MLAB 2401: Clinical Chemistry Keri Brophy-Martinez Analytical Techniques and Instrumentation Electromagnetic Radiation & Spectrophotometry 1

Introduction 2  How do we actually measure the concentrations of molecules that are dissolved in the blood?  Spectrophotometry Mix chemicals together to produce colored products, shine a specific wavelength of light thru the solution and measure how much of the light gets “absorbed”  Nephelometry and Turbidimetry Mix chemicals together to produce cloudy or particulate matter, shine a light thru the suspension and measure how much light gets “ absorbed” or “refracted”  pH Meters / Ion Selective Electrodes (ISE) Electrically charged ions effect potentials of electrochemical circuits  Electrophoresis Charged molecules move at different rates when “pulled” through an electrical field  Osmometers Dissolved molecules & ions are measured by freezing point depression and vapor pressure

Electromagnetic Radiation: Properties of light and radiant energy 3  Electromagnetic radiation is described as photons of energy traveling in waves  There is a relationship between energy and the length of the wave (wavelength)  The more energy contained, the more frequent the wave and therefore, the shorter the wavelength

Electromagnetic Radiation: Properties of light and radiant energy 4  This relationship between energy and light is expressed by Planck's formula:  E = hf Where: E= energy of a photon h = a constant f = frequency  The formula shows that the higher the frequency; the higher the energy or the lower the frequency, the lower the energy  We do not use this to perform any calculations. You only need to recognize Planck’s formula and its components

Electromagnetic Spectra 5

Electromagnetic Radiation: Properties of light and radiant energy 6  White light  Combination of all wavelengths of light  Diffract (bend) white light and all the colors become visible  The color you see depends on the wavelength of color(s) that are not being absorbed  Light that is not being absorbed is being transmitted

7 Electromagnetic Radiation: Properties of light and radiant energy  Wavelength  Measured in nanometers (nm) or meters.

Electromagnetic Radiation Properties of light and radiant energy 8  Interactions of light and matter  When an atom, ion, or molecule absorbs a photon, the additional energy results in an alteration of state (it becomes excited). Depending on the individual “species,” this may mean that a valence electron has been put into a higher energy level, or that the vibration or rotation of covalent bonds of the molecule have been changed.  Ultimately, as energy is released, an emission spectra is formed

Electromagnetic Radiation (Properties of light and radiant energy) 9  In order for a ray of radiation to be absorbed it must: 1. Have the same frequency of the rotational or vibrational frequency in the molecules it strikes, and; 2. Be able to give up energy to the molecule it strikes.

Electromagnetic Radiation 10  Many lab chemistry instruments measure either the absorption or emission of radiant energy /light.  Spectroscopy is based on the mathematical relationship between solute concentration & light absorbance  Beer’s law

Electromagnetic Radiation 11  Beer's Law  States the relationship between the absorption of light by a solution and the concentration of the material in the solution.  The absorption and/or transmission of light through a specimen is used to determine molar concentration of a substance.

Beer-Lambert law (Beer’s Law) 12

Beer-Lambert law (Beer’s Law) 13  A = 2 – log%T

Requirements for Beer’s Law  Keep light path constant by using matching sample cuvettes standardized for diameter and thickness  Solution demonstrates a straight line or linear relationship between two quantities in which the change in one (absorption) produces a proportional change in the other (concentration).  Not all solutions demonstrate a straight line graph at all concentrations.  If these rules are followed, we can calculate / determine an unknown’s concentration, by comparing a characteristic (its absorbance) to the same characteristic of the standard (whose concentration is known – by definition) Concentration unk = (A unk /A std ) * Concentration std 14

Percent transmittance 15

Photometry/Spectrophotometry 16  In photometry we measure the amount of light transmitted through a solution in order to determine the concentration of the light absorbing molecules present within.

Photometry/Spectrophotometry 17  Types -Simple photometers and colorimeters use a filter to produce light of one wavelength (monochromatic light).

Spectrophotometer / Spectrophotometry 18  Spectrophotometers differ from photometers in that they use prisms or diffraction gratings to form monochromatic light.

Spectrophotometer: Components 19  Light source/lamps  Vary according to need, but must be a constant beam, cool and orderly  Types  Tungsten or tungsten iodide lamps for visible and near infrared  Incandescent light (400 nm nm)  Deuterium or mercury-arc lamps required for work in U.V. range  Range nm

Spectrophotometer: Components 20  Monochromators  Promote spectral isolation  Operator selects specific wavelength  Isolate a single wavelength of light  Provides increased sensitivity & specificity  Types  Glass filters  Prisms  Diffraction gratings

Spectrophotometer: Components 21  Monochromator characteristic:  Bandpass/bandwidth –  Measures the success of the monochromator  Defines the width of the segment of the spectrum that will be isolated by the monochromator

Spectrophotometer: Components 22  Cuvet  Made of high quality glass or quartz  Glass – for work in the visible light range  Quartz or fused silica – for work in the UV range  Shape  Round cuvets are cheaper but light refraction and distortion occur  Square cuvets have less light refraction but usually more costly  Optically clean  No inconsistencies in composition  No marks, scratches, or fingerprints  Positioning  Orientation and placement into the instrument important. Each time must be the same so light passes through the cuvet at the same place.

Spectrophotometer: Component 23  Photodetectors  Purpose – to convert the transmitted light into an equivalent amount of electrical energy  Most common is the photomultiplier tube

Spectrophotometer: Component 24  Readout devices  Purpose – to convert the electrical signal from the detector to a usable form  Types  Meters/Galvanometers  Recorders  Digital Readout

Spectrophotometer: Quality Assurance 25  Wavelength calibration or accuracy is checked by using special filters with known peak transmission  Should be done periodically  Must be done if a parameter, such as a change in light / lamp has taken place.  Must be done if the instrument has been bumped or traumatized.  Wavelength calibration verifies that the wavelength indicated on the dial is what is being passed through the monochromator.

Spectrophotometer: QA 26  Stray light  any wavelength of light reaching the detector, outside the range of wavelengths being transmitted by the monochromator.  Spectrophotometers must be periodically checked for Stray Light  Causes insensitivity and linearity issues  Resolve by cleaning optical system

Spectrophotometer: QA 27  Linearity Check  A linearity check is made by reading the absorbance of a set of standard solutions (obtained commercially) at specified wavelength(s), or by using neutral density filters  Produces a graph similar in appearance to standard curve.

Spectrophotometer: Sources of Error 28  Lamp burnout – most frequent source of error  Hours of use can be logged by system  Watch for lamp to turn dark or smoky in color  Monochromator error  Poor resolution due to wide bandpass  Results in decreased linearity and sensitivity  Cuvet errors  Dirt, scratches, loose cuvet holder - all cause stray light  Air bubbles in specimen

Spectrophotometer: Sources of Error 29  Reagent make-up  some test procedures make a product that easily foams  Volume too low for light path  Electrical static (noise)  Dark current - from the detector. Leakage of electrons when no light passing through.

Nephelometer  Principle  Measures scattered light  Light “bounces” off insoluble complexes and hits a photodetector  The photodetector is at an angle off from the initial direction of the light.  This is a measure of ‘Light Scatter”  Clinical Applications  Protein measurements in serum, CSF, immunoglobulins, etc. 30 Most of the component parts are similar to those of the spectrophotometer. Major differences: The position of the detector and reduces stray light Light source/beam= LASER light

References  Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.  Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson. 31