4  Chromatography

Note

Last edited: 03NOV2023 NP

This is not a full protocol yet, just notes. Nora will build on it.

Link to full protocol (pdf)

4.1 Preparation

  • Air dry, pulverize, and sieve samples to 2mm

  • Label 125mL Erlenmeyer flasks with sample codes; measure 5g soil into each. We did not use the analytical balance for this. This can be done ahead of time!

  • Use Whatman #4 filter paper to cut wicks; Gaby has a piece of laminated square grid paper to get each piece to be 0.5x0.5in (I believe that is the measurement). Tightly roll each into a wick

    • Each sample needs 2 wicks (one for AgNO3, one for soil solution); if doing duplicates of each sample, each needs 4 wicks

  • Count out one #4 filter paper for each sample; poke a tiny hole in the middle for the wick to go in and label with pencil

    • Hole should be 1/16 inches in diameter; Gaby has a good tool for this

    • Make one small pencil mark 1.5 inches from the center and one 2 ⅜ inches from the center of each filter paper [edit: update these measurements for 18.5cm; 15cm filters should be used]

Figure 4.1: Making holes in filter paper

  • Make 0.5% AgNO3 solution. Should be stored in a brown bottle and not exposed to light

    • 0.5% solution = 5g AgNO3 / 1L H2O → 2.5g / 0.5 L → 1.25g / 250mL

    • Reasoning: 1L = 1000mL and the density of water is 1g/L, so there are 1000g/1L. 10% by weight would be 100g/1L, 1% would be 100g/1L, 1% would be 10g/1L, and 0.5% would be 5g/1L. Checked with Gaby 03MAY2023

    • This does not need to be made on on a stirrer; AgNO3 is H2O soluble

  • Make 1% NaOH solution

    • Using the extra pure solution (50% NaOH by weight), add 20mL solution per 1L H2O

    • We need to dilute the 50% NaOH solution 50 times

    • CAS: 1310-73-2. Kept in flammable liquid storage cabinet underneath fume hood in S434. Because it’s a liquid, just mix them in 1L Nalgene bottles.

  • Set out one large petri dish and one small one for each sample

Figure 4.2: Small petri dish in large petri dish (same height) 
::: callout-warning
Are you processing foreign soils? Tape off a counter and have plenty of ethanol/bleach spray. Trash should go in an autoclave waste bag, not in a trash can
:::

4.2 Procedure

  • 5g soil in Erlenmeyer flask → each gets 50mL 1% NaOH. Do not add until doing procedure
  • After NaOH is added, thoroughly mix the sample by swirling the flask. Swirl for 6 rotations, repeating 6 times, for 36 total rotations.
  • Let flasks stand undisturbed for 15 minutes. Repeat the swirling process.
  • Let flasks stand for 1 hour. Repeat the swirling process.
  • After samples are swirled, let sit for 5 more hours. After the 6th hour (including hour in step above; 6 total hours), then chromatography can begin.
    • Do not disturb the flasks at all during the resting period.
  • Saturate filter papers with AgNO3 to first line, using the large/small petri dish and wick setup.
  • Once saturated, they must dry completely with no light exposure. Consider making a sandwich with 1 piece printer paper, 1 paper towel, 1 filter paper, 1 paper towel, and 1 piece printer paper. See photo below. Store in a closed box.
Figure 4.3: Saturated filter papers in sandwich to dry

The following photos were taken during a chromatography run as examples of setup and workflow.

Figure 4.4: Beakers for rinsing pipette between samples
Figure 4.5: Counter setup - petri dishes spaced out
Figure 4.6: Running - flasks next to filters to keep track of codes
Figure 4.7: Running - plenty of empty small petri dishes to swap out
Figure 4.8: Running - finished chroma; at second line
Figure 4.9: Full counter setup
Figure 4.10: Full counter setup - note red tape (foreign soil)
Figure 4.11: Completed chromas drying on counter - with paper
Figure 4.12: Completed chromas curing on window

4.3 Post-Procedure

  • Samples should dry on a bench for 3 hours, then get hung in a window

  • Nora gently attached a paper clip to each filter paper, then taped the paper clip to the window. Didn’t want to risk ripping the filter paper when taking the tape off

  • Chromas should develop in the window for 7-8 days. Window should not get direct sunlight, although a little bit seems to be fine

  • Chromas should be scanned on a high quality scanner (UIC or SWAC printer?) within 1-2 days of being fully developed

  • Store chromas with a sheet of paper in between each