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chromatography.qmd
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chromatography.qmd
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# Chromatography
::: callout-note
Last edited: 03NOV2023 NP
This is not a full protocol yet, just notes. Nora will build on it.
Link to [full protocol](https://drive.google.com/file/d/1b1qVL2BmtHHxatD9wqjuA225A1mHu8vw/view?usp=sharing) (pdf)
:::
## 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 \[edit: update these measurements for 18.5cm; 15cm filters should be used\]
![Making holes in filter paper](chroma1.jpg){#fig-chromaholes}
- 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
![Small petri dish in large petri dish (same height)](chroma2.jpg){#fig-petridish}
::: 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
:::
## 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.
![Saturated filter papers in sandwich to dry](chroma3.jpg){#fig-chromasandwich}
The following photos were taken during a chromatography run as examples of setup and workflow.
![Beakers for rinsing pipette between samples](chroma4.jpg){#fig-rinsebeakers}
![Counter setup - petri dishes spaced out](chroma5.jpg){#fig-countersetup}
![Running - flasks next to filters to keep track of codes](chroma6.jpg){#fig-flasks}
![Running - plenty of empty small petri dishes to swap out](chroma7.jpg){#fig-smallpetri}
![Running - finished chroma; at second line](chroma8.jpg){#fig-finishedchroma}
![Full counter setup](chroma9.jpg){#fig-fullcounter}
![Full counter setup - note red tape (foreign soil)](chroma10.jpg){#fig-fullcounter2}
![Completed chromas drying on counter - with paper](chroma11.jpg){#fig-chromadrying}
![Completed chromas curing on window](chroma12.jpeg){#fig-chromacuring}
## 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