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Questions and Answers
What is the main purpose of a spectrophotometer in laboratory analysis?
According to Beer's Law, what does absorbance depend on?
Which component is not typically found in a spectrophotometer?
Which of the following statements best describes colorimetry?
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What is a critical maintenance task for ensuring accurate spectrophotometric measurements?
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What does the intensity of color measured in endpoint colorimetric reactions indicate?
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Which reaction is an example of an endpoint enzymatic method?
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In kinetic reactions, what is primarily measured?
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What kind of product often forms in endpoint enzymatic reactions?
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What measurement technique is used in the Jaffe reaction for creatinine analysis?
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Which coenzyme is produced in the hexokinase method for measuring glucose?
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Which of the following wavelengths is typically used for endpoint enzymatic reactions?
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Which of the following describes a key characteristic of the kinetic method?
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What is the primary reason NADP is used in spectrophotometry?
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In the kinetic method for measuring ALT, which substrate is NOT used?
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What type of relationship is generally established between absorbance and concentration in spectrophotometry?
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Which equation describes how to calculate the concentration of an unknown sample?
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How is the patient albumin concentration determined using the absorbance of patient and standard solutions?
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What is the absorbance of the patient sample if its albumin concentration is calculated to be 5.0 g/dL?
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Which statement best describes the role of coenzymes in spectrophotometric reactions?
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What is the first step in setting up the photometry module?
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What does percent transmittance (%T) represent in spectrophotometric measurements?
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Which of the following equations correctly relates absorbance (A) to percent transmittance (%T)?
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What term is synonymous with absorbance (A)?
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What is the range of values for absorbance (A) in spectrophotometry?
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In the context of Beer’s Law, what does the term 'molar absorptivity' signify?
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Which of the following is not a component of the transmittance ratio (T)?
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What happens to the absorbance (A) as the concentration of the analyte increases?
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What is the physical meaning of transmittance (T) being equal to 1?
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What happens to a chemical when it absorbs light?
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According to Beer’s Law, what do we know about absorbance?
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What is the relationship between wavelength and energy of light?
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What units are used to measure frequency?
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How does the absorption spectrum of a substance relate to the light it emits?
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What is the unit of Planck’s constant (h)?
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In the equation E = h, which parameter represents the energy of a photon?
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What parameter describes the height of a light wave's electric vector?
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What does a decrease in wavelength indicate about energy?
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Which constant represents the speed of light in a vacuum?
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If a photon has a high energy, what can be inferred about its frequency?
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What happens to the absorbance of a sample if the concentration of absorbing chemicals increases?
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Which of the following accurately describes the relationship between frequency and wavelength?
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What is the major factor in determining the type of light a chemical absorbs?
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In an experiment measuring absorbance, which parameter is NOT a factor according to Beer’s Law?
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Study Notes
Spectrophotometry
- Spectrophotometry is a technique that uses light to measure chemical concentrations.
- It's a colorimetric method that uses an instrument to determine the amount of analyte in a sample by its ability or inability to absorb light at a specific wavelength.
Types of Light
- Light waves consist of perpendicular, oscillating electric and magnetic fields.
- The wavelength of light is the distance from peak to peak, measured in units of nanometers (nm), centimeters (cm), or meters (m).
- The frequency of light is the number of complete oscillations that the waves makes each second.
- Energy (E) of light is proportional to its frequency.
- Energy (E) of light increases as frequency () increases or wavelength () decreases
Beer's Law
- Beer's Law states that the relative amount of a certain wavelength of light absorbed (A) that passes through a sample is dependent on the distance the light must pass through the sample, the amount of absorbing chemicals in the sample and the ability of the sample to absorb light.
- Absorbance (A) is the relative amount of light absorbed by the sample and is related to transmittance (T), which is the relative amount of light making it through the sample.
Spectrophotometric Methods
- Endpoint colorimetric reactions occur when there is a color change at the end of a reaction. The color intensity is proportional to the amount of solute in the sample.
- Endpoint enzymatic reactions use an enzyme to catalyze the chemical reaction. The final product often absorbs light strongly at lower wavelengths in the visible or near-UV spectrum.
- Kinetic spectrophotometry measures the rate of a reaction by monitoring the change in absorbance over time, which depends on the activity of an enzyme present.
Spectrophotometer Components
- Light Source: Emits a beam of light with a specific wavelength.
- Monochromator: Isolates a specific wavelength of light.
- Sample Cell (Cuvette): Holds the sample being tested.
- Detector: Measures the amount of light that passes through the sample.
- Readout Device: Displays the results of the measurement.
- Calibration Standards: Used to create a calibration curve, which relates absorbance to concentration.
- Control Samples: Used to assess the accuracy and precision of the method.
- Wavelengths: Different wavelengths of light are used for different analytes.
- Absorbance and Transmittance: Directly proportional to the concentration of the analyte.
Clinical Chemistry Applications for Spectrophotometry
- Spectrophotometry is used in a variety of clinical chemistry applications, such as:
- Glucose analysis
- Creatinine analysis
- Albumin analysis
- Alanine transaminase (ALT) analysis
- Protein analysis
- Enzyme activity analysis
- Drug analysis
- Hemoglobin analysis
- Bilirubin analysis
Spectrophotometer Maintenance and Calibration
- Cleaning: Regularly cleaning the cuvette, monochromator, and detector helps to ensure accurate results.
- Calibration: Calibration curves must be generated periodically to ensure the accuracy of the spectrophotometer.
- Validation: Daily validation must be performed to ensure the accuracy and precision of the analyses.
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Description
This quiz covers the fundamentals of spectrophotometry, including the types of light and Beer's Law. You'll learn about how light interacts with chemical substances and how to measure concentrations using light absorption techniques.
