Spectrophotometry Lecture Notes PDF

Spectrophotometry C L I N I C A L C H E M I S T R Y: P R I N C I P L E S , T E C H N I Q U E S , A N D C O R R E L AT I O N S (8TH ED.) - CHAPTER 5 (9TH ED.) – CHAPTER 4  Analytic Techniques 1. Spectometry Spectrophotometry, atomic absorption, mass spectrometry 2. Lumines...

Spectrophotometry C L I N I C A L C H E M I S T R Y: P R I N C I P L E S , T E C H N I Q U E S , A N D C O R R E L AT I O N S (8TH ED.) - CHAPTER 5 (9TH ED.) – CHAPTER 4  Analytic Techniques 1. Spectometry Spectrophotometry, atomic absorption, mass spectrometry 2. Luminescence Fluorescence, chemiluminescence 3. Electroanalytic Electrophoresis, potentiometry, amperometry 4. Chromatography Gas, liquid, thin layer  Wavelength Parameters Light is a type of radiant energy - it travels in waves Cycle - One complete waveform Frequency (v) = # cycles/sec (hertz) v= An inverse relationship exists between frequency & wavelength Short wavelengths - high energy, high frequency Long wavelengths - low energy, low frequency Velocity (c) - The distance travelled by a wave in one second.  Wavelength Wavelength (λ) - The distance from a point on a wave to the corresponding point on the next and is measured in nm (10-9 m). Amplitu de Amplitude - The magnitude of the peak of the wave  Radiation vs. Wavelength Type of Approximate Wavelength Radiation (nm) Gamma 25 x 107 Long λ  Colors of the Visible Spectrum The term visible light is used to describe radiant energy with wavelengths visible to the human eye or with wavelengths bordering on those visible to the human eye. (400 - 700 nm) Type of Approximate Wavelength Radiation (nm) Memory Aid: Visible Violet 380-440 Indigo 420-450 Roy G. Biv Blue 440-500 Green 500-580 Richard of York Gave Battles Yellow 580-600 Orange 600-620 In Vain Red 620-750 Colors of the Visible Spectrum When white light is passed through a prism, it is separated into its component colors. This is called dispersion.  Absorption/Transmission of Light The color of light seen in the visible spectrum depends on the wavelength(s) that are NOT absorbed. In a solution, if light is not absorbed, it is transmitted. When white light is passed through a colored solution, part of the light is absorbed by molecules and the remaining light is transmitted. The greater the # of absorbing molecules, the less light is transmitted (more is absorbed).  Absorption/Transmission of Light A Blue solution appears blue because blue wavelengths of light are transmitted (the rest of the wavelengths are absorbed). Blue light Eye White transmitt Light ed For a ray of electromagnetic radiation to be absorbed it has to have the same frequency as a rotational or vibrational frequency in the atom or molecule it hits. Fig 5.2 Characteristic Absorption or Emission Spectra Absorption of energy by atoms results in a line spectra. Molecules emit a bank of energy over a large region resulting in a band spectra. Incandescent solids (tungsten or deuterium) emit light in a continuous spectra.  Beer’s Law A law relating the absorption of light by a solution and the concentration of that solution. We will break down Beer’s Law into four parts.  Beer’s Law - Part 1 Equal thicknesses of an absorbing material will absorb a constant fraction of energy incident upon it. Transmitted Incident Light Light 1 cm 20% 80% transmitted absorbed Colored Glass 1 cm Transmitted Light 80% 20% absorbed transmitted  Beer’s Law - Part 2 The absorption of energy by an absorbing material is logarithmic. (Each equal layer will absorb a constant fraction) 100% T= T= T= T= 80% 64% 51.2% 41% A= A= A= A= 20% 20% 20% 20%  Beer’s Law - Part 2 The absorption of energy by an absorbing material is logarithmic. Figurefraction) (Each equal layer will absorb a constant 5-4: (A)Percent of original incident light transmitted by equal layers of light-absorbing solution. (B) Percent T versus concentration on linear graph paper. (C) Percent T versus concentration on semilog graph paper. (D) A versus concentration on linear graph paper.  Beer’s Law - Part 3 The thicker the absorbing material, the greater the absorbance. A 1 cm B 2 cm B will absorb more and transmit less than A  Beer’s Law - Part 4 The greater the number of absorbing molecules, the greater the absorbance. A B B is more concentrated and will absorb more light. i.e. the deeper the color of solution, the greater the absorbance.  Beer’s Law - Concentration and Path Length (depth) Absorbance is directly proportional to concentration x depth The more concentrated the solution and greater the depth (distance), the greater the absorbance. The less concentrated the solution and smaller the depth, the smaller the absorbance (and greater the transmittance)  Beer’s Law - Mathematically Absorbance is directly proportional to concentration x path length (depth) A=εxbxc Where: ε = molar absorptivity (fraction of a specific λ of light absorbed by a given type of molecule) b = length of the light path c = concentration of absorbing molecules Because the path length and molar absorptivity are constant for a given λ: A ̴c  Beer’s Law - Mathematically We tend to use a simplified version: A = C xWhere l A = absorbance C = concentration l = path length (depth) We can use this to derive a formula to find unknown patient concentrations: At = C t x l t Where: t = test (ex. patient serum) As = C s x l s s = standard (solution of known concentration used to The path calibrate length (depth) is always the same, a method) therefore lt = ls  Beer’s Law - Mathematically At = C t x We can rearrange these to get: lt = lt ls = As = C s x ls Since lt = ls , then it follows that = We can now rearrange the above to get a formula to solve for unknown patient (test) concentrations: CtAs = CsAt Ct = s  Example using Beer’s Law Using the information below, calculate the patient’s glucose concentration in mmol/L. Concentration of Standard = 7.5 mmol/L Absorbance of Standard = 0.290 Absorbance of Patient = 0.242 Ct = Cs = = 0.83 x 7.5 = 6.2 mmol/L  Filter Photometer Beer’s Law can be used in photometry and spectrophotometry to find the concentration of unknown samples. Photometry is the measurement of the intensity of light, independent of wavelength. Spectrophotometry is the measurement of the intensity of light at selected wavelengths. Instruments that use filters to select the wavelength are called filter photometers.  Parts of a Filter Photometer Light Filter Cuvette Radiant Source Energy Meter Detector Light Source Radiant Energy Provides white light Detector Converts light energy Filter into electrical energy selects the λ of light that will give the greatest Meter absorbance for the colored Displays current as solution Absorbance or %T  Spectrophotometry Instruments that use prisms or gratings to select the wavelength are called spectrophotometers. There are two classifications: 1. Single-beam Light Monochromat Source or Cuvett Meter Entrance Detector Exit Slite Slit The sample must be blanked using an appropriate reference solution that does not contain the compound of interest. Reagent Blank - corrects for the color contribution of  Spectrophotometry 2. Double-beam All components are doubled except for the light source. Allows automatic correction of sample and reference absorbance, so a reagent blank is not necessary The introduction of computerized, continuous zeroing, single-beam spectrophotometers has replaced the double- beam.  Absorbance and Transmittance of Light Measurement by spectrophotometry is based upon the reaction between a substance to be measured in the sample and a reagent (chemical) to produce a color. The amount of color produced depends upon the concentration of the substance in the sample. So, the intensity of color is proportional to the concentration of the substance. More substance = deeper color Deep color = ↑ concentration of substance And, the higher the concentration of the substance (molecules in solution), the more light is absorbed (the less light is transmitted)  Expressing Light Absorbed or Transmitted Absorbance is an expression of the amount of light absorbed by a solution. Values are directly proportional to the concentration of the solution. Percent transmittance is the amount of light that passes through a colored solution. As concentration increases, %T decreases. Both values are compared with the amount of light that passes through a reagent blank solution. The reagent blank contains all the reagents used in the procedure but does not contain the substance being  Photometer Scale Reads Transmittance and Absorbance Transmittance fraction of light transmitted expressed as %T (to avoid fractions) Transmittance and absorption are reciprocally related and logarithmically related A = 2 – log %T As absorbance increases, %T decreases  Calibration Graphs (Standard Curves) A standard curve is a graph with absorbance (A) or %T plotted on the y axis (vertical) and increasing concentrations of standards on the x axis (horizontal). Constructed after obtaining the A or %T readings from solutions of known concentrations (standards) used in a reaction or procedure. A or %T is plotted against concentration for each standard on graph paper Common types: Concentration vs Absorbance (linear paper) Concentration vs %T (semi-log paper)  Common Types of Calibration Graphs Concentration vs Absorbance (linear paper) Concentration vs %T (semi-log paper) If a straight line is formed = Follows Beer’s Law Ideal Calibration Curves An ideal curve covers a wide range of absorbance and concentrations. Curve B is ideal.  Preparation and Use of a Standard Curve Plotting a Standard Curve Plotted using Absorbance (or %T) readings from known standard solutions. (Absorbance (or %T) vs Concentration) Using a Standard Curve Absorbance (or %T) of unknown patient samples are read on spectrophotometer. Calibration graph is used to extrapolate sample concentration. Plotting a Standard Curve Abs vs. Concentration Concentra Absorban tion ce (g/L) 1.0 0.2 2.0 0.4 3.0 0.6 4.0 0.8 5.0 1.0 Standard Curves and Unknown Patient Samples Patient sample is run on the spectrophotometer and an Absorbance of 5.7 is obtained. To find the concentration we draw a line across until we hit the standard curve line. Draw a line down to find the patient’s  Notes Re: Standard Curves Construction of standard curves is not usually done manually in current clinical labs. Relationships between known standards and unknowns are now mostly automated in analyzers. Calibration of the automated procedure is still performed at certain intervals.  Parts of a Spectrophotometer Light Monochromat Cuvette Radiant Source or Energy Galvanome Detector ter  Light Source Provide wavelengths of light in the visible or near- infrared range Incandescent tungsten lamp Tungsten-iodide lamp Give a range from 300-1000 nm Provide wavelengths of light in the ultraviolet (UV) range Deuterium discharge lamp Mercury Arc lamp  Monochromator Eliminate unwanted wavelengths of light & allow the desired light to pass through the sample. Includes :Filters, prisms, diffraction gratings A good monochromator is determined by its bandwidth The smaller the bandwidth the better  Bandwidth (Bandpass) Bandwidth is the range of wavelengths where %T is ½ the peak of transmittance. To Determine Bandwidth: The bandwidth in this case is 445-475 or 30 nm  Monochromator - Filters Types of Filters: Glass Thin layer of colored glass containing metals; allows some colors to go through but not others Bandwidth = 25-50 nm Wratten Layer of colored gelatin Doesn’t withstand heat well; prone to leakage Bandwidth = 25-50 nm Interference Two metallic layers, two glass layers, and a dielectric layer (glass or vacuum) Use cut-off filters to absorb undesirable wavelengths Bandwidth = 10-17 nm  Monochromator - Diffraction Gratings Flat glass plates of an aluminum and copper alloy with thousands of etched grooves. Two types: Reflection - reflects spectrum from the surface Transmission - creates a spectrum as light passes through Both give a linear spectrum (equal space between the colors/wavelengths)  Monochromator - Prisms Separates white light into a spectrum by refraction. Longer wavelengths are bent less than shorter ones as they pass through Produce a non-linear spectrum (spacing not equal between the colors/wavelengths) Rotating the prism allows the desired wavelength to be selected.  Cuvettes (Sample Cells) The vessel to hold the solution to be measured (patient serum, controls, standards, etc.) Can be round, square, or cylindrical Can be constructed from: Glass - used for work in the visible range Silica (quartz) - used for wavelengths below 340 nm Plastic - problems with tolerance, cleaning, etching by solvents, and temperature deformation - disposable plastic cuvettes are usually used because of this  Photodetectors (Radiant Energy Detectors) Change light energy to electrical energy Include: Barrier Layer Cell (Photocell) Phototube Photomultiplier Tube Photodiode  Photodetector - Barrier Layer Cell Photosensiti AKA Photocell Composed of a light-sensitive ve Layer layer (selenium) on a plate of iron, with a layer of silver on top. Electrons in the selenium layer are excited when exposed to light and are released to the conductive silver layer. (No external voltage source needed) Galvanomet Current that is produced is er proportional to the amount of light falling on the photocell.  Photodetector - Phototube + - Cathode acts as a resistor in the dark but emits electrons when exposed to light Electrons are attracted to the positively charged anode. The transfer of electrons produces a current which is measured by a galvanometer. Current ̴ amount of light An outside voltage is required for operation.  Photodetector - Photomultiplier Tube Detects and amplifies low levels of light. Electrons are attracted to the anodes, called dynodes. Each dynode has a Dynod successively increasing e Signal ↑’d positive charge which gives 200x off an increasing number of secondary electrons. The accumulation of electrons produces a current which is proportional to the intensity of Figure 5-8: Dynode chain in a photomultiplier light.  Galvanometer Measures current When light hits the radiant energy detector, current is produced. The current is directly proportional to the amount of light transmitted. Spectrophotometer - Overview Copyright © 2016 by Mosby, an imprint of Elsevier Inc. Copyright © 2012, 2007, 1999, 1992, 1979, 1970, by Mosby, Inc., an affiliate of Elsevier Inc. NovaSpec III and III+ Spectrophotometers  Spectrophotometer Quality Assurance Instrument function needs to be validated and should include: 1. Checks on wavelength accuracy 2. Checks for stray light 3. Checks on linearity  Quality Assurance - Wavelength Accuracy The set wavelength on the spectrophotometer should match the actual wavelength that is passed through the monochromator. This is checked by using standard absorbing solutions or filters of known maximum absorbance. Didymium and holmium oxide in glass are commonly used as filters. Another method involves substituting a mercury lamp for the usual light source. If the wavelength was found to be inaccurate, the optics would be adjusted to calibrate the monochromator  Quality Assurance - Stray Light Scratches on cuvette surfaces and dust particles in the light path are the most common causes of stray light. Cutoff filters eliminate all wavelengths other than the one of interest and can be used to check for stray light. Figure 5-10: Spectrophotometer’s ability to measure high absorbance with stray light. (A)No stray light, with no deviation from the actual absorbance. (B)Some stray light within the instrument showing deviations from the actual at high absorbance. (C)A higher degree of stray light showing further deviation from the  Quality Assurance - Linearity A calibration curve should result in a straight line following Beers Law. Linearity is checked by using coloured solutions that are diluted and ensuring that Beers Law is followed. Solutions are available commercially.  Reading Assignment Clinical Chemistry: Principles, Techniques, and Correlations (8TH ED) Chapter 5 (p. 101-107)