Spectrophotometry

Spectrophotometry Overview

• Spectrophotometry is a technique used in clinical chemistry to determine the concentration of unknown samples
• Spectrophotometry measures the intensity of light at specific wavelengths
• Analytical techniques include spectrophotometry, atomic absorption, mass spectrometry, luminescence (fluorescence, chemiluminescence), electroanalytic (electrophoresis, potentiometry, amperometry), and chromatography (gas, liquid, thin layer)
• Light is a type of radiant energy that travels in waves
• A cycle is one complete waveform
• Frequency (v) = number of cycles/second (hertz), and is inversely related to wavelength
• Short wavelengths have high energy and high frequency, while long wavelengths have low energy and low frequency
• Velocity (c) is the distance a wave travels in one second
• Wavelength (λ) is the distance between corresponding points on a wave, measured in nanometers (nm)
• Amplitude is the magnitude of the peak of a wave
• The different types of radiation have different approximate wavelength ranges. This is shown in Radiation vs Wavelength table

Wavelength Parameters

• Short wavelengths carry more energy than longer wavelengths
• Visible light ranges from 380 to 750nm
• Violet light is at the shortest visible wavelength, and red is at the longest
• A mnemonic device for remembering the colours of the visible spectrum in order is "ROY G. BIV": Red, Orange, Yellow, Green, Blue, Indigo, Violet
• White light, when passed through a prism, separates into its component colors
• This is called dispersion
• Different substances absorb different wavelengths of light, which is how they appear different colors.

Absorption/Transmission of Light

• The color of light seen by the human eye depends on which wavelengths are not absorbed.
• Solutions that absorb specific wavelengths of light appear as that colour.
• In a solution, if light is not absorbed it is transmitted (passed through)
• The more absorbing molecules there are in a solution, the less light is transmitted
• The more concentrated a colored solution is, the more light is absorbed.
• For electromagnetic radiation to be absorbed, it must have the same frequency as a rotational or vibrational frequency in the atom or molecule it hits

Beer's Law

• A law relating the absorption of light by a solution to the concentration of that solution
• There are four parts to Beer's Law
• Part 1: Equal thicknesses of an absorbing material will absorb a constant fraction of energy incident upon it.
• Part 2: The absorption of energy by an absorbing material is logarithmic, each equal layer absorbs a constant fraction

Beer's Law - Concentration and Path Length

• Absorbance is directly proportional to concentration and path length.
• The more concentrated the solution and greater the path length, the greater the absorbance.
• The less concentrated the solution and smaller the path length, the smaller the absorbance (and greater the transmittance).

Beer's Law - Mathematically

• Absorbance is directly proportional to the concentration (C) of absorbing molecules and the path length (b) of the light through the solution (A∝ C x b)
• ε is the molar absorptivity (a constant for a specific wavelength of light and molecule), a measure of how strongly a substance absorbs light at a particular wavelength.
• The equation for Beer's Law is A = εbc
• Values for unknown concentrations can be calculated if A, ε ,and b values are known for the relevant standard.

Example using Beer's Law

• The example provided shows how to calculate an unknown concentration, using data for standard concentration, standard absorbance, and patient absorbance)

Filter Photometer

• Beer's Law is applied in photometry and spectrophotometry to determine the concentration of unknown samples
• Photometry measures light intensity regardless of wavelength
• Spectrophotometry measures light intensity at selected wavelengths
• Filter Photometers use filters to select wavelengths of light

Parts of a Filter Photometer

• Light Source (provides white light)
• Filter (selects the wavelength)
• Cuvette (holds the solution being measured)
• Detector (converts light energy to electrical energy)
• Meter (displays absorbance or %T)

Spectrophotometry - Classifications

• Spectrophotometers can either be single-beam or double-beam
• In the single beam spectrophotometer, the light passing through a reference solution is measured and then the light passing through the sample solution is measured
• In double-beam spectrophotometers, the light passing through the sample and the reference solutions is measured simultaneously

Absorbance and Transmittance of Light

• Measurement of light absorption in the sample depends on the reaction between the substance being measured and the reagent, which produces a colour change
• The intensity of the color depends on the concentration of the substance in the sample
• The intensity of color is proportional to concentration; more substance= deeper colour; deeper colour = increased concentration
• The more concentrated a substance solution, the more light that substance absorbs (and less transmitted)

Expressing Light Absorbed or Transmitted

• Absorbance is a measure of the amount of light absorbed by a solution, and is directly proportional to concentration.
• Percent transmittance (%T) is a measure of the amount of light that passes through a solution.
• Both absorbance and transmittance are compared to a reagent blank, which has all reagents but not the substance of interest.

Photometer Scale

• Transmittance is the fraction of light that passes through the solution, which is displayed as %T
• %T and absorbance are related, when absorbance increases %T decreases
• A = 2 – log %T

Calibration Graphs (Standard Curves)

• A standard curve is created by plotting absorbance or %T against increasing concentrations of standards
• It is necessary to obtain known readings from known solutions to create a calibration graph
• A or %T are plotted against concentration of each standard on graph paper
• Typical graphs include concentration vs absorbance or concentration vs %T

Ideal Calibration Curves

• Ideal calibration curves span a wide range of absorbances and concentrations
• A curve where absorbance readings are linear in relation to concentration is ideal

Preparation and Use of a Standard Curve

• Plotted using absorbance or %T readings from known standard solutions (absorbance vs. concentration).
• Use absorbance (or %T) of unknown patient samples read on the spectrophotometer to create the concentration of unknown samples

Parts of a Spectrophotometer

• Light Source, Monochromator, Cuvette, Detector, Galvanometer

Light Source

• Incandescent tungsten lamp for the visible and near-infrared range (300-1000nm)
• Deuterium and Mercury Arc lamps for the ultraviolet (UV) range

Monochromator

• Filters, Prisms, Diffraction Gratings, used to eliminate unwanted wavelengths
• Bandwidth is the range of wavelengths where %T is ½ the peak of transmittance

Types of Filters

• Glass, Wratten, Interference; varying bandwidths

Gratings

• Flat glass with etched grooves used for reflection and transmission, creating linear spectra

Prisms

• Separates light through refraction, creating nonlinear spectra, wavelengths bent differently

Cuvettes (Sample Cells)

• Vessels used to hold solutions (serum, controls or standards)
• Constructed from glass, silica (quartz) or plastic
• Disposable plastic cuvettes are usual, due to solvent and temperature issues with other materials

Photodetectors (Radiant Energy Detectors)

• Change light energy to electrical energy
• Examples: Barrier Layer Cells (Photocell), Phototube, Photomultiplier tubes, Photodiodes

Galvanometer

• Measures current, and current produced is proportional to amount of light transmitted

Spectrophotometer Overview

• Diagram showing the light source, diffraction grating, plate with slit, cuvette, photoelectric cell and galvanometer

NovaSpec III and III+ Spectrophotometers

• Examples of specific instruments used
• Parameters such as wavelength and absorbance reading may vary with the instrument used

Spectrophotometer Quality Assurance

• Instrument function needs validation including checks on wavelength accuracy, stray light and linearity

Quality Assurance - Wavelength Accuracy

• The set wavelength on the spectrophotometer should match the actual wavelength passed through the monochromator; use absorbing solutions and filters
• Mercury lamp used instead of usual light source for verification

Quality Assurance - Stray Light

• Scratches on cuvette surfaces and dust in light path cause stray light
• Cutoff filters can be used to validate measurement

Quality Assurance - Linearity

• Calibration curves should be straight and follow Beer's Law
• Linearity is checked by running diluted colored solutions

Reading Assignment

• Clinical Chemistry: Principles, Techniques, and Correlations (8th Edition)
• Chapter 5 (pages 101-107)