CC 1 OLFU INSTRUMENTATION.pdf

COLLEGE OF MEDICAL LABORATORY SCIENCE Photons CLINICAL CHEMISTRY 1 - Discrete packets of energy or INSTRUMENTATION particles Frequency Light W...

COLLEGE OF MEDICAL LABORATORY SCIENCE Photons CLINICAL CHEMISTRY 1 - Discrete packets of energy or INSTRUMENTATION particles Frequency Light Waves Theory - Is the number of vibrations of X Isaac Newton wave motion per second - Light was made of tiny particles PARTS OF A LIGHT WAVE - Light moves in a straight direction in a vacuum X Planck’s Formula: E=hv Where: E= is the energy of photon in Joules H= constant ( 6.626 x 10!"# erg sec) WAYS OF THE DIRECTION OF LIGHT V= frequency CAN CHANGE Reflection: light path bends back from a reflective surface ( e.g. mirror, surface of pond, table, chair, pen etc….) Refraction: light path bends at interface between two transparent media of X Christian Huygens different indices of refraction (densities) - Proposed a wave theory of life. Particles of light moves in a wave-like manner V= C 𝜆 C= Speed of light in a vacuum (3𝑥 10$% cm/s) 𝜆= wavelength in cm E= hc 𝜆 TERMINOLOGIES Energy - Is transmitted via electromagnetic waves that are characterized by their frequency and wavelength Wavelength - Is the distance between two successive peaks. AUBREY ANN M. INOY 1  COLLEGE OF MEDICAL LABORATORY SCIENCE Scattering - Light path changed by interaction with small particles (e.g. molecules, dust, etc,..) about same size as light wavelength Dispersion - Light paths are refracted by different amounts Diffraction - Light waves interfere constructively and destructively TYNDAL EFFECT x phenomenon of scattering of light by colloidal particles x when a fine beam of sunlight enters a smoke filled room, smoke particles become visible due to scattering of light by these particles. x when sunlight passes through a canopy of dense forest, tiny water REMEMBER: droplets in the mist scatter light. X The lower the wave frequency, the longer the wavelength X The wavelength is inversely related to frequency and energy; the shorter the wavelength, the higher the frequency and the energy and vice versa. COLORIMETRY The primary analytical utility of spectrophotometry of filter photometry is the isolation of discrete portions of the spectrum for purpose of ,measurement A. Spectrophotometric measurement - Is the measurement of light intensity in a narrower wavelength - Measurement of the intensity of light at selected wavelengths B. Photometric measurement - Measurement of light intensity AUBREY ANN M. INOY 2  COLLEGE OF MEDICAL LABORATORY SCIENCE SPECTROPHOTOMETRY Single beam spectrophotometer BEER’S LAW - Is the simplest type of absorption - States that the concentration of a spectrometer substance is directly proportional - It is designed to make one to the amount of the light measurement at a time at one absorbed and inversely specified wavelength proportional to the logarithm of Double beam spectrophotometer the transmitted light. - is an instrument that splits the Absorbance (A) monochromatic light into two - Is the amount of light absorbed components - Proportional to the inverse log of - one beam passes through the transmittance sample, and the other through a reference solution or blank A= abc= 2-log%T 2 TYPES OF DOUBLE-BEAM SPECTROPHOTOMETER Where: a= molar absorptivity X double-beam in space b= length of light through the solution - uses 2 photodetectors, for the c= concentration of absorbing sample beam and reference molecules/solution beam X double-beam in time Transmittance - uses one photodetector and - Is the ratio of the radiant energy alternately passes the transmitted (T) divided by the monochromatic light through the radiant energy incident on the sample cuvette using a chopper sample (I). or rotating the sector mirror. PARTS OF SPECTROPHOTOMETER %T= 1t X 100 Io C= ABS %T It = transmitted light thru the sample I0 = intensity of light striking the sample SPECTROPHOTOMETRY - It involves measurement of light transmitted by a solution to determine the concentration of the light-absorbing substances in the solution. AUBREY ANN M. INOY 3  COLLEGE OF MEDICAL LABORATORY SCIENCE FACTORS FOR CHOOSING A LIGHT SOURCE X range X spectral distribution within the range X the source of radiant production X stability of the radiant energy and temperature LIGHT/RADIANT SOURCE - it provides polychromatic light ALTERNATIVE FOR TUNGSTEN and must generate sufficient BULB radiant energy or power to MERCURY ARC VISIVLE AND UV measure the analyte of interest. DEUTERIUM UV - To give accurate absorbance LAMP (165nm) measurements throughout its HYDROGEN UV absorbance range and its LAMP response to chance in light XENON LAMP UV intensity must be linear. MERST IR GLOWER 2 TYPES OF LIGHT SOURCE GLOBAR IR X Continuum Source (SILICONE - Emits radiation that changes in CARBIDE) intensity; widely used in the ENTRANCE SLIT laboratory Minimizes unwanted or stray light and Eg prevents the entrance of scattered light Tungsten light bulb into the monochromator system Deuterium lamp Xenon discharge lamp X Line Source - Emits limited radiation and wavelength - Wide use in atomic absorption, molecular, and fluorescent spectroscopy Eg Mercury and sodium vapor lamps Hollow cathode lamp AUBREY ANN M. INOY 4  COLLEGE OF MEDICAL LABORATORY SCIENCE STRAY LIGHT FILTERS X refer to any wavelengths outside the X are simple, least expensive, not band transmitted by the monochromator; precise but useful it does not originate from the X are made by placing semi-transparent polychromatic light source silver films on both sides of a dielectric X most common cause of loss of such as magnesium fluoride linearity at high-analyte concentration X produced monochromatic light based MONOCHROMATOR on the principle of constructive It isolates specific or individual interference if waves wavelength of light X usually pass a wide band of radiant energy and have low transmittance of the selected wavelength PRISMS X are wedge-shaped pieces of glass, quartz or sodium chloride EXIT SLIT X it can be rotated, allowing only the - It controls the width of a light desired wavelength to pass through an beam (bandpass) exit slit - Allows only a narrow fraction of the spectrum to reach the sample cuvette BANDPASS - Is the total range of wavelengths transmitted DIFFRACTION GRATINGS - The degree of wavelength X are most commonly used; resolution isolation is a function of the type than prism of device used and the width of X are made by cutting grooves or slits the entrance the exit slits into an aluminized surface of a flat piece - Spectral purity of the of crown glass- wavelengths are bent as spectrophotometer is reflected by they pass a sharp corner the bandpass, that is, the narrower the bandpass, the greater the resolution AUBREY ANN M. INOY 5  COLLEGE OF MEDICAL LABORATORY SCIENCE CUVETTE TYPES OF DETECTORS It hold the solution whose concentration X barrier layer cell / photocell / is to be measured. photovoltaic cell it is simplest detector; least expensive; temperature sensitive it is used in filter photometers with a wide bandpass it is a basic photo-transducer that is used for detecting and KINDS OF CUVETTES measuring radiation in the visible x alumina silica glass- most commonly region used (350-2000nm) composed of selenium on a x quartz/plastic – used for plate of iron covered with measurement of solution requiring transparent layer of silver. visible and ultraviolet spectra Maximum sensitivity of 550 nm x borosilicate glass PHOTOTUBE x soft glass it contains cathode and anode - Silica cuvettes transmits light enclosed in a glass case effectively at wavelengths > it has a photosensitive material 220nm that gives off electron when light - The path length of cuvettes is 1 energy s it cm, although much smaller path lengths are used in automated systems - to increase sensitivity, some cuvettes are designed to have path lengths of 10cm, increasing the absorbance for a given solution by a factor of 10. PHOTODETECTOR It detects and converts transmitted light into photoelectric energy. It detects the PHOTOMULTIPLIER TUBE (PMT) amount of light that passes through the it is the most commonly used sample in the cuvette. detector it has excellent sensitivity and has a rapid response the response of the PMT begins when incoming photons strike a photocathode. These tubes are limited to measuring low power AUBREY ANN M. INOY 6  COLLEGE OF MEDICAL LABORATORY SCIENCE radiation because intense light NOTES TO REMEMBER: causes irreversible damage to the instrument deviation is commonly photoelectric surface a result of the finite band pass of the filter or monochromator turbidity readings on a spectrophotometer are greater in the blue region than in the red region of the spectrum an absorbance check is PHOTODIODE performed using glass filters or it is nor as sensitive as PMT but solutions that have known with excellent linearity absorbance values for a specific it measures light at a multitude of wavelength wavelengths-detect less amount of light THE LINEARITY OF A it has a lower dynamic range and SPECTROPHOTOMETER CAN BE higher noise compared to PMT DETERMINED USING OPTICAL it is most useful as a FILTERS OR SOLUTIONS THAT HAVE simultaneous multichannel KNOWN ABSORBANCE VALUES FOR detector A GIVEN WAVELENGTH BLANKING TECHNIQUE Means the blank contains serum but without the reagent to complete the assay Reagent blank corrects absorbance caused by the color of the reagents METER It displays output of the detection The absorbance of reagents is system automatically subtracted from each of unknown reading A blanking process may not be effective in some cases of turbidity, and ultracentrifugation may be necessary to clear the serum or plasma AUBREY ANN M. INOY 7