St. Lawrence College CHEM 1001 Lab 5: Quantification of Unknowns PDF

CHEM 1001 Lab 5: Quantification of Unknowns INTRODUCTION Instrumentation found in analytical chemistry laboratories are used to determine the amount of an analyte of interest in a sample. An example of this is the use of spectrophotometers and the determination of caffeine content in beverage sample...

CHEM 1001 Lab 5: Quantification of Unknowns INTRODUCTION Instrumentation found in analytical chemistry laboratories are used to determine the amount of an analyte of interest in a sample. An example of this is the use of spectrophotometers and the determination of caffeine content in beverage samples. When samples are analyzed with spectrophotometers, their absorbance readings are obtained. Without proper context, absorbance readings do not readily tell us how much analyte (caffeine in this example) is in the sample. Calibration Standards To determine the amount of analyte (e.g. caffeine) in a sample, the instrument response (absorbance) needs to be related back to known values. This is accomplished by analyzing calibration standards and measuring their absorbance readings. Calibration standards are prepared by the analyst and contain known concentrations of the analyte of interest. Using the caffeine in beverage example, prior to analyzing unknown samples an analyst would prepare between 3 – 5 calibration standards and take their absorbance readings in addition to the absorbance readings of unknown samples. Calibration Curves and the Equation of the Line Following analysis, the absorbance vs concentration data for the calibration standards is plotted to yield a calibration curve. 2 Absorbance 1.5 1 y = 0.1654x + 0.0296 0.5 R² = 0.9998 0 0 2 4 6 8 10 12 Caffeine Concentration (% (w/v)) Once plotted, a linear trendline is fitted to the calibration data and the resulting equation of the line can be obtained in the form of y = mx + b. Where: m = slope of the line, b = y intercept, x = amount of analyte (e.g. concentration) and y = instrument response (e.g. absorbance). The equation is rearranged to isolate and solve for x (concentration) as follows: x = (m – y)/b. Absorbance readings (y) of the unknowns are substituted into the equation and used to determine the concentration (x) of the unknowns. Overall, the purpose of the calibration standards is to establish the calibration curve, which relates the instrument response (e.g. absorbance) to a known amount of analyte (e.g. concentration). This ultimately generates the equation of a line which is used to determine the amount of analyte in a sample. Control Standard Analysts also prepare an additional standard of known concentration called a control standard. The control standard contains a known amount of the analyte of interest and is prepared from a separate source of the analyte of interest. This source can be another manufacturer of the analyte (i.e. caffeine purchased from another supplier) or another lot number for the analyte (i.e. caffeine prepared in a separate batch by the manufacturer). After the absorbance of the control standard is read and the equation of the line is generated from the calibration curve, the control standard concentration can be calculated by plugging the absorbance reading (y) into the equation of the line and solving for x (concentration). If the calibration standards have been prepared accurately and the instrument is working properly, the control standard concentration that is calculated from the absorbance readings should be close to the target (expected) control standard concentration. Typically, values between 80 – 120 % of the target values are considered acceptable which means the system (instrument and calibration standards) is fit for the analysis. If the concentration of the control standard is outside this window of acceptability, further investigation is required because the system is not giving accurate data and the results produced cannot be trusted to analyze unknown samples. Overall, the purpose of the control standard is to assess the quality of the calibration curve and performance of the instrument. It is used to determines if the system is fit for the analysis of unknowns. THIS STANDARD IS NOT INCLUDED WHEN PLOTTING THE CALIBRATION CURVE. 1  CHEM 1001 Lab 5: Quantification of Unknowns 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) PROTOCOL Part I: STANDARD/SAMPLE (PER PAIR): Calibration Standard Preparation 1. Prepare the following calibration standards outlined in the table below using the 40% (w/v) Red Dye #40 Calibration Stock Solution (C1).  You will be making the following concentrations (C2): 0.2% (w/v), 0.4% (w/v), 0.6% (w/v) and 0.8% (w/v).  The volume (V2) for each is 25mL.  Use C1V1=C2V2 to calculate the volume of stock (V1) required for each solution.  Fill in the following chart to help set up all the calibration standard dilutions. Calibration Standard Calibration Stock Volume Dye Stock Dye Calibration Standard Red Dye Calibration Number Required (V1) Concentration (C1) Concentration (C2) Standard Volume (V2) (mL) (% (w/v)) (% (w/v)) (mL) 1 2 3 4 2. Label four 25 mL volumetric flasks with 0.2%, 0.4%, 0.6% or 0.8%. Write on the clear glass portion of the flask. DO NOT write on the white portion of the flask as it is difficult to remove markings off this area. 3. Fill each volumetric flask approximately halfway with dH 2O. 4. Using a 1000 µL micropipette, add the required amount of 40% (w/v) Red Dye #40 Calibration Stock Solution to each flask. 5. Bring each flask up to the final volume using dH 2O. 6. Cap each volumetric with a stopper. 7. Hold the stopper in place with your thumb and mix by slow inversion (turning the flask upside down, then right-side up and repeat several times) 8. Label four test tubes with one of the following concentrations: 0.2%, 0.4%, 0.6% or 0.8%. 9. Transfer each calibration solution you have prepared to the corresponding test tube above by pouring directly from the volumetric flask into the test tube until it is approximately half full. 2  CHEM 1001 Lab 5: Quantification of Unknowns Additional Standard/Sample Preparation 1. Label three additional test tubes, one with ‘Blank’, one with ‘0.5% Ctrl’ and one with ‘Unknown’. Be sure to write around the top of the tube, so it does not interfere with the beam of light traveling through the bottom portion of the test tube. 2. You can pour directly from the volumetric flask to the test tube using the instructions below. Be sure to fill approximately half the volume of the tube with liquid to ensure that the beam of light travels through your solution and not the air above. 3. Remember, you can pour directly from the volumetric flasks into your test tubes. 5 mL will be approximately half the volume of the test tube. 4. Transfer ~5 mL dH2O to the tube labeled ‘Blank’ until it is approximately half full 5. Transfer ~5 mL of the 0.5% w/v Red Dye #40 Control Standard provided to the tube labeled ‘0.5% Ctrl’. 6. Transfer ~5 mL of the Red Dye #40 unknown sample provided to the tube labeled ‘Unknown’. Part II: STANDARD/SAMPLE ANALYSIS Spectrophotometer Example Spectrophotometer Used Manufacturer: Agilent Model: Cary 60 SLC Equipment Number: SLC-1234 1. Prior to analysis by spectrophotometry, ensure that the outside of the test tube (or cuvette) is wiped clean with a Kimwipe. 2. Set the wavelength on the spectrophotometer to: 500 nm (optimal wavelength for red dye). 3. Place the BLANK test tube into the spectrophotometer and close the lid. 4. Set the spectrophotometer’s absorbance to zero by pressing the button labelled: 0 ABS or 100%T. 5. Replace the BLANK test tube with your first calibration standard and close the lid. You do not need to blank your spectrophotometer between samples, as you will not be changing the wavelength. 6. Read and record the absorbances for all test tubes in the table below. Sample ID Absorbance 0.2% (w/v) Calibration Standard 0.4% (w/v) Calibration Standard 0.6% (w/v) Calibration Standard 0.8% (w/v) Calibration Standard 0.5% (w/v) Control Standard Unknown Sample WASTE DISPOSAL Waste Type Disposal Location/Container Liquid Waste container in the fumehood Glass Test Tubes Dispose in the bucket labeled: ‘Broken glass Only’ 3  CHEM 1001 Lab 5: Quantification of Unknowns Part III: DATA ANALYSIS 1. Refer to the ‘Plotting with Excel’ document provided for instructions and expectations when using Excel to make graphs. 2. Using the absorbance readings for the CALIBRATION STANDARDS ONLY produce a calibration curve by plotting Absorbance (y-axis) versus Concentration (x-axis). DO NOT PLOT THE ABSORBANCE VALUES OF THE UNKNOWN OR CONTROL STANDARD. THEY DO NOT BELONG IN THE CALIBRATION CURVE. They are used later in the protocol. 3. Add a linear trendline, equation of the line (y = mx + b) and R 2 to your calibration curve. 4. Using the equation of the line for your calibration curve generated by Excel, determine the actual concentration of the unknown and control standard. The target for the control standard is 0.5% (w/v) but the actual concentration will vary depending on your calibration curve and the absorbance measured for the control standard. The equation of the line is written as follows: 𝒚 = 𝒎𝒙 + 𝒃 m: slope of the line b: y intercept of the graph of the line x: concentration (% (w/v)) y: absorbance readings (𝐲 – 𝐛) The equation is rearranged to isolate and solve for x (concentration) as follows: 𝐱 = 𝒎 5. Using your absorbance readings (y) for the unknown and control standard and the re-arranged equation of the line, determine the concentrations (x) of the unknown and control standard. Unknown Sample: Control Standard: 6. Calculate the percent recovery of the control standard using the formula below. Typically, recoveries between 80 – 120 % are deemed acceptable for assessing data quality. If the control standard recovery falls within this range, then the system is considered fit for analysis. 𝑨𝒄𝒕𝒖𝒂𝒍 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 (𝒄𝒂𝒍𝒄𝒖𝒍𝒂𝒕𝒆𝒅 𝒂𝒃𝒐𝒗𝒆) % 𝑹𝒆𝒄𝒐𝒗𝒆𝒓𝒚 = 𝒙 𝟏𝟎𝟎% 𝑬𝒙𝒑𝒆𝒄𝒕𝒆𝒅 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 (𝒇𝒓𝒐𝒎 𝑴𝒂𝒕𝒆𝒓𝒊𝒂𝒍𝒔 𝒔𝒆𝒄𝒕𝒊𝒐𝒏) Control Standard Percent Recovery: 4  CHEM 1001 Lab 5: Quantification of Unknowns MARKING SCHEME FOR GRAPHS: Please refer to the marking scheme on Blackboard for expectations related to plots Lab #5 – Spectroscopy II – Results Summary /12 marks  One Blackboard submission per group  Due date on Blackboard  Refer to the ‘Submitting Assignments Via Blackboard’ instructions Results Summary Requirements  Submit one PDF document containing two pages: o Page 1:  Plot of absorbance vs concentration for your calibration standards (5 marks)  Summary table of your absorbance readings for your calibration standards, unknown and control standard (1 mark). o Page 2:  Concentration calculations for your unknown and control standard. Show your work (2 marks).  Percent recovery calculations for your control standard. Show your work (2 marks).  Comment on the quality of the calibration curve and the resulting control standard concentration/percent recovery. Is the system fit for analysis? Why or why not? (2 marks). 5