Spectrophotometer Updated PDF

Photometric Measurement SPECTROPHOTOMETRY Sumbul Bushra MSc, ASCP CM  Objectives 1. Describe the quantitative nature and clinical applications of spectrophotometric & electrochemical measurements. 2. State Beers law in terms of absorbance, transmittance, analyte...

Photometric Measurement SPECTROPHOTOMETRY Sumbul Bushra MSc, ASCP CM  Objectives 1. Describe the quantitative nature and clinical applications of spectrophotometric & electrochemical measurements. 2. State Beers law in terms of absorbance, transmittance, analyte conc., and absorptivity. 3. Given appropriate information, calculate analyte conc. Using Beers law. 4. Identify the basic functional components of a spectrophotometer and state the purpose of each. 5. Describe how maintenance and calibration of a spectrophotometer undertaken. 2 10/20/2024  UV-VIS SPECTROPHOTOMETER VIS SPECTROPHOTOMETER 3 10/20/2024  Fundamentals of Spectrophotometry Solutes are going to absorb light and the spectrophotmeter Introduction measures how much light is beiong transmitted throgh and what is the amount being absorbed 1.) Colorimetry An analytical technique in which the concentration of an analyte is measured by its ability to produce or change the color of a solution - Changes the solution’s ability to absorb light 2.) Spectrophotometry A technique that uses light to measure chemical concentrations A colorimetric method where an instrument is used to determine the amount of analyte in a sample by the sample’s ability or inability to absorb light at a certain wavelength. Colorimetry Instrumental Methods Non-Instrumental Methods (spectrophotometry) 4 10/20/2024  Fundamentals of Spectrophotometry Properties of Light 1.) Particles and Waves Light waves consist of perpendicular, oscillating electric and magnetic fields Parameters used to describe light - amplitude (A): height of wave’s electric vector - Wavelength (): distance (nm, cm, m) from peak to peak - Frequency (): number of complete oscillations that the waves makes each second ▪ Hertz (Hz): unit of frequency, second-1 (s-1) ▪ 1 megahertz (MHz) = 106s-1 = 106Hz 5 10/20/2024  Fundamentals of Spectrophotometry Properties of Light 1.) Particles and Waves Parameters used to describe light - Energy (E): the energy of one particle of light (photon) is proportional to its frequency E = h where: E = photon energy (Joules) = frequency (sec-1) h = Planck’s constant (6.626x10-34J-s) As frequency () increases, energy (E) of light increases 6 10/20/2024  Fundamentals of Spectrophotometry Properties of Light 1.) Particles and Waves Relationship between Frequency and Wavelength  = c  = c /  where: c = speed of light (3.0x108 m/s in vacuum)) = frequency (sec-1)  = wavelength (m) Relationship between Energy and Wavelength hc ~ E= = hc  where: ~ = (1/) = wavenumber 7 10/20/2024 As wavelenght () decreases, energy (E) of light increases  Fundamentals of Spectrophotometry Properties of Light 2.) Types of Light – The Electromagnetic Spectrum Note again, energy (E) of light increase as frequency () increases or wavelength () decreases 8 10/20/2024  Fundamentals of Spectrophotometry Properties of Light 2.) Types of Light – The Electromagnetic Spectrum 9 10/20/2024  The Electromagnetic Spectrum 10 10/20/2024  Fundamentals of Spectrophotometry Absorption of Light 1.) Colors of Visible Light Many Types of Chemicals Absorb Various Forms of Light The Color of Light Absorbed and Observed passing through the Compound are Complimentary 11 10/20/2024  Fundamentals of Spectrophotometry Absorption of Light 2.) Ground and Excited State When a chemical absorbs light, it goes from a low energy state (ground state) to a higher energy state (excited state) Energy required of photon to give this transition:  =  −  Only photons with energies exactly equal to the energy difference between the two electron states will be absorbed Since different chemicals have different electron shells which are filled, they will each absorb their own particular type of light - Different electron ground states and excited states 12 10/20/2024  Fundamentals of Spectrophotometry Absorption of Light 3.) Beer’s Law The relative amount of a certain wavelength of light absorbed (A) that passes through a sample is dependent on: - distance the light must pass through the sample (cell path length - b) - amount of absorbing chemicals in the sample (analyte concentration – c) - ability of the sample to absorb light (molar absorptivity - ) Increasing [Fe2+] 13 10/20/2024 Absorbance is directly proportional to concentration of Fe+2  Absorption of EM radiation P0 (radiant intensity) P (transmitted intensity)  When a beam of monochromatic light enter a solution:  Some of the light absorbed, the reminder passes through, strikes a light detector and is converted to an electric signal.  Spectrophotometric measurements are based on detection and quantification of energy that is transmitted after passing an incident beam of light through the solution being analyzed.  Transmitted energy is expressed in terms of percent transmittance (% T)=  is the ratio of the radiant energy transmitted (Ts) divided by the radiant energy incident on the sample (Io). % T = Ps/Po x 100 14 10/20/2024  Fundamentals of Spectrophotometry Absorption of Light 3.) Beer’s Law The relative amount of light making it through the sample (P/Po) is known as the transmittance (T) P T= Po  P  Percent transmittance %T = 100     Po  15 10/20/2024 T has a range of 0 to 1, %T has a range of 0 to 100%  Fundamentals of Spectrophotometry Absorption of Light 3.) Beer’s Law Absorbance (A) is the relative amount of light absorbed by the sample and is related to transmittance (T) - Absorbance is sometimes called optical density (OD) - The relationship is expressed by the following equation: A = 2 – log %T P  A = − log   = − log (T ) = − log (%T / 100 )  Po  16 10/20/2024 A has a range of 0 to infinity  Fundamentals of Spectrophotometry Absorption of Light 3.) Beer’s Law Absorbance is useful since it is directly related to the analyte concentration, cell pathlength and molar absorptivity. This relationship is known as Beer’s Law A = bc where: A = absorbance (no units)  = molar absorptivity (L/mole-cm) b = cell pathlength (cm) c = concentration of analyte (mol/L) Beer’s Law allows compounds to be quantified by their ability to absorb light, Relates directly to concentration (c) 17 10/20/2024   4. There three types of spectra:  Absorption or emission of energy by atoms result in a line spectrum.  Molecules produce band spectra  Light emitted by incandescent solid (tungsten or deuterium) is in a continuous spectra. 18 10/20/2024  Think  If we have a reading of 70 %T for the highest standard and 98 %T for the sample, what would it indicate regarding the analyte concentration in the sample: 19 10/20/2024   The amount of light absorbed at a particular wavelength depends on molecular and ion types present and may vary with conc., pH or temperature.  Unknown concentrations are determined from a calibration curve that plots the absorbance at a specific wavelength versus concentration for standards of known concentration.  By choosing a wavelength of light that is optimal for the absorbance of the substance, absorption units represent the light that is absorbed by that substance and no other substances.  The increase or decrease of light that is absorbed is proportional to the concentration of the substance.  Percentage transmittance (%T) versus concentration is a nonlinear function.  When %T versus concentration is plotted on linear graph paper, a curvilinear response is obtained. 20 10/20/2024  Figure 3–6 illustrates this relationship. %T is used only when plotted on semi-log paper to obtain a straight line. The straight line obtained can be used to determine the concentration of unknown samples. Absorbance versus concentration is a linear function, as shown in Figure 3–7. The straight line obtained from this relationship can be used to determine the concentration of unknown samples. Nonlinear relationship between linear relationship between %T and concentration. absorbance and concentration. 21 10/20/2024  Table 3-2. SPECTROPHOTOMETRY NOMENCLATURE Name Symbol Definition Absorbance A -log T or log Io/I Absorptivity a A/bc (c in g/L) Molar absorptivity  A/bc (c in mol/L) Path length b Internal diameter of cuvet (cm) Transmittance T I/Io* Wavelength unit nm 10-9 m Absorption maximum max Wavelength at which a maximum absorption occurs *I/Io is the ratio of the intensity of transmitted light to incident light. %T T A 100 1 0 10 0.1 1 1 0.01 2 0.1 0.001 3 Avoid measurements where A > 2! It means that T < 0.01 or 1%. The intensity of light reaching the detector is too low for 22 accurate readings. 10/20/2024  Example: If a substance has an absorptivity of 4300 at 340 nm and the concentration is 1 mg/L. What would the absorbance be in a 1 cm path cuvet? A = abc A = 4300 x 1 x 0.001 g/L A = 4.3 Is this OK, or should the sample be concentrated or diluted? 23 10/20/2024  Components of the Spectrophotometer These are the components of a spectrophotometer: a. Power supply b. Light source c. Entrance slit d. Monochromator e. Exit slit f. Cuvet /sample cells g. Photodetector h. Readout Devices i. Recorders 24 10/20/2024  Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design An instrument used to make absorbance or transmittance measurements is known as a spectrophotometer 25 10/20/2024  Fundamentals of Spectrophotometry Spectrophotometer 1.) Basic Design Light Source: provides the light to be passed through the sample - Tungsten Lamp: visible light (320-2500 nm) Low pressure (vacuum) - based on black body radiation: heat solid filament to glowing, light emitted will Tungsten Filament be characteristic of temperature more than nature of solid filament - Deuterium Lamp: ultraviolet Light (160-375 nm) D2 or H2 Gas In presence of arc, some of the electrical energy is Filament absorbed by D2 (or H2) which results in the Electric Arc Sealed Quartz Tube 40V disassociation of the gas and release of light Electrode D2 + Eelect → D*2 → D’ + D’’ + h (light produced) Excited state 26 10/20/2024  1) Type of lamps:  a) Tungsten lamp (350 -950 nm): most common, used visible and near ultraviolet regions.  b) Quartz lamp: intense beam, used in the near ultraviolet region. (185-375 nm).  c) Hydrogen lamp: used in the ultraviolet region (185-375 nm).  d) Mercury lamp: uneven emission spectrum, not widely used.  e) Xenon lamp: brilliant emission; too bright for routine work.  f) Hydrogen, mercury, and xenon are all vapor lamps.  G) deuterium discharge lamp, mostly used for UV work.  For a light source the most important factor is the, range, spectral distribution within the range, the source of radiant production, stability of the radiant energy & temperature. 27 10/20/2024 2. Monochromators  Is used to eliminate unwanted wavelengths of light and allow the desired light to reach the sample.  Beer’s law holds true only for monochromatic light;  Therefore, accurate measurements require that the desired wavelength be separated from other wavelengths in polychromatic light.  Wavelength isolation is accomplished by a monochromator.  A monochromator is composed of an entrance slit, wavelength dispersing device and an exit slit.  Entrance slit: focuses a narrow beam of polychromatic light from the energy source on the dispersing device. Slit is fixed in position and size.  Exit slit: selects the bandpass and allows light of particular wavelengths to pass through the sample cuvet onto the detector, by eliminating or blocking unwanted wavelength from reaching the detector.  Types of Monochromators: A. Glass filter-absorption filters B. Interferences filters. C. Prisms D. Diffraction granting-most common one 28 10/20/2024 Types of Monochromators: A. Glass filters  Simplest and least expensive: thin layers of colored glass, colored gelatin sandwiches between two glass plates.  Used for transmitting visible and near visible light. 29 10/20/2024 Types of Monochromators: B. Interference filters  Interference filters are made of two glass pieces, each with a layer of silver on one side separated by a dielectric material (an insulating material that doesn’t allow electric current to flow), usually magnesium fluoride.  Only specific wavelengths or multiples-not for all wavelengths such as prisms. 30 10/20/2024 Types of Monochromators: C. Prisms a. A narrow beam of light focused on a prism is reflected as it enters the more dense glass (quartz, fused silica, or sodium chloride, or other material that permits transmission of light). b. Shorter wavelengths are refracted (bent) more than long wavelengths (violet refracted the most, red the least), resulting in a dispersion of white light into a continuous spectrum. c. Glass prisms: visible region; quartz or fused silica; ultraviolet. 31 10/20/2024 Types of Monochromators: If we change the light bulb we have to align everyrthing because if the alignment D. Diffraction gratings changes then there is an alignment error then we will get an incorrect wv length so for example if the exit slit is down we will get a higher wv length than expected  a. Most common monochromator used for wavelength selection.  b. Reflectance: lines are engraved on the surface of a mirror, which may consist of either a polished metal slab or a glass plate on which a thin metallic film has been deposited.  c. Usually a thin layer of a aluminum copper alloy on the surface of a flat glass plate that has many small parallel grooves etched on to the surface (15,000 – 30,000/ inch) with a diamond stylus. 32 10/20/2024  Band pass  a. The range of wavelengths passed through the monochromator and exit slit is called the band pass, that is, the range of wavelengths that a monochromator can isolate between two points of a spectral scan where the transmittance is one-half of the peak transmittance.  Spectral band pass reflects the quality of the monochromator.  b. Bandpass widths from narrowest to widest:  1) Prisms and diffraction gratings ( 50 nm) 33 10/20/2024  f. Photo-detectors  1. A detector converts the electromagnetic radiation (light) transmitted by a solution into an electrical signal.  The more light that is transmitted, the more energy, the greater the electrical signal that measures light intensity going through the sample.  2. Three types of photo-detectors:  a. Barrier layer cell (photocell)  B. Photomultiplier tubes (PMT)  C. photodiode array: used to monitor light at multiple wavelength simultaneously. 34 10/20/2024  Components of the Spectrophotometer (cont)  A sample holder holds the cuvet containing the test solution.  Readout Devices: Electrical energy from a detector is displayed on some type of meter or readout system.  Recorders 1. Spectrophotometers may be equipped with recorders in addition to or instead of digital display of values. 2. Recorders are synchronized to provide line traces of transmittance or absorbance as a function of either time or wavelength. Cuvette:  Round or square  Commonly the width is 1 cm  For wavelength in the visible range, optical plastics or glass cuvettes are used.  Quartz cuvettes… UV range (340 nm and below). 35 10/20/2024  Fundamentals of Spectrophotometry Sample Cell: sample container of fixed length (b). - Usually round or square cuvet - Made of material that does not absorb light in the wavelength range of interest 1. Glass – visible region 2. Quartz – ultraviolet 3. NaCl, KBr – Infrared region 36 10/20/2024  Calibration and maintenance procedures for spectrophotometers:  Setting transmittance/absorbance limits  Zeroing: set 0 %T  Blanking: set 100%T (A=0)  Wavelength calibration  Photometric linearity checks  Stray light correction.  Stray light is defined as energy reaching the detector through the monochromator and exit slit that is not of the selected wavelength. 37 10/20/2024  QUESTION ?? 38 10/20/2024  Three Types of Spectrophotometric Methods  The spectrophotometer measures the absorbance of light of one of the components of a chemical reaction.  Three examples of common types of chemical reactions that are measured by the spectrophotometer are: 1) endpoint colorimetric, : at the end of the reaction there will be a color and we are gonna measure the intensity of this color which is directly proportional to the amount of solute in the sample 2) endpoint enzymatic, : there will be enzyme in a reaction and we will measure the increase or decrease in the enzymes 3) kinetic reactions. : the amount of reactions and change in product happening per min 39 10/20/2024  Examples of Spectrophotometric Methods  The Jaffe reaction for creatinine is an example of an endpoint colorimetric reaction.  The hexokinase reaction with glucose is an example of an endpoint enzymatic reaction in which enzymes catalyze the reaction to measure the analyte.  An example of a kinetic method, which employs substrates and coenzymes to measure the activity of the enzyme, is that of measurement of alanine transaminase (ALT) using alanine, alpha-ketoglutarate, and pyridoxyl-5’-phosphate. 40 10/20/2024  Endpoint Colorimetric Spectrophotometry  Simple endpoint spectrophotometric reaction.  The Jaffe reaction for creatinine analysis is an example of this method.  In an endpoint spectrophotometric method, a colored product or chromogen forms that is measured by its ability to absorb visible light.  Its reaction sequence is described with the other two reaction sequences in Table 3–8. 41 10/20/2024  Endpoint Enzymatic Spectrophotometry  Use an enzyme to catalyze the chemical reaction.  The final product is often a coenzyme that absorbs light strongly at lower wavelengths in the visible or near-UV spectrum.  The hexokinase method for the measurement of glucose in body fluids is such a procedure;  the analyte in this complex reaction is glucose.  That is, glucose is the substance that is to be measured.  Glucose absorbs light at many wavelengths.  chemical reactions such as the hexokinase method have been developed in which a chemical reaction involving glucose, the enzyme, and other substances is used to produce NADP.  NADP is the substance measured by the spectrophotometer because it absorbs light uniquely at 340 nm and is not subject to many types of interferences.  The concentration of glucose is proportional to the consumption of the coenzyme in the reaction. 42 10/20/2024  Kinetic Spectrophotometry  An example of a kinetic method, which employs substrates and coenzymes to measure the activity of the enzyme, is that of measurement of alanine transaminase (ALT) using alanine, alpha-ketoglutarate, and pyridoxyl-5’-phosphate. 43 10/20/2024 44 10/20/2024  Creating a Concentration (Calibration) Curve  Concentration of an analyte in a patient or quality control sample is determined after a series of standard solutions are analyzed,  a graph of absorbance versus concentration is found to be a linear relationship.  Solutions of unknown concentration are tested for absorbance; these absorbance results are read from the curve to determine concentration.  The equation used for determining concentration of the patient or another unknown (unk) sample is as follows:  Concentration of unknown = (Concentration of standard/Absorbance of standard) x Absorbance of unknown CUnk= (Aunk/Astd) x Cstd 45 10/20/2024   For example, the absorbance results from a patient sample and an albumin standard in albumin analysis are shown below. Given these standard solution results, the concentration of the patient albumin can be determined.  2.5 g/dL albumin standard conc.  Absorbance of std 0.250  Absorbance of Patient1 sample 0.500  CUnk= (Aunk /Astd) x Cstd  Patient’s albumin conc. = (0.500 /0.250) x 2.5 = 5.0 g/dL 46 10/20/2024  Common Clinical Chemistry Spectrophotometric Reactions 47 10/20/2024  Cell Holder Compartment  Instrument> initialization To set up method & wavelength  Setting Up PHOTOMETRY Module  ASpect UV starts and establishes the connection to the SPECORD PLUS. The monochromator of the SPECORD PLUS moves and the message "Initialization" is shown on the monitor.  If the connection to SPECORD PLUS could not be established: establish the connection later by selecting INSTRUMENT → INITIALIZATION from the menu  Open the PHOTOMETRY module by clicking on the button in the launch bar of the main window:  Open the method by clicking on [METHOD SETUP].  Configure the method on the screens of the window PHOTOMETRY - SETTINGS.  Enter the parameters on the screens of the Method window 50 10/20/2024 51 10/20/2024 52 10/20/2024  Sample Setting  1. Click on [ADD SAMPLE] and set a reference at the start of the sample table: 53 10/20/2024   Click on [ADD SAMPLE] once again and add 3 samples to the end of the sample table: 54 10/20/2024   Enter "Reference” in the NAME field of the reference line and confirm with ENTER.  In the 2nd line, enter "Sample 1" as the name and then confirm with ENTER. Highlight the three successive sample name fields, incl. the "Sample 1” field, with the mouse button held down. Right- click on the highlighted choice and in the context menu, select FILL WITH ASCENDING VALUES  Sample positions that are not required may be removed from the sample table: 55 Click on [REMOVE SAMPLES]. 10/20/2024   Exit the parameter input by clicking on [OK] in the PHOTOMETRY - SETTINGS 56 window and return to the document window. 57 10/20/2024 58 10/20/2024  Reference  Christenson, R.H., Gregory, L.C., Johnson, J.L.; APPLETON & LANGES OUTLINE REVIEW CLINICAL CHEMISTRY1st edition, ISBN 0070318476. Publisher: McGraw-Hill Medical Publishing Division-Release date 2001. 59 10/20/2024