Analytical Chemistry Calibration Curves

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)

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.