Hello! I have been preparing synthetic urine at various pHs. I am struggling to prepare a synthetic urine at pH 9 with a Tris-HCl buffer, because there is always precipitate! I have tried the following & gotten the corresponding results:
Trial 1: 100 mM Tris-HCl buffer in synthetic urine
ingredients: 1.34g urea, 800mg NaCl, 164mg KCl, 242.5mg monobasic sodium phosphate, 263mg potassium sulfate, 29mg potassium bicarbonate, 150mg creatinine, 78mg magnesium sulfate, 51mg citric acid, 3.80 uL of 25% ammonium hydroxide, 1.1013g Tris, 151.5uL 6N HCl.
outcome: precipitate formed (cloudy); heating & stirring only made it worse (cloudier).
Trial 2: 100 mM Tris-HCl buffer in synthetic urine with some reduced organic matter (i.e. 1/3 the concentration of urea)
ingredients: 0.0932g urea, 800mg NaCl, 164mg KCl, 242.5mg monobasic sodium phosphate, 263mg potassium sulfate, 29mg potassium bicarbonate, 150mg creatinine, 78mg magnesium sulfate, 51mg citric acid, 3.80 uL of 25% ammonium hydroxide, 1.1013g Tris, 151.5uL 6N HCl.
outcome: precipitate formed (cloudy); heating & stirring only made it worse (cloudier).
Trial 3: 50mM Tris-HCl buffer in synthetic urine with highly reduced organic matter (i.e. 1/3 urea concentration, 1/4 creatinine concentration, and 1/4 citric acid concentration)
ingredients: 0.0932g urea, 800mg NaCl, 164mg KCl, 242.5mg monobasic sodium phosphate, 263mg potassium sulfate, 29mg potassium bicarbonate, 104mg creatinine, 78mg magnesium sulfate, 30mg citric acid, 3.80 uL of 25% ammonium hydroxide, 0.5507g Tris, 75.8uL 6N HCl.
outcome: precipitate formed (just a few granules of salts remained undissolved!); heating & stirring only made it worse (became cloudy!).
My next trial would be to repeat Trial 3 without heating or stirring and just letting the solution sit. However, I don't think 50mM will be concentrated enough of a solution for me. I've tested the buffering capacity at 40mM and it wasn't enough. If anyone can suggest any tips or recommend a different buffer/formulation for preparing BASIC synthetic urine, I would greatly appreciate it!
- The mass of the ingredients is not the relevant factor, but the concentration (mass/volume).
- In particular, you have to consider the co-solubility of bivalent metal ions like Mg2+ with anions like carbonate or phosphate (solubility product). In particular in the presence of NH4+, Mg will precipitate as mixed salt, NH4MgPO4.
- At higher pH, Mg2+ will precipitate as hydroxyde. This is particularly true is you change the pH of buffer by adding base, locally produced precipitates may not re-dissolve once formed.
- Urine also may contain precipitates or crystals.
I am wondering about the use of 25% ammonium hydroxide. What is your pH? it must be rather high.
Should this perhaps be just 2.5% or even less?
Sorry for delayed response. The magnesium was most definitely the culprit! Thanks Engelbert Buxbaum !
Achim Recktenwald , the pH came out to ~8.31, but this was only after adding the Tris in the Tris-HCl buffer. Before adding the Tris, the pH was actually quite acidic from all the HCl, citric acid, etc.
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