conditions: 1. Very elevated concentrations are measured- will not follow beers law, we will have to dilute 2. incident light is not monochromatic- any test is a very specific wavelength for accurate results 3.  the solvent absorption is significant compared with the solute absorbance, if the solution that sample is in absorbs a lot it is useless 4.  The sides of the cell (cuvette) are not parallel- square cant use round, causes bended light and stray light will cause false high reading 5.    radiant energy (light) is transmitted by other mechanisms (stray light) 6.   Fluorescence: If the absorbance of a fluorescent solution is being measured, Beer’s Law may not be followed. (this is light that is emitted by a compound) for beers law we do not use fluorescence 7. If two or more chemical species are absorbing the wavelength of incident radiant energy, each with a different absorptivity, Beer’s Law will not be followed.- you wont get accurate, one wavelength per compound 8. Stray radiation (stray light) is a radiant energy that reaches the detector at wavelengths other than those indicated by monochromatic setting. All radiant energy that reaches the detector with or without having passed through the sample will be recorded.- too much stray light will decrease accuracy, we have to do a lot of maintenance and qc on analysers 9. As the amount of stray light increases (or monochromicity decreases), deviation from Beer’s law also increases (that is, linearity decreases). Design of Spectrometric Methods The analyte (chemical of interest) absorbs at a unique wavelength, can’t have two things absorbing the same wavelength The analyte reacts with a reagent to produce a product that absorbs at a unique wavelength (a chromophore- part of a molecule responsible for color) The analyte is involved in a reaction that produces a chromophore Spectrophotometer components Diagram: Light Source Wavelength Isolator (filters, prisms, monochromator)- narrow what wavelength Detector- measures amount of light transmittance Read out Device (meter) Light Source: Light sources come in 2 forms, either a lamp or a laser- very expensive Lamps: Tungsten or Tungsten-iodide for visible range & near infrared. Quartz-halogen- visible most common Hydrogen deuterium – U.V. Mercury for emission lines @ a specific wavelength. Also for uv and fluorometer Lasers: Light Amplification by Stimulated Emission of Radiation provide intense radiation of a narrow wavelength. They are expensive and typically used for fluorescence and specialized testing. Use Amax for achieving the most in sensitivity. Use Tmax for achieving a wider range of values, but low sensitivity. The most important factors for a light source are range, spectral distribution within the range. The source of radiant production, stability of the radiant energy, and temperature. Excitor lamp must give an intense reasonably cool consant beam of light (no dimming or strobe flickering) Must exactly align when replaced. Must be highly reproducible Temperature of the lamp is important. Different temperatures cause different spectrum. Wavelength Isolator (light separation devices): Lsers don’t need a monochrometer Unless a laser is used, a device is needed to restrict and isolate wavelength Three devices are used to separate white light into individual bands: Filter Prism Gratings Filters: know other name Absorbance Filters are made of one or more layers of coloured glass and work by absorbing all other wavelengths that are not of interest Also known as “Wratten Absorption Filter”. – know this! Glass: one or more layers of coloured glass known as “Wratten Absorption Filter”. They have limited usefulness because of the wide band pass (35-50 nm). Not precise, but simple and inexpensive. Interference Filters (Fabry-Perot): semi-transparent silver films on both sides of a thin transparent layer of magnesium fluoride. Used for a “fixed wavelength”. Also known as fabry-perot- know this Interference filters produce monochromatic light based on the principle of constructive interference of waves. When radiant energy penetrates the film and is reflected from the front, while other radiant energy penetrates the film and is reflected by the surface on the other side. If the two reflected rays are in phase, their resultant intensity is doubled. If they are out of phase, they destroy each other. This kind has a narrow band pass. Prisms: A narrow beam of light focused on a prism is refracted as it enters the denser glass. Short wave lengths are refracted more than long wavelengths, resulting in dispersion of white light into a continuous spectrum. The prism can be rotated to allow only the desired wavelength to pass through an exit slit. Glass prism for visible light; quartz for UV. Focusing lens is used to condense the light on the prism Major disadvantage of using the prism is that it gives nonlinear separation of bands. The lower bands (violet) bend more upon exiting the prism than does the higher (red) band. Therefore for proper wavelength calibration, several wavelengths must be measured because of this nonlinear refraction of light Not as commonly used in spec anymore because it bends light, we want straight beams. Visible light we will use glass Advantages: Inexpensive, offers a good means of separating light into bands. Disadvantages: gives a non-linear separation of bands. The lower bands (violet) bend more upon exiting the prism than does the higher (red) bands. Proper wavelength calibration would require several wavelengths to be measured. Gratings: Transmittance: made of glass Reflection: made of aluminum Advantage: Offer linear dispersion of light over the entire UV and visible range. Modern Holographically produced grating are made using a laser, making them very accurate, minimizes stray radiant energy (light), widely used. Modern Holographically produced (replicated), therefore minimizes stray radiant energy. Diffraction grating consists of a highly polished reflecting surface with many equally spaced parallel grooves (15,000 or 30,000 per inch) with sharp corners. It provides a narrow band pass. The resolution depends on the number of grooves. By changing the angle at which the radiation strikes the grating, it is possible to alter the wavelength reflected Monochromator: A group or band of spectra can be isolated and sent from the monochromator into the sample. Monochromator is made up of entrance slit dispersion device and exit slit. Entrance slit focuses the light onto the dispersion device in the monochromator to be isolated into individual bands that are then to be passed through an exit slit into the sample compartment. Other features: Slits: 2 types- helps narrow the light into one path Entrance: focuses the light on the grating or prism. Exit: isolate the selected narrow band of wavelength. Increase the exit slit width, the band of emerging light is broadened, which increases the intensity, but decreases spectral purity. Bandpass: The bandpass of a monochromator defines the range of wavelengths transmitted and is calculated as width at over half the maximum transmittances. Example: Bandpass = 10 nm If wavelength setting is 455nm, then the range at which light is transmitted is 455 + 5nm or 450 nm – 460 nm. The point at which bandpass is measured is at one half the peak transmittance. The bandpass is one of the most important specification of the monochromator and is indicated in nm (20, 10, 5, 2 nm, and so on). It describes the purity of the light passing through the sample. Factors that determine bandpass: 1. the light intensity output of the source 2. the efficiency of the system to isolate wavelength bands. How good your monochromator the sensitivity of the detector. How good the analyzer is Narrow bandpass offers greater resolution and sensitivity, but there is decrease in intensity. Sample Holder Also known as a cuvette or cell. Can be made of quarts (UV testing, uv doesn’t penetrate glass), glass or transparent plastic (visible) The glass and disposable plastic cuvettes are satisfactory for use in the visible region. All disposable For ultraviolet radiation quartz or fused silica cuvettes are necessary because glass can absorb UV light. Available in different sizes, the volume they can hold can vary depending on the test Cuvettes can be round or square (square are optimal because they are straight so there is less stray light, round bends and causes stray light) Cuvettes are sold in matched sets. Square cuvettes have plane-parallel optical surfaces and a constant light path. They have advantage over round cuvettes in that there is less error from the lens effect, orientation in the spectrophotometer and refraction. Cuvettes with scratches on their optical surface scatter light and should be discarded, bubbles should be removed Factors affecting results: Orientation: 0.25% error for every 5° deviation (round cuvettes). Sample size: Increasing sample size, increases sensitivity. Cell path length: Normally = 1 cm. Longer path length is required for very dilute samples. Why would this help dilute samples, by increases the [ath length the concentration gets higher because you have more sample in there so you get a higher reading Types: Quartz or fused silica - U.V. (not glass as lime in glass may absorb U.V. light) Plastic – visible Must be matched, no scratches or bubbles. Detectors: The purpose of the detector is to convert the transmitted radiation energy into an equivalent amount of electric energy, detect the light that went through the sample We need to see how much light was transmitted, we can then find out how much was absorbed The more intense the light the greater the electric signal. Four types of detectors: 1. Barrier layer cell 2. Silicon photodiode 3. Phototube 4. Photomultiplier Tube Barrier Layer Cell: Good for basic, simple spectrophotometers Light ↓ Cathode (silver) Selenium (semiconductor) Anode (iron or copper) Light hitting cathode will pop electrons off of selenium layer , this current produced is proportional to the amount of light that hit the layer cell. How it works: A semi-conducting layer of selenium and a light layer of metal that act as a collector electrode This cell generates its own electromotive force, which can be measured. Light strikes the transparent electrode, it induces electron flow in the semiconductor, which is measured by an amp-meter. The current generated is a direct function of the quantity of light striking the photocell. Photocells require no external voltage source but rely on internal electron transfer to produce a current in an external circuit. Limitations: Response times is slow Signal is difficult to amplify, therefore suitable for higher intensity. We need a lot of light to get a signal, not good for lab Exhibit fatigue effect, resting time is required between readings. Used mainly in filter photometers with a wide bandpass producing a fairly high level of illumination so that there is no need to amplify the signal. Phototube Similar to a barrier-layer cell (it has photosensitive material that gives electrons when energy strikes it), but….outside voltage is required for operation we have to plug in to use Photomultiplier Tube (PMT): photon hits pmt and is emitted into electrons, these electrons will be attracted to the dinodes, when it hits it will split into two and then four etc, exponential growth. When it gets to the end it will convert the amount of elctrons into a concentration value. The more light the more electrons the less absorbance the less concentrations It detects and amplifies radiant energy. Incident light strikes the coated cathode, emitting electrons. The electrons are attracted to a series of anodes, known as dynodes, each having a successively higher positive voltage. When a dynode is hit by an electron it will give off many secondary electrons. Initial electron emission at the cathode thus triggers a multiple cascade of electrons within the PMT itself. Because of this amplification, the PMT is 200 x more sensitive than the phototube. PMT used in instruments that are very sensitive to very low light levels, and light flashes of very short duration. The accumulation of electrons striking the anode produces a current signal, measured in amperes, that is proportional to the initial intensity of the light. The analog signal is converted first to a voltage and then to a digital signal through the use of an analog-to-digital (A/D) converter. Digital signals are processed electronically to produce absorbance readings. PMT requires high voltage power supply, PMT is used in spectrophotometer with narrow bandwidths Good for double beam spectrophotometers. Detects and amplifies radiant energy. 200 x more sensitive than the phototube. Photo Diode & Diode Arrays Each are sensitive to specific wavelengths, therefore do not have the ability to change the wavelength. Know that this is a type of detector Read Out Devices Amplifies and mathematically manipulates the electrical signal and converts it into a convenient format Direct Reading = meter Null Point System = direct digital readouts The use of computers has largely replaced all other devices Know parts of the spec types pof specs and what ;oight waves for different spectrums (ligh sources) Blanking the instrument: try to get rid of any unwanted readings from the regent or the sample itself Distilled water blank: the response obtained from or due to non-analyte species in the sample. We don’t want response from anything but the analyte we are testing for Reagent blank: a test species used that corrects for colour due to reagent colour used in the method assay. Serum/Sample blank: corrects for colour due to all non-analyte colour of species. (if there is colour in patient sample that will interfere, blank using their sample, ex hemolysed) QC of the spectrophotometer- needs to work as it should Wavelength accuracy Linearity Stray light: example for 5% increase in stray light, the result for observed absorbance is almost ½ the actual true absorbance. Instrument function should be validated by performing at least the following checks: Wavelength Accuracy means that the wavelength indicated on the control dial is the actual wavelength of light passed by the monochromator. It is most commonly checked using standard absorbing solutions (potassium dichromate) or filters with absorbance maximum of known wavelength. Didymium or holmium oxide in glass are stable and are frequently used as filters. The filter is placed in the light path, and the wavelength control is set at the wavelength at which maximal absorbance is expected. The wavelength control is then rotated in either direction to locate the actual wavelength that has maximal absorbance. If these two wavelengths do not match, the optics must be adjusted to calibrate the monochromator correctly. Instruments with narrow bandpass use a mercury vapor lamp to verify wavelength accuracy. (when we set for a specific wavelength we need to make sure that that is the only wavelength hitting the detector. Stndards are of known concentration Linearity: This is checked with a series of stable solution of increasing concentration of analyte known to follow Beer’s Law (straight line calibration curve). Neutral density filters and sets of various coloured and concentrated solutions are commercially available. Either can be used to check for linearity over a range of wavelengths (use density filters for linearity over different wavelengths or standards Stray light: refers to any wavelengths outside the band transmitted by the monochromator. Not coming from the monochrometer, most commonly caused from dust and scratches on the cuvette Most common causes of stray light: Reflection of light from scratches on the optical surfaces or from dust particles anywhere in the light path. 2. leakage of extraneous light- no light when closing the lid 3. high solute concentration- the molecules in sample can reflect light sometimes, the spec has limits for how m,uch it can detect, you ould have to dilute the sample The major effect is absorbance error, especially in the high absorbance range. Stray light manifests itself as gradual negative deviation from Beer’s Law at the high end of known linear range. Transmission caused by stray light should be less than 1% Stray light is detected by using cutoff filters which eliminate all radiation at wavelengths beyond one of interest. Example to check for stray light in the near UV region, insert a filter that does not transmit in the region of 200 to 400 nm. If the instrument reading is greater than 0% T, there is stray light present. Certain liquids, such as NiSO4, Na NO2, and acetone absorb strongly at short wavelength and can be used in the same way to detect stray light. Kniw how to QC spec Other Spectrophotometers: Single-Beam Spectrophotometer: type we use It has a single position for sample and reference cell. Components: monochromator, cuvette, cuvette holder, exit slit, detector and read-out device. Double Beam Spectrophotometer: Has the same components as a single beam with the addition of both a reference beam and a sample beam. This is to correct for variation in light source intensity. One light source, two spots one for sample one for blank or reference Double beam In-Space: 2 of everything splits at the mirror ater the light source Two equal intensity beams formed by splitting a single light source. Two separate, matched detectors, readout device compares the signal output from each detector. Ratio of two signals is computed by read-out device. Double Beam In-Time: after the monochromator is where it splits A rotating chopper or mirror is used to focus the light exiting from references and sample cuvettes on a single detector. Light is detected as pulses. Double beam spectrophotometers are useful when spectral scans are needed. One will be a straight beam of light, the other one will be pulsed so detector can differentiate between the two Other light measuring systems: Refractometry: travels faster in air then in water delas with density of whatever the light is moving in Based on refractivity of light. The change in direction of a light ray as it passes from one medium into a second or as it passes through regions of non-uniform density within a single medium. The velocity becomes altered. When light passes from one medium into another, the path of the light beam will change direction at the boundary surface if its speed in the second medium is different from that in the first. This bending of light is called refraction. Refraction depends on the difference of the speed of light between 2 different mediums. Application: Measurement of total serum protein or specific gravity of urines. (Refractometer) Can be used for urine specific gravity, how concentrated is the urine. Water is the blank because the sg is 1.000 Reflectance: or reflection The change in direction of a light ray or wave, due to its interacting with a smooth plane reflection surface. The reflected ray will always be in the same plane as the incident ray and always the opposite side of the perpendicular (normal). Reflectance Photometry: Reflectance pf light from reaction products on solid surfaces Example: glucometer, urinalysis instruments Diffuse Reflectance: The amount of light reflected by a solution dispersed onto a white granular or fibrous surface. Example: urinalysis dipsticks and urinalysis instruments. Converts it into semi concentration, usually there is a blank reference so the urinalysis would know what total relfe ction would be The relationship between the concentration of the test species and the amount of light reflected is a non-linear equation and can be calculated as follows(beers law does not apply): we don’t need but here it is Result: [Sample] is proportional to the light intensity when compared to a reference that reflects ALL the incident light. RD be corrected for stray light. A blank standard with the same surface characteristics as the test sample can be used to give a value for maximum absorbance. Any reflection read by the instrument under these conditions will be stray light. It can be subtracted from the test value to correct for stray reflection. Reflectance allows quantitative measurement of reactions on surfaces such as dipstick or dry film. NON-linear relationship!! Atomic Absorption Spectroscopy (AAS)- absorption from atoms for lead or arsenic for poisoning. If we wanted to test for zinc we would need a filament made out of zinc Based on the principle of the absorption of energy, in the form of light, by atoms in their ground state. AAS uses a hollow cathode lamp (HCL)(neon or argon gas inside) containing a filament of the same metal to be tested and an inert gas. When voltage is applied, the gas is ionized. Ions attracted to the cathode collide with the metal, knock atoms off, and cause the metal atoms to be excited. When they return to the ground state, light energy is emitted. Samples to be analyzed must contain the reduced metal in the atomic vapourized state. This is achieved by using the heat of a flame to break the chemical bonds and form free, unexcited atoms Nebulizer( very hot) vapourizes the sample to have free floating samoles, the atoms in the sample are going to hang out in the flame. There is a monochromater after the sample and then a pmt and a read out device Take sample and nebulized it into gesous form, we gwt grounf state atoms, these can absorb energy like light. They become more excited. Atoms don’t like to stay excited- when they go back to ground state they will emit the light, every element will only emit a specific wavelength. We want to see how much of the e,ement is in the sample. We shine light on the ground state atoms. When light turns on the gas flows around and hits a filament made of the same element and nocks off some excited atoms for eaxamle zinc, the zinc atoms go back to ground state and will emit light. They go through tehe chopper which is changing straight light into pulse light. The ground state zinc atoms absorb the pulse light, they are noe excited they want ground state and will reemit the kight absorbed, will also be oulsed. Some wont be reemitted some will hang around. It will go through the monochromator (specific wavelength we want) because the taoms are hanging out in flame they will become excited from the heat but it wont be pulsed it will be straight. They will pass through the monochromater as will the pmt will onlt detct the pulse light (the chopper helps determine the difference from the pulse light and the straight light) only the poulsed light that originated from the light will be read by the detector. And then will be read as absorbance and proportional to concentration Start with heating the sample in nebulizer ground stat ehanging in flae Turn on light the gas neon and zinkc, becomes excited and hits the metal filament and nock off excited zinc atoms from the filament they go back to ground and emit straight light, go through chopper and gets pulsed, the zinc in flame will get excited and reemit pulsed light not all will be emitted some will be absorbed, some of the flame atoms will get excited on their own and will emit a straight lkight, the pmt will only read the pulse. The reading device reads aborbance and then can tell you the concentration. Flame = sample cell (rather than a cuvette) When a ground state atom absorbs light energy, an excited atom is produced. This atom will return to the ground state and emit light of the same energy it absorbed. Therefore, the flame sample contains atoms both absorbing and emitting radiant energy Light from HCL passes through the sample of ground state atoms in the flame. Amount of light absorbed is proportional to the concentration The detector must be able to distinguish between light emitted by the HCL and that emitted by the atoms in the flame. To do this, a rotating chopper is placed between the light and the flame Because the light being absorbed enters the sample in pulses, the transmitted light will also be in pulses. However, there will be less light in the transmitted pulses as some will have been absorbed Therefore, the detector will accept only the pulsed light signal from the HCL Light then travels to the monochromator, where only the desired wavelength is isolated. The detector is the final component of the AAS. It will measure the amount of light transmitted by the sample. Beer’s Law can then be used to determine amount of light absorbed. Only metals can be atomized easily so used to measure calcium, magnesium, zinc, copper, and lead (used for trace and toxic metals, not for metals found in larger quantities in plasma such as Na or K) Know the parts, what kind of testing it is used for. Essential components: Hollow cathode lamp Chopper Flame and nebulizer Entrance slit Monochromator Exit slit Detector Meter Flameless Burners Carbon rod or graphite furnace Advantages: Uniform heating Increased sensitivity Lower detection limit (read lower limits) What is Magnetic (Zeeman) Correction? Read in textbook, will be on the midterm Interferences: Spectral absorption by other closely absorbing atomic species- type of method where the wavelength specific to the element absorption by molecular species- we are testing atoms, molecules get in the way sometimes scattering by nonvolatile salt particles or oxides, scatter of light sometimes other particles causes stary light and we will end up with a false result background emission Non-spectral: May either be specific or non-specific. Non-specific interferences affect the nebulization by altering the viscosity, surface tension, or density of the analyte solution, and consequently the sample flow rate. Certain contaminants also decrease the atomization efficiency by lowering the atomization temperature. All atoms need to be in a vapor state or else it cannot be tested Specific Interferences: Chemical interference: With some elements the presence of certain anions in the sample results in the formation of compounds that are not completely dissociated in the flame. The result is a decrease in the number of ground-state atoms present in the flame (example: formation of calcium phosphate). The effect of tightly complexing anions can be minimized or eliminated by adding lanthanum to the sample to displace calcium from the complex. Ionization interference: when atoms in the flame become ionized (A+) instead of remaining in the ground state (A0), they will not absorb the incident light. This effect will result in an apparent decrease in analyte concentration. This can be corrected by the addition of excess substance that is more easily ionized, to provide free electrons. The excess free electrons shift the reaction to the formation of ground-state. It can also be minimized by operating the flame at lower temperature. We want them to be in ground state not ionized or excited. Matrix interference: differences in the matrix between the sample and the standard can result in error. Composition of standard varies greatly from test sample (difference in viscosity, solvent composition due to presence of protein) This can be things n our solvet that could be accidentally analyzed. Its good to correct with a blank. Know the parts Fluorometry Fluorescence: occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength Fluorophore: atom or molecule that fluoresces Basic Principle: Use of a photometer that measures light emitted by a source of short wavelength. The molecule absorbs the shorter wavelength. (usually UV) The electrons are excited to higher electronic states (energy level). These molecules are unstable, collide and return to ground state emitting at a longer wavelength, usually in the visible range. Much more sensitive than absorption methods. We’re measuring the lower energy, we want the wavelength that is emitted Mathematical relationship of concentration and fluorescent intensity: Derived from the Beer-Lambert Law and expressed as: F = Φ Io abc Where: F = relative intensity Φ = fluorescence efficiency Io = initial excitation intensity a = molar absorptivity b = volume element defined by geometry of the excitation and emission slits. c = concentration (mol/L) This equation indicates that fluorescence intensity is directly proportionate to the concentration of the fluorophore and the intensity of the excitation- don’t need to know The more fluorescence that is measured tells you how much concentration there is, higher fluorecense means higher concentration Instrumentation: Basic Components: Excitation source Excitation monochromator Cuvette Emission monochromator Detector Fluorescence Polarization-immunoassay (antigen antibody reactions) Light waves passed through polarizers (crystalline material) which orient in a single plane. Fluorescence polarization is used to quantitate analytes following immunological reactions. FPIA is a competitive immunoassay FPIA combines 3 principles: fluorescence, rotation of molecules in solution, and polarized light Fluorescein is the label of choice for this method The Ab-Ag-fluorescein molecules are large and rotate slowly in solution when excited by polarized light The smaller Ag-fluorescein rotate more rapidly in solution When the solution is exposed to polarized light, the smaller antigen-fluorescein molecules emit light in a different plane from that which was absorbed When the larger Ab-Ag-fluorescein molecule is exposed to polarized light, it emits light in the same plane as the absorbed light energy There is an inverse relationship between the signal and the concentration of analyte in the sample Factors Affecting Fluorescence: Several factors influence the intensity of fluorescence: 1. concentration 2. background effects (Rayleigh- no change in wavelength excitation and emission are so close there is no change and Raman scattering- longer wavelength emitted from solvent, we don’t want. Ethanol can fluoresce at certain wavelengths and can give readings that are false ) 3. solvent effects (quenching)- any process that decreases fluorescent intensity of a sample ( we don’t want from solvent we want from analyte 4. sample effects- anything that fluoresces other than what we want 5. temperature effects- if it gets too hot fluorescence will go down and give falsely decreased results 6. photodecomposition (bleaching)- too intense light will bleach fluorophore and it will no longer fluoresce Flow Cytometry Cytometry refers to the measurement of physical and/or chemical characteristics of cells. Flow cytometry: process in which such measurements are made while the cells or particles pass in a single file through a measuring apparatus in a fluid stream. A cytometer is used for counting and measuring the physical and chemical characteristics of cells and other biological particles. A flow cytometer is different from other cytometers in that a single-cell suspension is passed through it in a fluid stream. A flow cytometer is a complex combination of optics, fluidics, and electronics. Some instruments can separate cells that meet certain preselected criteria. Therefore, the flow cytometer is widely used in research as well as in clinical immunology and hematology to perform rapid immunophenotyping, cell sorting, and DNA analysis. A labeled(fluorescence labelled antigen) cell is forced through the system, causing the cell to scatter light and emit fluorescence. This output is sensed by photodetectors and then amplified and converted to digital signals for storage in computers. This stored data can be displayed or used for further analysis. Principles included in Flow Cytometry 1. cell counting 2. cytochemical staining 3. fluorometry Luminescence Is the emission of light or radiant energy when an electron returns from an excited or higher energy level to a lower energy level Chemiluminescence: excitation event caused by a chemical reaction Bioluminescence: excitation event caused by a biochemical reaction- happens naturally Electrochemical luminescence: excitation caused by an electrochemical reaction. Luminometer Consists of a sample cell in a “light tight” chamber, injection system to add reagents and a detector (photomultiplier tube). Reactions are oxidative involving oxygen or peroxide molecules Limitations: Light leaks, light piping, and high background luminescence from assay reagents and reaction vessels (e.g., plastic tubes exposed to light) are common factors that degrade the analytical performance of luminescence measurements The ultrasensitive nature of chemiluminescence assays requires stringent controls on the purity of reagents and the solvents used to prepare reagent solutions Light Scattering Techniques Basic Concepts Radiant energy collides with a molecule resulting in light being scattered in all directions The wavelength of the scattered light is the same as the incident light Antigen antibody reactions use these techniques Effects of Particle Size Small Particles: light is scattered in equal intensity in both forward and reverse directions. Back and forward equally Larger Particles: Scattering of light is dissymmetrical (lesser in reverse direction as compared to forward direction). Particle diameter is larger than the wavelength of radiation: amount of forward scattering is greater than the reverse. Measurement of Scattered Light Turbidimetry- cant see through it Measures turbidity or cloudiness of a solution by measuring the amount of light PASSING THROUGH the solution. (antigen and antibodies come together to make bigger molecules (agglutination) these can cause cloudiness) Soluble antigen and antibody join and once they join in sufficient amounts precipitate, results in cloudiness. The cloudier the solution, the less light can pass through. The cloudier the solution the higher the concentration for whatever we are testing Beer’s Law does not apply because this is not a clear solution Light in straight line Nephelometry The light reflected is detected at an angle from the source. Measures only the scattered light and is a function of size, molecular weight, and number of particles present. It is a more quantitative measurement. Measures SCATTERED light bouncing off antigen-antibody complexes Usually 90 degrees sometimes 45 degrees Light at an angle Clinical Applications Biological fluids used: Serum CSF Urine Used to measure plasma proteins; IgG, IgA, IgM, complement components C3 and C4, and albumin. Hormones, drugs (gentamycin, phenobarb, phenytoin, theophylline) Rheumatoid factor and anti -streptolysin 0.