2.2: Week 1

Calibration Standard "B"

You may collaborate and share this solution with your teammates or with other teams - however you are each individually responsible for the accuracy of this solution preparation and for recording how it was created in your notebook. It is essential that the concentration of calibration standard B solution is accurate.

• For convenience, use a micropipette and a volumetric flask: Use a 1.000 mL automatic micropipette to quantitatively transfer 1.000 ml of stock solution to a 25.00 mL volumetric flask. It is critical that this transfer is precise and accurate — check the function of the pipette first, and then weigh your transferred liquid to be sure. Then dilute with the matrix.
• When using the volumetric flasks, carefully dilute the solution up to the mark using 0.01 M sodium acetate solution and mix well. Put some of this solution into a screw cap vial labeled "B" so that you can access the solution with a pipette in the next step. Each team will need at least ~20 mL of this solution or more. If you need to re-do the solution prep you will need even more.  You can store the leftover solution B in a sealed vial.
• Record triple replicate absorbance of Solution B on at least two spectrometers (to be sure the spectrometers are giving consistent readings).

Calibration Standards C1-F1 — serial dilution, Class A volumetric glassware (Calibration Set 1) 

One person in your team should create standard solutions for calibration set 1 (C1-F1) by serial dilution, using Class A volumetric glassware:

1. Start with solution B.  Prepare solution "C1" using a 15 ml volumetric pipette, quantitatively transfer 15 ml of solution B to a clean 25 ml volumetric flask. Carefully dilute the solution up to the mark using 0.01 M sodium acetate solution. Mix well by inverting 20 times. Transfer solution C1 to a labeled vial before the next step. 
2. In the same manner, perform 3 more serial dilutions to create solutions D1-F1 (see the example tables provided below).
   • Transfer and store each solution to a labeled vial and keep vials closed to avoid evaporation.
3. Collect absorbance data using the "Data Collection" instructions below. Make sure to also collect the absorbance of the Blank, the Validation Standard, and the Sample.
4. Use the data to determine if your calibration is valid. If it is not, troubleshoot with the help of your instructors, and try again to create the solutions.

Calibration Standards C2-F2  — serial dilution, automatic volumetric pipettes (pipetters)

One person in your team shouid create standard solutions for calibration set 2 (C2-F2) by serial dilution, using automatic pipettes: 

1. Prepare solution "C2" using a 1.000 mL volumetric pipette, quantitatively transfer 1.200 mL of sodium acetate matrix to a clean tube. Then, add 1.800 mL of solution B and mix well. These steps can be accomplished by dialing the pipette to half of the desired volume, and making two careful transfers to double the volume. 
   Important points:
   • Use the pipette to gently mix this solution by slowly pipetting up and down, but be careful not to "shoot" the solution up into the barrel of the pipette by releasing the plunger too quickly. If the solution goes up too high, it will become contaminated, and the pipette should be cleaned - consult your instructor if this happens and start over.
   • Note that the error for all transfers must be propagated! 
   • Due to the small volume of this solution, record the absorbance of the C2 solution before creating the next one. See "Data Collection" instructions below.
2. Use the "Data Collection" instructions below to record the absorbance of solution C2. Then use solution C2 to create solution D2, and repeat this process for E2, and F2.
   • Use the pipette to mix each solution, then re-use your tip for the next solution.
   • Reserve a second tip exclusively for transferring the "clean" sodium acetate matrix. 
   • Store all leftover solutions in labeled tubes and keep tubes closed to avoid evaporation.
   • Make sure to also collect the absorbance of the Blank, the Validation Standard, and the Sample.
3. Use the data to determine if your calibration is valid. If it is not, troubleshoot with the help of your instructors, and try again to create the solutions.

Tips for data collection

• Use one cuvette (a "working cuvette") for all measurements and keep a second separate cuvette for all blanks ("blank cuvette"). The blank cuvette should be filled with 0.01 M sodium acetate solution.
  • After the instrument is zeroed with the blank cuvette, measure the absorbance of the working cuvette after filling it with the 0.01 M sodium acetate solution - this is your absorbance at zero concentration of analyte.
  • Be very sure that both cuvettes are clean before making these measurements.
• Measuring sequence and working with the cuvettes:
  • Use the same working cuvette for all measurements, and the same blank cuvette for all zeroing.
  • It is essential that residual solution from the previous measurement does not dilute or contaminate your next sample!
    • You can clean and dry the cuvette between measurements - but this is not recommended. It is tedious and ineffective unless you have access to a cuvette washer. Do not stuff towels into a cuvette to dry it, and never blow compressed air into a cuvette. 
    • To prepare the cuvette for the next measurement:
      1. Empty the cuvette and tap the cuvette gently upside down on a soft towel to remove as much liquid as possible.
      2. Rinse the cuvette with a very small amount (a few drops) of the next solution to be measured, and do this three times.
      3. Add the bulk of the next solution and make the measurement.
  • If you are measuring multiple solutions sequentially, work starting with the least concentrated solution and proceed in order of increasing concentration. However, this is not possible using the automatic pipette protocol due to limited volume.
  • The outside of the cuvette should always be clean and dry.
• Take three measurements for each solution (use same liquid for each measurement; take cuvette out and wipe down then replace in instrument) and calculate the average absorbance and standard deviation from three replicate readings for each solution. Usually, you should get exactly the same absorbance reading for all three replicate measurements. The three readings should not differ by more than \(\pm 0.003\) absorbance units or 1% of the absorbance (see uncertainty associated with the spectrometer readings described in the operating instructions.)
• Between each measurement, the sample cuvette should emptied and then rinsed twice with small (ca. 1 mL or less) aliquots of the next solution to be assayed.
  ***Work carefully because you have limited quantities of each solution.
• Between measurements, check the stability of the instrument by inserting the blank and making sure it still reads zero absorbance.