MLAB 2401: Clinical Chemistry

Keri Brophy-Martinez

Analytical Techniques and Instrumentation

Electromagnetic Radiation & Spectrophotometry

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Introduction

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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

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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

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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

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Electromagnetic Radiation: Properties of light and radiant energy

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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

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Electromagnetic Radiation:Properties of light and radiant energy

Wavelength Measured in nanometers (nm) or 10-9

meters.

Electromagnetic Radiation Properties of light and radiant energy

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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)

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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

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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

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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)

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Beer-Lambert law (Beer’s Law)

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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 = (Aunk /Astd) * Concentration std

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Percent transmittance

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Photometry/Spectrophotometry

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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

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Types -Simple photometers and colorimeters use a filter to produce light of one wavelength (monochromatic light).

Major components of a simple photometer.Major components of a simple photometer.

Spectrophotometer / Spectrophotometry

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Spectrophotometers differ from photometers in that they use prisms or diffraction gratings to form monochromatic light.

Spectrophotometer: Components

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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 - 700 nm)

Deuterium or mercury-arc lamps required for work in U.V. rangeRange 160-375 nm

Spectrophotometer: Components

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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

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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

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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

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Photodetectors

Purpose – to convert the transmitted light into an equivalent amount of electrical energy

Most common is the photomultiplier tube

Spectrophotometer: Component

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Readout devices

Purpose – to convert the electrical signal from the detector to a usable form

Types Meters/Galvanometers Recorders Digital Readout

Spectrophotometer: Quality Assurance

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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

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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

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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

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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

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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.

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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 .

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