Analytical Chemistry Calibration Curves
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Questions and Answers

What is the primary purpose of creating a calibration curve using absorbance readings?

  • To calculate the absorbance of broken glass
  • To dispose of waste materials properly
  • To establish a control standard for future experiments
  • To determine the concentration of the unknown sample (correct)

    • Which materials should NOT be included in the calibration curve plot?

  • 0.5% (w/v) Control Standard (correct)
  • Unknown Sample (correct)
  • 0.4% (w/v) Calibration Standard
  • 0.2% (w/v) Calibration Standard

    • What is indicated by the target concentration of 0.5% (w/v) for the control standard?

  • It automatically calibrates the unknown sample measurements.
  • It is the exact concentration that must be achieved regardless of measurements.
  • It represents the only valid concentration for calibration standards.
  • It acts as a reference point for evaluating the calibration curve against absorbance. (correct)

    • Which statement about the waste disposal process is accurate?

    <p>Glass test tubes should be disposed of in a bucket labeled ‘Broken glass Only’.</p> Signup and view all the answers

    What mathematical representation is required to be included with the calibration curve?

    <p>A linear trendline and the equation of the line</p> Signup and view all the answers

    What is the concentration of the Red Dye #40 Calibration Stock Solution (C1)?

    <p>40% w/v</p> Signup and view all the answers

    What is the final volume required for each calibration standard?

    <p>25 mL</p> Signup and view all the answers

    Which pipette is used to add the required amount of Red Dye #40 Calibration Stock Solution into the volumetric flasks?

    <p>1000 µL micropipette</p> Signup and view all the answers

    What is the purpose of holding the stopper in place with your thumb before mixing the solution?

    <p>To prevent spillage</p> Signup and view all the answers

    What action should NOT be taken when labeling the volumetric flasks?

    <p>Mark on the white portion of the flask</p> Signup and view all the answers

    Which of the following is NOT part of the materials needed for the quantification of unknowns?

    <p>Ultrasonic cleaner</p> Signup and view all the answers

    What must be done to each volumetric flask after filling it with the solution?

    <p>Cap each volumetric with a stopper</p> Signup and view all the answers

    What calculation method is used to determine the volume of stock solution required for each calibration standard?

    <p>Dilution equation C1V1=C2V2</p> Signup and view all the answers

    What is the primary purpose of calibration standards in an analytical chemistry laboratory?

    <p>To provide known concentrations to relate instrument response to analyte amount</p> Signup and view all the answers

    In the equation of the line for a calibration curve, which variable represents the instrument response?

    <p>y</p> Signup and view all the answers

    How many calibration standards are typically prepared before analyzing unknown samples?

    <p>3 – 5</p> Signup and view all the answers

    What is the significance of the slope (m) in the calibration curve equation?

    <p>It measures the change in absorbance per unit change in concentration</p> Signup and view all the answers

    What is the rearrangement of the equation y = mx + b to solve for concentration (x)?

    <p>x = (m - y) / b</p> Signup and view all the answers

    What type of data is plotted to create a calibration curve?

    <p>Absorbance vs concentration of calibration standards</p> Signup and view all the answers

    What role does the absorbance reading of unknown samples play in analytical chemistry?

    <p>It is used to calculate the concentration of the analyte present in the samples</p> Signup and view all the answers

    What does the R² value of a calibration curve signify?

    <p>How closely the data points fit the line</p> Signup and view all the answers

    What is the primary purpose of pouring the calibration solutions into the test tubes until they are approximately half full?

    <p>To ensure the beam of light passes through the solution</p> Signup and view all the answers

    Which labeling is NOT correct for the test tubes during preparation?

    <p>Sample A</p> Signup and view all the answers

    What should be done to the spectrophotometer before placing the Blank test tube inside?

    <p>Wipe the cuvette clean with a cloth</p> Signup and view all the answers

    How much dH2O should be transferred to the 'Blank' test tube?

    <p>~5 mL</p> Signup and view all the answers

    What wavelength should the spectrophotometer be set to for optimal measurement of red dye?

    <p>500 nm</p> Signup and view all the answers

    After measuring the blank, what is the next action to take regarding the calibration standard?

    <p>Replace the blank with the first calibration solution</p> Signup and view all the answers

    What is the function of pressing the button labelled '0 ABS or 100%T' on the spectrophotometer?

    <p>To set the absorbance value to zero</p> Signup and view all the answers

    Is it necessary to blank the spectrophotometer between each sample when using the same wavelength?

    <p>No, as the wavelength remains unchanged</p> Signup and view all the answers

    What does the variable 'm' represent in the equation of the line?

    <p>The slope of the line</p> Signup and view all the answers

    How is the concentration (x) calculated from the absorbance readings (y)?

    <p>x = (y - b) / m</p> Signup and view all the answers

    What is considered an acceptable range for the percent recovery of a control standard?

    <p>80% - 120%</p> Signup and view all the answers

    What would indicate that the system is considered fit for analysis?

    <p>A control standard recovery between 80% and 120%</p> Signup and view all the answers

    When calculating the percent recovery, which values are used?

    <p>Actual Concentration and Expected Concentration</p> Signup and view all the answers

    In the equation y = mx + b, what does 'b' represent?

    <p>Y-intercept of the line</p> Signup and view all the answers

    What is the correct formula for percent recovery?

    <p>% Recovery = (Actual Concentration / Expected Concentration) * 100%</p> Signup and view all the answers

    What is indicated by the absorbance vs concentration plot?

    <p>The linear correlation between absorbance and concentration</p> Signup and view all the answers

    What is required to be added to the calibration curve generated in Excel?

    <p>Linear trendline and the equation of the line</p> Signup and view all the answers

    What must be excluded from the calibration curve when plotting absorbance readings?

    <p>Absorbance values of the control standard</p> Signup and view all the answers

    Where should broken glass test tubes be disposed of?

    <p>In a bucket labeled 'Broken glass Only'</p> Signup and view all the answers

    What is the target concentration for the control standard in the analysis?

    <p>0.5% (w/v)</p> Signup and view all the answers

    Which statement regarding the absorbance of the calibration standards is accurate?

    <p>Absorbance values directly correlate to concentration.</p> Signup and view all the answers

    What is the primary function of a control standard in analytical chemistry?

    <p>To evaluate the quality of the calibration curve and instrument performance</p> Signup and view all the answers

    What is considered an acceptable range for the concentration of a control standard after measurement?

    <p>80% - 120% of the target value</p> Signup and view all the answers

    What should be done if the measured concentration of a control standard falls outside the acceptable range?

    <p>Investigate potential issues with the calibration standards and instrument</p> Signup and view all the answers

    Which of the following statements about the preparation of a control standard is true?

    <p>It can be sourced from another manufacturer or different lot number</p> Signup and view all the answers

    Why is the control standard not included when plotting the calibration curve?

    <p>It is only used for validation after the calibration is established</p> Signup and view all the answers

    What does the variable 'b' represent in the equation of the line?

    <p>Y-intercept of the graph</p> Signup and view all the answers

    Which formula should be used to calculate the percent recovery of the control standard?

    <p>$ rac{Actual Concentration}{Expected Concentration} imes 100$</p> Signup and view all the answers

    What range is typically deemed acceptable for the percent recovery of a control standard?

    <p>80 – 120 %</p> Signup and view all the answers

    In the rearranged equation to solve for concentration (x), which variable represents the slope?

    <p>m</p> Signup and view all the answers

    When plotting absorbance vs concentration for calibration standards, which aspect is NOT required?

    <p>Legends indicating sample types</p> Signup and view all the answers

    What should be the next action after measuring the blank with the spectrophotometer?

    <p>Measure the absorbance of calibration standards</p> Signup and view all the answers

    Which of the following indicates the actual concentration of a solution?

    <p>Absorbance reading</p> Signup and view all the answers

    What is the primary purpose of the control standard in this analysis?

    <p>To validate the accuracy of absorbance readings</p> Signup and view all the answers

    What is the correct volume to transfer to each labeled test tube during preparation?

    <p>5 mL</p> Signup and view all the answers

    Why is it important to ensure the outside of the test tube is clean before using the spectrophotometer?

    <p>To ensure proper light transmission</p> Signup and view all the answers

    What should be done after placing the BLANK test tube in the spectrophotometer?

    <p>Set the absorbance to zero</p> Signup and view all the answers

    Which of the following should be labeled on the test tubes during preparation?

    <p>Unknown, Control and Blank</p> Signup and view all the answers

    What is the primary role of the spectrophotometer when analyzing the prepared solutions?

    <p>To measure the intensity of light absorbed by the solutions</p> Signup and view all the answers

    What is the optimal wavelength setting on the spectrophotometer for measuring red dye?

    <p>500 nm</p> Signup and view all the answers

    Which action is unnecessary when analyzing samples if the wavelength remains unchanged?

    <p>Blanking the spectrophotometer</p> Signup and view all the answers

    How should the test tubes be filled to ensure accurate measurement of absorbance?

    <p>Until they are approximately half full</p> Signup and view all the answers

    What does the slope (m) represent in the calibration curve equation?

    <p>The relationship between absorbance and concentration</p> Signup and view all the answers

    In the context of a calibration curve, what is the importance of the R² value?

    <p>It shows how well the calibration curve fits the data</p> Signup and view all the answers

    When calculating the concentration (x) of an unknown from absorbance (y), which equation is used?

    <p>x = (y - b)/m</p> Signup and view all the answers

    What must be prepared before measuring the absorbance of unknown samples?

    <p>Multiple calibration standards with known concentrations</p> Signup and view all the answers

    How is the absorbance reading of an unknown sample used after calibration?

    <p>To calculate the concentration of the unknown using the calibration curve</p> Signup and view all the answers

    What is the primary role of calibration standards in analytical chemistry?

    <p>To relate instrument response to known analyte concentrations</p> Signup and view all the answers

    What does the variable 'b' represent in the equation of the line for a calibration curve?

    <p>The y-intercept of the calibration curve</p> Signup and view all the answers

    Which action is crucial before taking absorbance readings with a spectrophotometer?

    <p>Blanking the spectrophotometer</p> Signup and view all the answers

    Study Notes

    Introduction

    • Spectrophotometers are used in analytical chemistry to measure the amount of an analyte in a sample
    • Absorbance readings are obtained from spectrophotometers
    • Calibration standards are solutions with known concentrations of the analyte
    • Calibration curves plot the absorbance readings of a set of calibration standards against their corresponding known concentrations

    Calibration Curve and Equation of the Line

    • Calibration curves facilitate the determination of the concentration of an analyte
    • Calibration curves can be plotted using Microsoft Excel
    • Absorbance data for calibration standards is plotted to generate a calibration curve
    • A linear trendline is fitted to the plotted data
    • The equation of the line for the calibration curve is in the form y = mx + b
    • y = instrument response (e.g, absorbance), x = analyte amount (e.g., concentration), m = slope of the calibration curve, and b = y intercept
    • The equation of the line is used to determine the concentration of unknown samples

    Materials

    • 40% (w/v) Red Dye #40 Calibration Stock Solution
    • 0.5% (w/v) Red Dye #40 Control Standard
    • Red Dye #40 Unknown Sample
    • Distilled water
    • Spectrophotometer
    • 1000 µL micropipettes and tips
    • Volumetric flasks (4x 25mL)
    • Stoppers (4x)
    • Test tubes (7x Size: 13x100mm)

    Calibration Standard Preparation

    • The calibration standards are prepared using the 40% (w/v) Red Dye #40 Calibration Stock Solution
    • The calibration standards are prepared in volumetric flasks
    • The target concentrations of the calibration standards are 0.2%, 0.4%, 0.6%, and 0.8% (w/v)
    • The volume of each calibration standard is 25 mL
    • The formula C1V1 = C2V2 is used to calculate the volume of calibration stock solution needed for each concentration
    • Each volumetric flask is filled with distilled water approximately halfway
    • The calculated volume of stock solution is added to each flask using a micropipette
    • Each flask is filled to the 25 mL mark with distilled water
    • Each flask is capped and mixed by inverting several times
    • Four test tubes are labeled with the concentrations of the calibration standards (0.2%, 0.4%, 0.6%, and 0.8%)
    • Each calibration solution is transferred to the corresponding test tube

    Additional Standard/Sample Preparation

    • Three additional test tubes are labeled: ‘Blank’, ‘0.5% Ctrl’, and ‘Unknown’
    • The ‘Blank’ test tube is filled with distilled water
    • The ‘0.5% Ctrl’ test tube is filled with the 0.5% (w/v) Red Dye #40 Control Standard
    • The ‘Unknown’ test tube is filled with the Red Dye #40 Unknown Sample

    Spectrophotometer Analysis

    • The spectrophotometer wavelength must be set to 500 nm
    • The ‘Blank’ test tube is measured in the spectrophotometer and the absorbance is set to zero
    • The remaining test tubes are analyzed individually in the spectrophotometer
    • The absorbance readings are recorded for all test tubes

    Waste Disposal

    • Liquid waste is disposed of in a waste container in the fume hood
    • Glass test tubes are disposed of in the broken glass only container
    • Dispose of all chemicals according to the safety regulations

    Data Analysis

    • A calibration curve is plotted in Excel using the absorbance readings of the calibration standards
    • The absorbance values of the ‘Unknown’ and ‘Control Standard’ are not included in the calibration curve
    • A linear trendline is added to the calibration curve
    • The equation of the line is determined
    • The equation of the line is used to calculate the concentration of the ‘Unknown’ and ‘Control Standard’
    • The percent recovery of the ‘Control Standard’ is calculated
    • Acceptable recovery ranges between 80-120%
    • If the control standard recovery falls within this range, the system is considered fit for analysis

    Results Summary Requirements

    • Each group submits one PDF document containing two pages
    • Page 1: (1) Calibration curve plot (5 marks) and (2) Summary table of absorbance readings (1 mark)
    • Page 2: (1) Concentration calculations for the ‘Unknown’ and ‘Control Standard’

    Introduction

    • Spectrophotometers are used to determine the amount of an analyte in a sample by measuring absorbance
    • Absorbance readings do not tell us how much analyte is present unless they are related to known values

    Calibration Standards

    • Calibration standards contain known concentrations of the analyte
    • Absorbance readings of calibration standards are measured to relate instrument response to known values
    • Calibration standards are prepared by analyzing between 3-5 standards and taking the absorbance readings

    Calibration Curves

    • A graph of absorbance vs concentration of calibration standards is called a calibration curve
    • A linear trendline fitted to the data gives the equation of the line, y=mx+b where:
      • m = slope of the line
      • b =y intercept
      • x = amount of analyte
      • y = instrument response
    • Equation can be rearranged to solve for concentration of unknown samples: x=(y-b)/m

    Control Standard

    • Control standard is a separate standard of known concentration prepared from a different source of the analyte
    • Control standard is not part of the calibration curve
    • The control standard concentration is calculated using the equation of the line and absorbance reading
    • A control standard concentration between 80-120% of the expected value indicates that the instrument and calibration standards are working properly and fit for analysis

    Spectrophotometer

    • Spectrophotometer manufacturer: Agilent
    • Spectrophotometer model: Cary 60
    • Spectrophotometer SLC number: SLC-1234

    Additional Standard/Sample Preparation

    • Label test tubes with: Blank, 0.5% Ctrl, Unknown
    • Fill each test tube approximately halfway with their respective solution
    • Blank: 5 mL dH2O
    • 0.5% Ctrl: 5 mL of 0.5% (w/v) Red Dye #40 Control Standard
    • Unknown: 5mL of Red Dye #40 unknown sample

    Standard/Sample Analysis

    • Clean the outside of the test tubes before analysis
    • Set the wavelength of the spectrophotometer to 500 nm
    • Place the Blank test tube in the spectrophotometer and set absorbance to zero by pressing the button labelled ‘0 ABS or 100% T’
    • Replace the Blank with the first calibration standard and close the lid
    • Do not re-zero the spectrophotometer between samples
    • Record the absorbance readings for all samples

    Waste Disposal

    • Liquid waste: Dispose in the waste container in the fumehood
    • Glass test tubes: Dispose in the bucket labeled ‘Broken Glass Only’

    Data Analysis

    • Use the ‘Plotting with Excel’ document for guidance
    • Plot the absorbance readings of the calibration standards only on a graph. Do not plot the absorbance values for the unknown or control standard.
    • Add a linear trendline, equation of the line (y=mx+b), and R2 to your calibration curve
    • Calculate the concentration of the Unknown and Control Standard using the equation of the line (y=mx+b)
    • Rearrange equation to solve for concentration: x = (y-b)/m
    • Calculate the percent recovery of the Control Standard using the formula: (Actual Concentration/Expected Concentration) x 100%

    Marking Scheme

    • One Blackboard submission per group
    • Refer to the ‘Submitting Assignments Via Blackboard’ instructions
    • One PDF document containing two pages:
      • Page 1:
        • Plot of absorbance vs concentration of calibration standards (5 marks)
        • Summary table of absorbance readings for calibration standards, unknown, and control standard (1 mark)
      • Page 2:
        • Concentration calculations for unknown and control standard (6 marks)

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    Description

    This quiz covers the fundamentals of calibration curves in analytical chemistry, focusing on their creation, functionality, and equations. Learn how to plot absorbance data, understand the significance of the slope, and interpret the calibration curves for effective analyte concentration determination.

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