Spectrophotometry Principles & Techniques
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Questions and Answers

What principle does spectrophotometry rely on to measure concentration in solutions?

  • Absorption patterns of light by atoms and molecules (correct)
  • Chemical reactions that change color in solutions
  • Refraction of light through different mediums
  • Differences in molecular weight of the substances

    • How does the color of a solution relate to its absorbance?

  • Absorbance is independent of the color present in the solution
  • Colored solutions absorb some light while clear solutions allow light to pass (correct)
  • The darker the solution, the lower its absorbance
  • Clear solutions absorb more light than colored solutions

    • Which of the following instruments would NOT be used for performing basic measurements in this lab?

  • Spectrophotometer
  • Thermometer (correct)
  • Micropipettes
  • Graduated cylinders

    • What does the absorbance scale of a spectrophotometer indicate?

    <p>The amount of light absorbed compared to a clear solution</p> Signup and view all the answers

    In preparing a percentage volume per volume (% v/v) solution, which component is crucial to calculate accurately?

    <p>The amount of solute and solvent used</p> Signup and view all the answers

    What type of solution would be expected to have the greatest absorbance?

    <p>A highly pigmented colored solution</p> Signup and view all the answers

    Which of the following statements about light in the visible spectrum is true?

    <p>Different wavelengths correspond to different colors</p> Signup and view all the answers

    Which of the following skills is emphasized in the lab objectives?

    <p>Performing metric conversions and measurements</p> Signup and view all the answers

    What is the initial step in calibrating the Vernier SpectroVIS Spectrophotometer?

    <p>Double click on the LoggerPro 3.8.2 icon on the computer desktop.</p> Signup and view all the answers

    What should be done with the cuvette after collecting data for the blue dye solution?

    <p>It should be set aside for future use.</p> Signup and view all the answers

    What is required before clicking 'Finish Calibration' on the spectrophotometer?

    <p>Wait for the lamp to warm up for 60 seconds.</p> Signup and view all the answers

    In order to determine the wavelength for maximum absorbance of blue dye, which collection mode should be selected?

    <p>Absorbance vs. Wavelength</p> Signup and view all the answers

    What solution should the cuvette be filled with for calibration?

    <p>A blank solution, typically water.</p> Signup and view all the answers

    When preparing to analyze the blue dye solution, what should be done with each solution?

    <p>Mix each solution with a vortex mixer.</p> Signup and view all the answers

    What is the expected outcome after clicking 'Collect' on the spectrophotometer?

    <p>A full chromatogram of the blue dye will be displayed.</p> Signup and view all the answers

    Which parameter is set to measure the concentration of the blue dye?

    <p>Absorbance vs. Concentration</p> Signup and view all the answers

    What is the purpose of clicking on 'Clear Selection' before choosing a wavelength?

    <p>To avoid data from previous experiments affecting the results</p> Signup and view all the answers

    What should you enter as the concentration for the blank sample?

    <p>0%</p> Signup and view all the answers

    What should be the expected size of droplets when measuring 500 µl?

    <p>The droplets should all be approximately the same size.</p> Signup and view all the answers

    What action should you take after inserting sample #1 into the cuvette?

    <p>Wait 5 seconds before clicking 'Keep'</p> Signup and view all the answers

    When performing a 1:5 dilution, what volume of solvent is required for 20 ml of stock solution?

    <p>80 ml of solvent</p> Signup and view all the answers

    How can you determine if your sample preparation and calibration were accurate?

    <p>By checking the correlation value for linearity</p> Signup and view all the answers

    What does a dilution fold of 1:20 signify?

    <p>0.5 ml of solute in 9.5 ml of solvent</p> Signup and view all the answers

    What does a higher dye concentration typically indicate about light absorbance?

    <p>Higher dye concentration increases light absorbance</p> Signup and view all the answers

    What is the significance of the slope value when performing a linear fit?

    <p>The closer it is to 1, the better the results</p> Signup and view all the answers

    Which equipment is used for discarding the tip in pipetting?

    <p>A tip ejection key</p> Signup and view all the answers

    What is the first step after measuring 150 µl with a micropipette?

    <p>Add the measured liquid to a container.</p> Signup and view all the answers

    Which of the following steps follows after clicking 'Collect' with the blank cuvette?

    <p>Click 'Keep' after waiting for a short period</p> Signup and view all the answers

    What is a key indicator that your dye samples have been prepared correctly?

    <p>A straight line is produced in the graph</p> Signup and view all the answers

    What is the correct way to describe the dilution achieved by mixing 1 ml of concentrated solution with 99 ml of solvent?

    <p>1:100 dilution</p> Signup and view all the answers

    What characteristic should the droplets exhibit when using a 150 µl volume?

    <p>They should be uniformly smaller than 500 µl droplets.</p> Signup and view all the answers

    How much total volume results from a 1:5 dilution using 20 ml of solute?

    <p>100 ml</p> Signup and view all the answers

    Study Notes

    Spectrophotometry Principles

    • Spectrophotometry measures the concentration of dissolved chemicals by analyzing their light absorption patterns.
    • Atoms, molecules, and chemical bonds absorb unique wavelengths of light.
    • Visible light wavelengths correspond to different colors and energy levels.
    • Colored solutions absorb some light, while clear solutions allow most light to pass through.
    • Absorbance (A) quantifies the amount of light absorbed by a solution. Higher absorbance indicates a darker solution.

    Measurement Techniques

    • Practice using glass pipettes, micropipettes, and graduated cylinders for accurate measurements.
    • Learn to calculate the amounts of solute and solvent needed for specific volume/volume (% v/v) solutions.
    • Micropipettes are used to measure and deliver precise small volumes (e.g., 500 µl, 150 µl).

    Simple Dilutions

    • Simple dilution involves combining a concentrated solute (stock solution) with a solvent to achieve a desired concentration.
    • Dilution fold is calculated as: amount of stock solution : total final volume (e.g., 1:5 dilution means 1 part solute to 4 parts solvent).
    • Example: A 1:20 dilution is created by mixing 0.5 ml of solute with 9.5 ml of solvent.

    Spectrophotometer Operation

    • The Vernier SpectroVIS Spectrophotometer is used without an on/off switch; only USB connection required.
    • Calibration involves filling a cuvette with a blank solution (water), inserting it into the chamber, and using the LoggerPro software to calibrate the spectrophotometer. A 60-second warm-up is needed.
    • Absorbance vs. Wavelength mode allows determination of the wavelength with maximum absorbance.
    • Absorbance vs. Concentration mode is used to measure absorbance at a specific wavelength, creating a graph of absorbance versus concentration. The slope of a linear fit should approach 1 for accurate results.

    Blue Dye Experiment

    • Students prepared blue dye solutions with varying concentrations.
    • A spectrophotometer was used to measure the absorbance of each blue dye solution at its maximum wavelength.
    • The experiment confirmed the relationship between dye concentration and absorbance. Higher concentration leads to higher absorbance.

    Data Analysis (Specific Data Points depend on experiment)

    • Sample Absorbance Values were recorded.
    • Correlation (R) value from the linear fit was obtained. Higher R values indicate better fit to concentration.
    • The sample with the highest concentration had the highest absorbance reading.
    • The relationship between dye concentration and absorbance is directly proportional (higher concentration = higher absorbance).

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    Related Documents

    Lab 5: Spectrophotometry PDF

    Description

    Explore the fundamental principles of spectrophotometry, including how light absorption relates to the concentration of chemicals in solutions. The quiz also covers practical measurement techniques using pipettes and graduated cylinders. Additionally, learn the basics of simple dilutions for achieving desired concentrations.

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