The method described is a modified semi-micro Kjeldahl procedure (Blakemore et al. 1987) in which digestion occurs in 50 mL calibrated test tubes in a drilled Al metal block on a hotplate. Concentrated H2SO4 plus anhydrous sodium sulfate (Na2SO4) is used to raise the distillation temperature to ≈375–400°C, with Cu as a catalyst. The effect over a few hours is to convert complexed N in soil samples to NH4-N. Use of a heating block with a maximum temperature option of 400°C helps prevent possible N loss from overheating.
Figure 7.4. Soil total N automated colour continuous segmented flow sheet.
Ammonium and any free amino acid in the digest solution (after making to volume) are determined by the colorimetric Berthelot reaction utilising FIA. Such systems typically operate at high sensitivity. This automated technology involves introducing, under the force of a peristaltic pump, a precisely measured volume of liquid sample from the sampler into a continuously flowing carrier stream. An injection valve with a fixed-volume sample loop or an injection valve with a fixed time period determines the injected volume of sample. A carrier stream sweeps the sample out of the loop and into the manifold where it disperses. The concentration gradient formed is detected continuously by (in this case) a colour reaction that occurs in the presence of continuously flowing reaction solutions. Refer to Section 4130 of APHA 2005 for further details of the technology.
Specifically, NH4+ reacts with hypochlorite ions that are generated in situ by alkaline hydrolysis of sodium dichloroisocyanurate. This reaction forms monochloramine, which then reacts with salicylate ions in the presence of sodium nitroprusside to form a blue, indophenol-type compound that absorbs strongly at 660 nm. A citrate buffer is employed to chelate metals that would otherwise form insoluble hydroxides or carbonates. Free amino groups that can react are most likely to be present in soils that have been fumigated or dried at elevated temperatures (Burton et al. 1989).
A reasonable but incomplete estimate of total soil P can also be determined following Kjeldahl digestion, as used in this method. Accordingly, standard solutions include P as well as N. Potassium can also be included; see Note 1.
Digestion Tubes
Heat resistant, borosilicate glass tubes 2.5 cm dia and carefully calibrated at 50 mL.
Solid Al metal block (30 cm × 30 cm × 5 cm) drilled with seven rows of six holes (2.7 cm dia, 3.5 cm depth), or a convenient alternative.
Hotplate
One option is a domestic stove-style single radiant element hotplate about 20 cm dia and an output of about 2 kW, mounted on a steel frame and fitted with a simmerstat for temperature control. The unit or the control setting should not permit temperatures in digest solutions >400°C.
FIA Instrumentation
A Lachat™ system is described (Quikchem Methods Manual, undated). As with segmented flow instrumentation, the analyst must be guided by the operational instructions and directions given by the particular FIA manufacturer. This could require changes to the specified reagents and to the flow diagram (Figure 7.5) provided for this method.
Digest Acid
Commercial grade sulfuric acid (H2SO4; 18 M).
Kjeldahl Copper Catalyst Tablets
BDH Cat No. 33064. Each tablet contains a mixture of 1 g anhydrous sodium sulfate (Na2SO4) and 0.1 g anhydrous copper sulfate (CuSO4).
Kjeldahl Matrix Solution
Carefully add 120 mL of 18 M H2SO4 to 1500 mL of water in a 2 L conical flask. Add 40 g anhydrous sodium sulfate (Na2SO4), 4 g anhydrous copper sulfate (CuSO4), and dissolve. Make to 2.0 L with deionised water.
Buffer Solution
To a plastic 1.0 L bottle add 65 g sodium hydroxide (NaOH), 50.0 g potassium sodium tartrate tetrahydrate (NaKC4H4O6.4H2O), and 14.2 g anhydrous sodium phosphate dibasic (Na2HPO4) and make to 1.0 L with deionised water. Shake to fully dissolve.
Salicylate–Nitroprusside Solution
To an amber glass bottle add 150 g sodium salicylate [C6H4(OH).COONa] and 1.0 g sodium nitroprusside [Na2Fe(CN)5NO.2H2O] and make to 1.0 L with deionised water. Shake to fully dissolve. Prepare fresh monthly.
Hypochlorite Solution
To a plastic 1 L bottle add 30 mL of commercial bleach (≈5.25% NaOCl) and make to 500 mL with deionised water. Prepare fresh weekly and keep cool when not in use.
Kjeldahl Ammonium, Phosphate and Potassium Primary Standard
1 mL contains 0.5, 0.1 and 0.5 mg of NH4-N, PO4-P, and K (as KCl), respectively; see Note 1.
Carefully weigh and dissolve separately in Kjeldahl Matrix Solution: (i) 2.3585 g ammonium sulfate [(NH4)2SO4; previously dried at 100°C for 4 h]; (ii) 0.4583 g disodium hydrogen phosphate [Na2HPO4; previously dried at 105°C for 24 h], and (iii) 0.9534 g potassium chloride [KCl; previously dried at 110°C for 2 h]. Add each while mixing into a 1.0 L standard flask and make to volume, again with Kjeldahl Matrix Solution. Add 2–4 drops of chloroform (CHCl3) to suppress biological activity and store in a borosilicate container for up to 6 months.
Kjeldahl Combined N, P and K Working Standards (KCWS)
Carefully and accurately transfer into 250 mL volumetric flasks 2.5, 5.0, 12.5, 25.0, and 50.0 mL of Kjeldahl Combined Ammonium, Phosphate and Potassium Primary Standard and make each to volume with Kjeldahl Matrix Solution. These KCWS contain 5.0, 10.0, 25.0, 50.0 and 100 mg N (as NH4) and mg K/L, and 1.0, 2.0, 5.0, 10.0, and 20.0 mg P/L (as PO4).
Weigh 0.50 g (0.1–0.2 g for peats or litters) of finely ground (<0.5 mm) air-dry (≈40°C) sample into clean, dry 50 mL calibrated test tubes, and note the actual sample weight. Include reagent (digest) blanks (see Note 1, Method 7A1) and control samples (known TSN content) at a frequency of two of the former to one of the latter for each batch of around 40–50 digest tubes.
Moisten the weighed soil samples with a few drops of deionised water, allow the moisture to penetrate, then add one Kjeldahl Copper Catalyst Tablet, 3.25 mL 18 M H2SO4, then swirl gently to mix and place on the preheated Al heating block. (Soils high in OM need careful watching to prevent loss of sample by frothing. If this occurs remove tube from rack to allow froth to settle then replace and resume digestion.)
Boil the Kjeldahl digestion mixture until it decolorises (usually a pale greenish-brown or off-white colour) and then carry on with the digestion for a further 20–30 min.
Next remove all digestion tubes from the Al heating block then 5–6 min later (to permit some cooling), carefully add 10–15 mL deionised water and swirl to dissolve any partly dried digest mixture that may have adhered to sides of the digestion tubes. When cooled to room temperature, make to volume (50 mL) with deionised water, stopper (this stopper should be inert), and shake vigorously to fully mix/dissolve the water-soluble contents then allow any solids to settle out.
In preparation for FIA, transfer a sub-sample from each digestion tube (volume will be determined by specifications of the autosampler; e.g. 15 mL).
Set up the FIA equipment as shown in the flow diagram (Figure 7.5), noting instrument selections and settings should be in accord with manufacturer’s instructions. For example, switch on the computer, load the software, load the sampler, inject/pump water through all reagent lines and check for leaks and smooth flow. Switch to reagents and allow the system to equilibrate for a few minutes. Ensure buffer flow is well established before pumping Salicylate–Nitroprusside Solution (it can precipitate unless it goes into an alkaline stream). Run Working Standards and sample digest solutions as necessary. Degas reagents if necessary.
where:
a = N in sample digest, expressed as mg/L
b = N in digest blank (mg/L)
V = final volume of sample digest (mL, usually 50 mL)
Figure 7.5. Soil total-N automated FIA flow sheet.
W = sample weight (g)
10–4 = factor to convert mg/kg to g/100g (%)
For a 0.5 g air-dry sample and a 50 mL final digest volume:
TSN (% N) = [(a – b) × 0.01]
Report TSN (%N) on an oven-dry basis. Use the air-dry moisture to oven-dry moisture ratio to make the oven-dry conversion. Refer to Method 2A1 for guidance with regard to this soil moisture calculation.
1. While K is included in both the Primary Standard and in Working Standards, this does not infer the resultant estimate of Total Kjeldahl K is a good approximation of total soil K. Indeed, recoveries can be as low as 5–10% of the true total.
