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CHEM 370

HPLC Procedure

Table of Contents
  1. Standard Preparation
  2. Running Standards
    1. Sample Preparation
  3. Running Samples
    1. Waste Disposal and Cleanup

Standard Preparation

  1. Make 50 ml of 100-ppm (w/v) acetaminophen stock solution using 98% 4-acetamidophenol in 1:3 methanol:water.
  2. Perform serial dilutions to create a set of 5 standards with concentrations ranging from 0.5 - 25 ppm. You will need 2 ml of each standard for HPLC analysis, but it won’t hurt to have a little extra.
  3. Filter your standards into autosampler vials for analysis using a 0.45$\mu$m syringe filter.

Running Standards

  1. Warm up the HPLC according to the HPLC SOP. Use the CHEM370_Acetaminophen.M method.
  2. Place your standards in the HPLC autosampler.
  3. Prepare a sequence based on the autosampler locations of your samples (refer to HPLC SOP for more info). Prepare your sequence in this order:
    1. Zero Standard/Rinse Blank (water)
    2. Low Standard
    3. Medium-low Standard
    4. Medium Standard
    5. Medium High Standard
    6. High Standard
    7. Rinse Blank
    8. QC Sample
  4. After the instrument has warmed up (stable baseline), run your sequence.
  5. Analyze your standard curve, blanks, and QC.
  • A passing blank will show no signs of contamination.
  • A passing standard curve will have $R^2 \ge 0.9900$.
  • A passing QC will be within $\pm$ 10% of the known concentration.
If any of these is not passing you must repeat the analysis and/or remake your standards until they pass!

Sample Preparation

  1. Determine the mass of all the contents of your vials.
  2. Grind your sample into a fine, homogenous powder using a mortar and pestle.
  3. Cone and quarter your sample (on weighing paper) until you have just over 100 mg.
  4. Dissolve 100.0 mg of the tablet in 1:3 methanol:water, and bring the final volume to 100.0 ml.
  5. Perform a 1:100 dilution of you sample in 1:3 methanol:water.
  6. Filter you final dilution through a 0.45 $\mu$m syringe filter using a 20 ml syringe. Make sure you:
    • Don’t pull up on the plunger with the filter attached to the syringe (you may tear the filter).
    • Discard the first 10 ml of filtrate.
    • Place an aliquot of the last 10 ml of filtrate in an HPLC autosampler vial for analysis.

Running Samples

  1. Warm up the HPLC according the the HPLC SOP (skip if already warm).
  2. Place an aliquot of your sample dilution in the HPLC autosampler.
  3. Prepare a sequence based on the autosampler locations of your samples (refer to HPLC SOP for more info). Prepare your sequence in this order:
    1. Standards & QC (see Standard Preparation section)
    2. Up to 10 samples
    3. QC Sample (same as previous QC; always finish with QC)
  4. After the instrument has warmed up, run your sequence.
  5. Analyze your standards and samples (you may skip the standards again if you’ve run them in the past hour – just start with a QC instead).
  6. Use a t-test or confidence intervals to determine if the mass of nicotine is significantly different from the expected mass. (See Comparing Means for more information on comparing means.)

Waste Disposal and Cleanup

  1. Place all liquid and/or powder wastes in the HPLC waste container for the lab.
  2. Place all (cleaned) disposable glass vials in the broken class bin.
No chemicals should go in broken glass!