Analytical Chemistry: Spectrophotometry Basics

Introduction

• Analytical chemistry laboratories use instruments to determine the amount of an analyte in a sample.
• Spectrophotometers are used to determine the caffeine content in beverages by measuring absorbance readings.
• Calibration standards are prepared with known concentrations of the analyte to relate the instrument response (absorbance) to known values.

Calibration Curves

• Absorbance vs. concentration data of calibration standards is plotted to create a calibration curve.
• A linear trendline is fitted to the data, producing an equation of the line (y = mx + b).
• The equation is rearranged to isolate and solve for the concentration (x): x = (m – y)/b.

Control Standard

• A control standard, prepared from a separate source of the analyte, is analyzed to assess the quality of the calibration curve.
• The control standard concentration is calculated by plugging in the absorbance reading into the equation of the line.
• Values within 80-120% of the target concentration are considered acceptable, indicating a reliable system.

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
• Micropipettes and tips
• Volumetric flasks
• Stoppers
• Test tubes

Protocol: Calibration Standard Preparation

• Prepare calibration standards at concentrations of 0.2%, 0.4%, 0.6%, and 0.8% w/v Red Dye #40.
• Use C1V1 = C2V2 to calculate the volume of stock solution needed for each standard.
• Label volumetric flasks with the corresponding concentrations.
• Add distilled water to each flask, followed by the calculated volume of stock solution.
• Bring each flask up to the final volume with distilled water and mix well.

Protocol: Additional Standard/Sample Preparation

• Label test tubes with ‘Blank’, ‘0.5% Ctrl’, and ‘Unknown’.
• Transfer approximately 5 mL of distilled water to the ‘Blank’ test tube.
• Transfer 5 mL of the 0.5% w/v Red Dye #40 Control Standard to the ‘0.5% Ctrl’ test tube.
• Transfer 5 mL of the Red Dye #40 Unknown Sample to the ‘Unknown’ test tube.

Protocol: Standard/Sample Analysis

• Wipe the outside of each test tube clean.
• Set the spectrophotometer to 500 nm.
• Place the BLANK test tube in the spectrophotometer and set the absorbance to zero.
• Replace the BLANK with each calibration standard and record the absorbance readings.
• Measure and record the absorbance readings for the ‘0.5% Ctrl’ and ‘Unknown’ samples.

Data Analysis

• Plot a calibration curve using the absorbance values of the CALIBRATION STANDARDS ONLY (y-axis) against their concentrations (x-axis).
• Add a linear trendline, equation of the line, and R² value to the calibration curve.
• Use the equation of the line generated from the calibration curve to determine the actual concentrations of the control standard and unknown sample.

Waste Disposal

• Liquid waste should be disposed of into the designated waste container in the fume hood.
• Glass test tubes should be disposed of in the "Broken glass only" bucket.

This quiz covers the fundamental concepts of analytical chemistry related to spectrophotometry, focusing on the determination of caffeine content in beverages. Key topics include calibration curves, absorbance measurements, and control standards to ensure accurate analysis. Test your understanding of how calibration impacts the quality of results in analytical procedures.