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Questions and Answers
Which method can be used to ensure the wavelength accuracy of a spectrophotometer?
Which method can be used to ensure the wavelength accuracy of a spectrophotometer?
What indicates a failure in the spectrophotometer's optical system that might affect readings?
What indicates a failure in the spectrophotometer's optical system that might affect readings?
What is the primary relationship described by Beer’s Law in terms of a spectrophotometer's function?
What is the primary relationship described by Beer’s Law in terms of a spectrophotometer's function?
In a spectrophotometer, what component is primarily responsible for producing a current in response to light intensity?
In a spectrophotometer, what component is primarily responsible for producing a current in response to light intensity?
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Which of the following procedures is essential for validating a spectrophotometer's functionality?
Which of the following procedures is essential for validating a spectrophotometer's functionality?
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What is the effect of stray light on absorbance measurements in a spectrophotometer?
What is the effect of stray light on absorbance measurements in a spectrophotometer?
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How is transmittance related to absorbance according to the principles governing spectrophotometry?
How is transmittance related to absorbance according to the principles governing spectrophotometry?
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What characterizes a good monochromator in terms of bandwidth?
What characterizes a good monochromator in terms of bandwidth?
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Which type of filter is least resistant to heat and prone to leakage?
Which type of filter is least resistant to heat and prone to leakage?
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What is the role of the cuvette in a spectrophotometer?
What is the role of the cuvette in a spectrophotometer?
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How does a prism produce a spectrum from white light?
How does a prism produce a spectrum from white light?
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What is the primary function of the monochromator in a spectrophotometer?
What is the primary function of the monochromator in a spectrophotometer?
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Which of the following wavelength ranges does a deuterium discharge lamp provide?
Which of the following wavelength ranges does a deuterium discharge lamp provide?
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What does Beer’s Law relate to in a spectrophotometric context?
What does Beer’s Law relate to in a spectrophotometric context?
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Which type of monochromator component uses etched grooves to provide a linear spectrum?
Which type of monochromator component uses etched grooves to provide a linear spectrum?
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What is the definition of bandwidth in the context of a spectrophotometer?
What is the definition of bandwidth in the context of a spectrophotometer?
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What is the main purpose of using a standard solution in Beer’s Law calculations?
What is the main purpose of using a standard solution in Beer’s Law calculations?
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Which component of a filter photometer is responsible for selecting the specific wavelength of light?
Which component of a filter photometer is responsible for selecting the specific wavelength of light?
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In spectrophotometry, what is the role of the blank reference solution?
In spectrophotometry, what is the role of the blank reference solution?
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How does transmittance differ from absorbance in context of light measurement?
How does transmittance differ from absorbance in context of light measurement?
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Which formula correctly represents the relationship in Beer’s Law for determining unknown patient concentrations?
Which formula correctly represents the relationship in Beer’s Law for determining unknown patient concentrations?
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What characteristic distinguishes a single-beam spectrophotometer from a double-beam spectrophotometer?
What characteristic distinguishes a single-beam spectrophotometer from a double-beam spectrophotometer?
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What is the purpose of the detector in a filter photometer?
What is the purpose of the detector in a filter photometer?
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What is typically the outcome if the path length of the sample and standard are not equal in Beer’s Law experiments?
What is typically the outcome if the path length of the sample and standard are not equal in Beer’s Law experiments?
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In the context of standard curve construction, what key factor is critical when establishing the curve?
In the context of standard curve construction, what key factor is critical when establishing the curve?
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Study Notes
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)
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