Appendix II. Barton's reagent:

Transcription

1 Appendix II SOLUTIONS AND REAGENTS Barton's reagent: A. Dissolved 25 g ammonium molybdate in 400 ml de-ionized water (15 ). B. Dissolved 1.25 g ammonium metavanadate in 300 ml of boiling de-ionized water (15 ), cooled and then added 250 ml concentrated HNO 3. The solution A and B were mixed and volume made 1 litre with de-ionized water (15 ). Kovac s reagent: Isoamyl alcohol : ml p-dimethylaminobenzaldehyde : 10.0 g Hydrochloric acid : 50.0 ml Dissolved the aldehyde in alcohol and slowly added acid. Shaken gently before use. Methyl-red reagent: Dissolved 0.1 g methyl red in 300 ml ethanol and volume made to 500 ml with de-ionized water (15 ). Stored at room temperature (25±2 C). Phosphate standard solution (50 ppm): Dissolved g KH 2 PO 4 (AR Grade) in de-ionized water (15 ) to 1 litre. Chrome Azurol Sulphonate (CAS) reagent: CAS stock solution (2 mm) : CAS : g Fe stock solution (1 mm) : Ferric chloride anhydrous : 1.62 g Hydrochloric acid : 36.5 ml De-ionized water (15 ) : up to 100 ml Piperazine buffer: Piperazine : De-ionized water (15 ) : 30 ml ph : 5.6 with HCl HDTMA solution: HDTMA : g

2 Mixed 1.5 ml iron solution with 7.5 ml CAS solution and added to HDTMA solution in a 100 ml measuring cylinder. Added piperazine solution and the volume made to 100 ml with de-ionized water (15 ). Stored in amber coloured bottle at 4 C. Hoagland nutrient solution: Calcium nitrate : 4.7 g Magnesium sulphate : 2.6 g Potassium nitrate : 3.3 g Ammonium dihydrogen phosphate : 0.6 g Solution A : 5.0 ml Solution B : 0.5 ml De-ionized water (15 ) : 500 ml Solution A: Boric acid : 280 mg Manganous sulphate : 340 mg Copper sulphate : 10 mg Zinc sulphate : 22 mg Ammonium molybdate : 10 mg Solution B: Sulphuric acid (95-98%) : 0.5 ml Salkowski reagent: Perchloric acid (35%) : 49 ml Ferric chloride (0.5 M) : 1.0 ml Ferric chloride (0.5 M): Ferric chloride anhydrous : 8.11 g De-ionized water (15 ) : ml Prepared fresh when required. 1-aminocyclopropane-1-carboxylate (ACC, 0.5 M): Dissolved 1.62 g ACC in 10 ml de-ionized water (15 ) and stored at -20 C. 2,4-dinitrophenylhydrazine reagent: Dissolved 0.2 g 2,4-dinitrophenylhydrazine in 2 N hydrochloric acid. Stored in amber coloured bottle at 4 C. X

3 Hydrochloric acid (HCl, 1 N): Added 8.33 ml of 12 N HCl (standard HCl) in 50 ml de-ionized water (15 ) in a 100 ml measuring cylinder and volume made to 100 ml with de-ionized water. Stored at room temperature (25±2 C). HCl (0.56 N): Added 4.67 ml of 12 N HCl (standard HCl) in 50 ml de-ionized water (15 ) in a 100 ml measuring cylinder and volume made to 100 ml with de-ionized water. Stored at room temperature (25±2 C). Tris HCl (0.1 M, ph 7.5): Mixed g Tris base (hydroxymethyl)-aminomethane) with 800 ml de-ionized water (15 ) and slowly added 65 ml 12 N HCl. Adjusted ph to 7.5 and volume made 1 litre with deionized water (15 ). Sodium hydroxide (NaOH, 2 N): Dissolved 80 g NaOH in de-ionized water (15 ) and volume made to 1 litre. NaOH (0.1 N): Dissolved 4 g NaOH in de-ionized water (15 ) and volume made to 1 litre. Nitrite-molybdate reagent: Dissolved 10 g sodium nitrite and 10 g sodium molybdate in 50 ml de-ionized water (15 ) in a 100 ml measuring cylinder and volume made to 100 ml with de-ionized water. Ferric perchlorate reagent (Atkin s reagent): Dissolved g ferric chloride in 100 ml de-ionized water (15 ) and added 1.43 ml perchloric acid. Stored in amber coloured bottle at room temperature (25±2 C). Reagents for Fatty Acid Methyl Ester (FAME) analysis Reagent 1 (Saponification): Sodium hydroxide : 45 g Methanol (HPLC Grade) : 150 ml De-ionized water (15 ) : 150 ml Reagent 2 (Methylation): Hydrochloric acid (6 N) : 325 ml XI

4 Methanol (HPLC Grade) : 275 ml Reagent 3 (Extraction Solvent): Hexane (HPLC grade) : 200 ml Methyl tert-butyl ether (HPLC grade) : 200 ml Reagent 4 (Base Wash): Sodium hydroxide : 10.8 g De-ionized water (15 ) : 900 ml IPTG (Isopropyl- -D-thiogalactopyranoside) stock solution (0.1 M): To 1.2 g IPTG added de-ionized water (15 ) to 50 ml final volume. Filter sterilized and stored at 4 C. X-Gal (5-bromo-4-chloro-3-indolyl- -D-galactopyranoside): Dissolved 100 mg X-Gal in 2 ml N, N -dimethyl-formamide. Covered with aluminium foil and stored at -20 C. Reagents for total and available N, P and K contents of soil and plant material Potassium permanganate (KMnO 4, 0.32%): Dissolved 3.2 g KMnO 4 in de-ionized water (15 ), volume made to 1 litre in volumetric flask and mixed well. Stored at room temperature (25±2 C). Sodium hydroxide (NaOH, 2.5%): Dissolved 25 g NaOH flakes in de-ionized water (15 ), volume made to 1 litre in a volumetric flask and mixed well. Stored in a plastic container at room temperature (25±2 C). Sulphuric acid (0.02 N or N/50): standard sulphuric acid Boric acid (H 3 BO 3 ) indicator: Dissolved 20 g pure H 3 BO 3 in 700 ml hot de-ionized water (15 ) and transferred the cooled solution to 1 litre volumetric flask containing 200 ml ethanol and 20 ml mixed indicator. After mixing the contents of the flask, added 0.05 N NaOH cautiously until the colour turned reddish purple. Diluted the solution to 1 litre volume with de-ionized water, mixed thoroughly and stored at room temperature (25±2 C). Mixed indicator: Dissolved 0.07 g methyl red with 0.1 g bromocresol green in 100 ml 95% ethanol. XII

5 Olsen s reagent (sodium bicarbonate 0.5 M, ph 8.5): Dissolved 42 g sodium bicarbonate in 950 ml de-ionized water (15 ). Adjusted the ph to 8.5 using 1 N sodium hydroxide (4 g NaOH/100 ml) and volume made to 1000 ml with de-ionized water. Stored in a glass bottle at room temperature (25±2 C). Reagent A: Dissolved 12 g ammonium molybdate in 250 ml de-ionized water (15 ). Dissolved g antimony potassium tartrate in 100 ml de-ionized water (15 ). Added the two solutions to 1000 ml 2.5 M sulphuric acid, mix thoroughly and volume made to 2000 ml with de-ionized water (15 ). Stored in an amber coloured bottle at 4 C. Reagent B: Dissolved g ascorbic acid in 200 ml reagent A and mixed well. Prepared daily as required. Sulphuric acid (2.5 M): Added 140 ml sulphuric acid to 700 ml de-ionized water (15 ) in 1 litre measuring cylinder and volume made to 1 litre with de-ionized water. Stored in a glass bottle at room temperature (25±2 C). Standard stock phosphorus solution: Dissolved potassium dihydrogen phosphate in 500 ml de-ionized water (15 ). Added 25 ml 7 N sulphuric acid and made to 1 litre with de-ionized water. This gives 100 ppm P standard stock solution. From this, a 2 ppm solution is made by diluting it 50 times. Ammonium acetate (1 N, ph 7.0): To 700 ml de-ionized water (15 ), added 57 ml 99.5% glacial acetic acid and 69 ml ammonium hydroxide. Diluted to a volume of 900 ml, adjusted ph to 7.0 using 3 N ammonium hydroxide and volume made to 1 litre. Stored in a Pyrex bottle at room temperature (25±2 C). Standard potassium solution: Dissolving g potassium chloride in de-ionized water (15 ) and volume made to 1 litre. This gives 1000 ppm K standard stock solution. Prepared 100 ppm K solution by diluting 100 ml of 1000 ppm stock solution to 1 litre with de-ionized water. Stored in a glass bottle at room temperature (25±2 C). XIII

6 Sulphuric-salicylic acid: 1 g salicylic acid dissolved in 30 ml sulphuric acid. Sulphate mixture (digestion mixture): 20 parts potassium sulphate and 1 part catalyst mixture (20 parts copper sulphate and 1 part selenium powder). ACC and X-gal were from Sigma, all other chemicals were from Hi-Media. All chemicals used were of AR Grade unless stated otherwise. XIV