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?

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

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

A linear trendline and the equation of the line Signup and view all the answers

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

40% w/v Signup and view all the answers

What is the final volume required for each calibration standard?

25 mL 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?

1000 µL micropipette Signup and view all the answers

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

To prevent spillage Signup and view all the answers

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

Mark on the white portion of the flask Signup and view all the answers

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

Ultrasonic cleaner Signup and view all the answers

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

Cap each volumetric with a stopper Signup and view all the answers

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

Dilution equation C1V1=C2V2 Signup and view all the answers

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

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

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

y Signup and view all the answers

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

3 – 5 Signup and view all the answers

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

It measures the change in absorbance per unit change in concentration Signup and view all the answers

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

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

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

Absorbance vs concentration of calibration standards Signup and view all the answers

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

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

What does the R² value of a calibration curve signify?

How closely the data points fit the line 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?

To ensure the beam of light passes through the solution Signup and view all the answers

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

Sample A Signup and view all the answers

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

Wipe the cuvette clean with a cloth Signup and view all the answers

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

~5 mL Signup and view all the answers

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

500 nm Signup and view all the answers

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

Replace the blank with the first calibration solution Signup and view all the answers

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

To set the absorbance value to zero Signup and view all the answers

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

No, as the wavelength remains unchanged Signup and view all the answers

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

The slope of the line Signup and view all the answers

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

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

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

80% - 120% Signup and view all the answers

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

A control standard recovery between 80% and 120% Signup and view all the answers

When calculating the percent recovery, which values are used?

Actual Concentration and Expected Concentration Signup and view all the answers

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

Y-intercept of the line Signup and view all the answers

What is the correct formula for percent recovery?

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

What is indicated by the absorbance vs concentration plot?

The linear correlation between absorbance and concentration Signup and view all the answers

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

Linear trendline and the equation of the line Signup and view all the answers

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

Absorbance values of the control standard Signup and view all the answers

Where should broken glass test tubes be disposed of?

In a bucket labeled 'Broken glass Only' Signup and view all the answers

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

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

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

Absorbance values directly correlate to concentration. Signup and view all the answers

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

To evaluate the quality of the calibration curve and instrument performance Signup and view all the answers

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

80% - 120% of the target value Signup and view all the answers

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

Investigate potential issues with the calibration standards and instrument Signup and view all the answers

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

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

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

It is only used for validation after the calibration is established Signup and view all the answers

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

Y-intercept of the graph Signup and view all the answers

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

$rac{Actual Concentration}{Expected Concentration} imes 100$ Signup and view all the answers

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

80 – 120 % Signup and view all the answers

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

m Signup and view all the answers

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

Legends indicating sample types Signup and view all the answers

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

Measure the absorbance of calibration standards Signup and view all the answers

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

Absorbance reading Signup and view all the answers

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

To validate the accuracy of absorbance readings Signup and view all the answers

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

5 mL Signup and view all the answers

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

To ensure proper light transmission Signup and view all the answers

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

Set the absorbance to zero Signup and view all the answers

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

Unknown, Control and Blank Signup and view all the answers

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

To measure the intensity of light absorbed by the solutions Signup and view all the answers

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

500 nm Signup and view all the answers

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

Blanking the spectrophotometer Signup and view all the answers

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

Until they are approximately half full Signup and view all the answers

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

The relationship between absorbance and concentration Signup and view all the answers

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

It shows how well the calibration curve fits the data Signup and view all the answers

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

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

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

Multiple calibration standards with known concentrations Signup and view all the answers

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

To calculate the concentration of the unknown using the calibration curve Signup and view all the answers

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

To relate instrument response to known analyte concentrations Signup and view all the answers

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

The y-intercept of the calibration curve Signup and view all the answers

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

Blanking the spectrophotometer 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.