Permanganate Oxidizable Carbon (Poxc)

Method of data collection and data needed to compute the method:

The procedure for determining POXC in detail.

I. Instrumentation and Materials  

• Spectrophotometer capable of reading absorbance at 550 nm
• Weighing balance capable of accurately weighing ~2.50g of soil to two decimal places (0.01 g)
• pH meter calibrated for measurement in the range of ~6.0-8.0 pH and NaOH for pH adjustment
• Oscillating (or horizontal) shaker capable of at least 240 oscillations per minute (or 120 rpm)
• Magnetic stir plate and stir bars
• Adjustable 10 mL pipettor and tips
• Adjustable 100-1000 µL pipettor and tips
• (2) Adjustable bottle-top dispensers fitted to a bottle of deionized water and calibrated to deliver 18.0 mL and 49.5 mL
• 50 mL disposable polypropylene centrifuge tubes with caps (Falcon tubes)
• Laboratory glassware for reagent preparation and waste collection
• Labeling supplies such as permanent markers and tape
• Reagent grade Potassium Permanganate (KMnO_⁴; FW=158.03 g mol^-1)
• Reagent grade Calcium Chloride, Dihydrate (CaCl₂·2H₂O; FW=147.01 g mol^-1)
• Soil standard (sieved and air-dried KBS topsoil for use as a lab reference sample)
• Timer capable of tracking time for two and ten minute intervals      

II. Reagent Preparation:   

KMnO4 Stock Solution 0.2 M (makes 1 liter, 2ml use per soil sample):

1. Weigh 147 g of CaCl₂ and place in a 1000 mL beaker. Add approximately 900 mL of deionized water and stir till dissolved. Transfer to a 1000 mL volumetric flask or graduated cylinder. Bring to volume with deionized water.
2. Weigh 31.60 g of KMnO₄ into a 1000 mL beaker and add approximately 900 mL of the CaCl₂ solution. Place on the magnetic stir plate (minimize exposure to light) with gentle heat and stir until dissolved completely.  Note: Dissolution may be very slow.
3. Once dissolution is complete, place the probe from a calibrated pH meter into the solution (with continued stirring) and measure the pH. Adjust the pH to 7.2 – 8.5 by adding 0.1 N NaOH, 1 drop at a time (endpoint approaches rapidly). Note that it can be challenging to adjust pH, and note in the record what pH that is achieved. With the CaCl₂ solution, adjust volumn in a 1000 mL volumetric flask or graduated cylinder. Transfer to a brown glass bottle and store in a dark place (stable 3-6 months).

III. Standard preparation:

Four standard concentrations (0.005, 0.01, 0.015 and 0.02 M) prepared from the KMnO₄ stock solution. The standard preparation involves first making a standard stock solution and then diluting each standard stock solution to a final working standard. The following materials will be needed:

• 50 mL disposable polypropylene centrifuge tubes
• Adjustable 1.0-10.0 mL pipettor and tips
• Adjustable 100-1000 µL pipettor and tips
• Adjustable bottle-top dispensers fitted to a bottle of deionized water and calibrated to deliver 49.5 mL

Part 1- Standard Stock Solutions: Use the table below to prepare standard stock solutions. These stock solutions can be prepared in centrifuge tubes or in small brown glass bottles and used for three days (stored in glass and in the dark).

Part 2- Dilution Step: Dilute each standard stock solution to a working standard by adding 0.5 mL of each stock solution to 49.5 mL of deionized water in 50 mL centrifuge tubes. These tubes contain the working standards and should be prepared fresh daily.

IV. Sample Preparation: Sample preparation involves a two part process: a sample reaction and sample dilution, as illustrated below.

A soil standard and solution standard are prepared in the same manner as the unknown samples. The soil standard serves as a laboratory reference sample. It is recommended to homogenize a large batch of air-dried soil for long-term use. The soil standard allows for a quality control check across POXC analyses performed on different batches, over multiple days, or with different reagents. The solution standard serves as another quality control reference. It is prepared in the same manner as the unknown soil samples, but without the soil. The solution standard will reveal if reagents or labware have been contaminated with oxidizing agents or carbon and thus serves as a true blank.

It is important that the timing of each step be consistent, particularly the shaking and settling times. The permanganate will continue to react with the soil as long as it remains in contact. Hence, working quickly with small batches of 10 samples or less is advised.

The following materials will be needed:

• (2) 50 mL disposable polypropylene centrifuge tubes with caps for each sample
• Adjustable 1.0-10.0 mL pipettor and tips
• Adjustable 100-1000 µL pipettor and tips
• (2) Adjustable bottle-top dispensers fitted to bottles filled with deionized water and calibrated to deliver 18.0 mL and 49.5 mL
• Labeling supplies such as permanent markers and tape
• Oscillating shaker capable of at least 240 oscillations per minute (or 120 rpm) and fitted with a lidded box that will hold at least ten 50 mL centrifuge tubes
• Timer capable of tracking time for two and ten minute intervals
• Soil standard (pulverized, homogenous soil as lab reference sample)

Sample Reaction

1. Label two 50 mL centrifuge tubes for each sample. Weigh 2.50 grams (± 0.05 g) of sieved, air-dried soil into one of the centrifuge tubes (may be done in advance). A soil standard should also be prepared. Place the other set of tubes aside.
2. Add 18.0 mL of deionized water to each of the centrifuge tubes containing the soil. Using the 1.0-10.0 mL pipettor, add 2.0 mL of 0.2 M KMnO₄ stock solution to each tube.
3. Prepare a solution standard by adding 18.0 mL of deionized water and 2.0 mL of 0.2 M KMnO₄ stock solution to a tube (no soil) and process in the same manner as the unknown soils.
4. Working quickly, cap tubes tightly and hand-shake each tube vigorously for 2 seconds to assure soil dispersion within the solution.
5. Place tubes on shaker and shake at 240 oscillations per minute for 2 minutes.
6. After 2 minutes, remove samples from shaker and swirl or shake the tube vigorously to ensure that there is no soil clinging to the sides or cap of the tube. At this point, remove caps to avoid further disturbance of soil after settling. Place the samples in a dark area and allow soil to settle for ten minutes. Settling time is a critical step so a timer is essential.

Sample Dilution

1. While samples are settling, add 49.5 mL of deionized water to the second set of centrifuge tubes (may be done in advance).
2. Once the ten minute settling period has passed, quickly transfer 0.5 mL of supernatant (avoiding any particulate matter) to the second tube containing 49.5 mL of water. Note: This step should be performed as quickly as possible as the permanganate will continue to react with soil as long as it remains in contact.
3. Cap second set of tubes and invert to mix. These are the final sample solutions for analysis. They are stable for up to 24 hours if stored in the dark.

Reading Samples on Spectrophotometer

1. This method has been shown to perform well on both single cuvette machines and 96-well plate reading spectrophotometers. If available, a 96-well plate reader is recommended to save time (see steps 2-5 below).
2. Clear polystyrene flat-bottom cell culture plates (or equivalent) work well, so more expensive UV-transparent plates are not necessary. Fill each well with 200µl of solution.
3. It is recommended to replicate all standards on a plate, including blanks of deionized water. Running each standard three or more times and taking the average typically yields good results.
4. Determine and record the absorbance (optical density) of standards and unknowns at 550nm using spectrophotometer software.
5. Subtract out average of deionized water blanks from all absorbance values (if not automatically performed by software). The intercept of the standard curve should be very close to zero.

Clean-up and Disposal

Leaving the centrifuge tubes capped but on the bench top for a week or more will allow the permanganate to completely react with the soil and lose all purple pigmentation. Liquid can then be safely disposed of down the sink and tubes with soil thrown out or cleaned and reused. The second dilution of samples and standards contains very little KMnO4 and may be safely flushed down the drain with copious amounts of water; however, check with your environmental health and safety department to ensure compliance with your department’s procedures.

 

Unit of analysis:

The amount of carbon oxidized is a function of the quantity of permanganate reduced. Consequently, the higher the POXC values the lower the absorbance (intensity of the color of the solution). Calculating “Mass of POXC for Unknown Soil Samples” can be done using the following equation, after Weil et al. (2003):

POXC (mg kg^-1 soil) = [0.02 mol/L - (a + b × Abs)] × (9000 mg C/mol) × (0.02 L solution/Wt)

Where: 0.02 mol/L = initial solution concentration; a = intercept of the standard curve; b = slope of the standard curve; Abs = absorbance of unknown; 9000 = mg of carbon oxidized by 1M of MnO4 changing from Mn⁷⁺ --> Mn⁴+; 0.02 L = volume of stock solution reacted; Wt = weight of air-dried soil sample in kg.

This produces the regression line: y = 0.0502x - 0.00004; R² = 0.999

Unknown sample absorbance: 0.3087; unknown sample soil weight: 2.48g

 

Limitations regarding estimating and interpreting:

TThis method requires significant equipment and supplies. The procedure is detailed, requiring someone with experience in wet chemistry. Results from this method should only be compared among soils from the same site under different management practices – it is not yet clear that it can be used to compare soils between sites.