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Questions and Answers
How does atomic absorption spectrophotometry (AAS) determine the concentration of a sample?
How does atomic absorption spectrophotometry (AAS) determine the concentration of a sample?
- By calculating the reflectance of the sample
- By measuring the emitted light after atomization
- By detecting the amount of UV light transmitted
- By measuring the light absorbed by an atom (correct)
What is a primary clinical application of flame emission spectrophotometry (FES)?
What is a primary clinical application of flame emission spectrophotometry (FES)?
- Determining molecular weight of proteins
- Measuring electrolytes like Na and K (correct)
- Analyzing trace metals in tissues
- Detecting impurities in colorless solutions
What light source is used in atomic absorption spectrophotometry?
What light source is used in atomic absorption spectrophotometry?
- Xenon flash lamp
- Incandescent bulb
- Hollow-cathode lamp (correct)
- Laser diode
Which of the following best describes the Beer-Lambert law?
Which of the following best describes the Beer-Lambert law?
What principle does infrared spectrophotometry primarily measure?
What principle does infrared spectrophotometry primarily measure?
In flame emission spectrophotometry (FES), how is concentration determined?
In flame emission spectrophotometry (FES), how is concentration determined?
Which technique is generally considered obsolete due to the development of ion selective electrodes (ISE)?
Which technique is generally considered obsolete due to the development of ion selective electrodes (ISE)?
Which type of spectrophotometer is primarily used for measuring colorless solutions?
Which type of spectrophotometer is primarily used for measuring colorless solutions?
What is the wavelength range of visible light?
What is the wavelength range of visible light?
Which type of cuvette is most suitable for a UV light spectrophotometer?
Which type of cuvette is most suitable for a UV light spectrophotometer?
What is the purpose of the photomultiplier tube (PMT) in a spectrophotometer?
What is the purpose of the photomultiplier tube (PMT) in a spectrophotometer?
Which spectrophotometer type uses one beam of light and measures only one sample at a time?
Which spectrophotometer type uses one beam of light and measures only one sample at a time?
How does a double beam spectrophotometer improve accuracy?
How does a double beam spectrophotometer improve accuracy?
What does the readout system in a spectrophotometer provide?
What does the readout system in a spectrophotometer provide?
Which color corresponds to the wavelength range of 651-700 nm?
Which color corresponds to the wavelength range of 651-700 nm?
What does lambda max (λmax) signify in spectrophotometry?
What does lambda max (λmax) signify in spectrophotometry?
What is the principle behind photoelectric colorimetry?
What is the principle behind photoelectric colorimetry?
How is the concentration of an unknown solution in a photoelectric colorimetry setup calculated?
How is the concentration of an unknown solution in a photoelectric colorimetry setup calculated?
Which of the following solutions can a spectrophotometer analyze that a photoelectric colorimeter cannot?
Which of the following solutions can a spectrophotometer analyze that a photoelectric colorimeter cannot?
What does the Beer-Lambert Law relate to in spectrophotometry?
What does the Beer-Lambert Law relate to in spectrophotometry?
What type of light sources can spectrophotometry utilize that photoelectric colorimetry cannot?
What type of light sources can spectrophotometry utilize that photoelectric colorimetry cannot?
In spectrophotometry, what is the role of the monochromator?
In spectrophotometry, what is the role of the monochromator?
What is the primary difference between a spectrometer and a colorimeter?
What is the primary difference between a spectrometer and a colorimeter?
What aspect of a solution is measured by a spectrophotometer to determine concentration?
What aspect of a solution is measured by a spectrophotometer to determine concentration?
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Study Notes
Band of Visible Light
- Visible light ranges from 350 to 700 nm.
- Color spectrum includes:
- Violet (350-430 nm)
- Blue (431-475 nm)
- Green (506-555 nm)
- Yellow (576-600 nm)
- Orange (601-650 nm)
- Red (651-700 nm)
- Specific color wavelengths:
- Green-blue (476-495 nm)
- Blue-green (496-505 nm)
- Yellow-green (556-575 nm)
Spectrophotometer Components
-
Sample Cells (Cuvettes):
- Made of glass or quartz, hold colored solutions.
- Must be scratch-free for accurate light passage.
- Glass cuvettes are used for visible light; quartz for UV light.
-
Light/Photo Detector:
- Converts transmitted light energy into electrical energy.
- Common types: photocell, phototubes, photomultiplier tubes (PMT).
- PMT is preferred for its sensitivity and rapid response.
-
Readout System:
- Displays output from the photodetector.
- Formats include meters, galvanometers, LED displays.
Types of Spectrophotometers
-
Single Beam Spectrophotometer:
- Uses one light beam; measures at wavelengths from 325 nm to 1000 nm.
- Reads test samples and blanks sequentially.
-
Double Beam Spectrophotometer:
- Operates from 185 nm to 1000 nm; splits light into two beams.
- One beam for reference and the other for sample, minimizing error.
Applications of Spectrophotometers
- Analyzes solution concentration qualitatively and quantitatively.
- Requires unknown concentration to fall within the standard curve range.
Standard Curve
- Example table shows concentration (mg/dl) vs. absorbance:
- Concentrations of 60, 70, 80, 90, 100, 110 correlating with specific absorbance values.
Finding Unknown Concentration
- Formula: Concentration of unknown = (Au/As) x Concentration of standard
- Au: absorbance of unknown
- As: absorbance of standard
Photoelectric Colorimetry
- Principle:
- White light through a filter only allows specific wavelengths.
- Colored solution absorbs some light; transmitted light strikes a photoelectric cell which converts it to electrical energy.
- Outputs percent transmittance or absorbance.
Spectrophotometer Overview
- Combination of spectrometer and photometer; measures light absorption to determine concentration.
- Can analyze colored and non-colored solutions using visible, UV, and infrared light.
Spectrophotometer Measurement Principle
- Light passes through a monochromator, creating a spectrum.
- Sample absorbs light; remaining light strikes a detector to quantify absorbance or transmittance.
Additional Spectrophotometric Applications
- Analyzes reaction rates (e.g., enzyme analysis).
- Determines molecular weights of substances.
- Detects impurities in solutions.
Atomic Absorption Spectrophotometry (AAS)
- Measures concentration based on light absorption by atoms, not molecules.
- Uses hollow-cathode lamp; atomizes samples via flame.
- Sensitive technique, ideal for trace metals (e.g., magnesium, copper, lead, calcium).
Flame Emission Spectrophotometry (FES)
- Measures emitted light from excited atoms post-atomization by flame.
- Emission color correlates to specific metal ions (e.g., red for lithium, yellow for sodium).
- Primarily measures electrolytes (Na, K, Li).
Infrared Spectrophotometry
- Measures vibrational spectra of samples using infrared radiation.
- Records absorbed frequencies and their extent.
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