Calculate the factor F from the expression:
E. SPECIAL REAGENTS 1. DE-IONIZED WATER
6. OXIDIZING REAGENT
Add 0.5 ml of sodium hypochlorite solution to 100 ml of alkaline sodium citrate.
This solution should be prepared immediately before use and not stored for more than 3 hours. Prepare multiples of the above solutions according to the number of samples to be analysed per batch (10 ml of oxidizing reagent per sample).
11.8. AMMONIA PLUS AMINO ACIDS 7. ACIDIFYING SOLUTION.
Dilute analytical reagent quality concentrated hydrochloric acid with an equal volume of de-ionized water.
Add to a 125-ml Erlenmeyer flask about 50 ml of distilled water, pipette into this 10.0 ml of oxidizing agent, and add 2 ml of sodium arsenite solution. Add one or two drops of thymol blue indicator solution (0.1% solution in distilled water) and titrate the mixture carefully with the diluted hydrochloric solution until the color changes from blue to pink (ca. pH 1.7). Carry out titrations in duplicate. Titrations should agree to better than 0.1 ml of acid and the mean volume should be recorded to the nearest 0.05 ml. If x ml of acid is used (about 5-6 ml) dilute 200x ml of acid to exactly 2000 ml with deionized water using a measuring flask. This solution must be prepared fresh whenever a new alkaline sodium citrate solution is used.
8. SULPHANILAMIDE SOLUTION.
Prepare as described in Part 11.6, E.4.
9. N- (l-NAPHTHYL)-ETHYLENEDIAMINE DIHYDROCHLORIDE SOLUTION.
F. EXPERIMENTAL
Section G2 should be consulted before commencing this determination.
PROCEDURE
1. Add 50 ml of sample to an Erlenmeyer flask from a 50 ml measuring cylinder.
Add 10 ml of oxidizing reagent from a pipette, swirl the solution, and allow the flask to stand at a temperature between 20 and 25°C for 10 minutes (note a).
2. Add 2 ml of sodium arsenite solution from an automatic pipette and mix the contents of the flask (note b). Add 10 ml of acidifying solution from a pipette and mix (note c).
3. After as short an interval as possible, to lessen the chances of atmospheric con-tamination, add 1 ml of sulphanilamide solution from an automatic pipette. Swirl the contents of the flask. After a further 3-8 minutes add 1.0 ml of naphthylethylene-diamine solution from an automatic pipette and mix immediately (note d). Between 10 min and 2 hours afterwards, measure the extinction of the solution in a 10-cm cell against distilled water at a wavelength of 5430 A. If the extinction exceeds about 1.3 (rarely) measure the extinction with a 5-cm cell and double the reading so obtained.
Unless adjacent samples are known to have extinction values within about 25% of each other, the absorptiometer cell should be rinsed with each new solution before filling.
4. Correct the measured extinction by that of a reagent blank (Sect. G) and calculate the ammonia-nitrogen concentration (plus some amino-acid nitrogen (see note e) from the expression:
0.70 X
I
N /liter F [E
F'
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84 A PRACTICAL HANDBOOK OF SEAWATER ANALYSIS
where E is the corrected extinction, F is the factor obtained as described in Section H below, C is the concentration of nitrite in the same sea water, expressed in µg-at N/liter, obtained by a separate analysis as described in Part 11 .7 . F' may be assumed to be equal to 2.1 with little error if a spectrophotometer is used (note f) .
NOTES
(a) The time required for maximum oxidation depends on salinity and temperature . The oxidation requires a full ten minutes after which stable results are obtained . The sensitivity in dis-tilled water is slightly less than in sea water but the difference is sufficiently small that it can be neglected when making a`blank' determination . Bromide acts as a catalyst and must be added to distilled water or synthetic bromide-free sea water .
(b) Arsenite is added to destroy excess hypochlorite without reducing nitrite . The reaction is very rapid but for safety about 2 minutes should be allowed for the reaction to be completed before adding the acidifying solution .
(c) If sulphanilamide is present at this stage, as in the original Richards and Kletsch method and the version of this method given in the 2nd Edition of Bulletin 125 (Fish. Res. Bd . Canada, 1965), an appreciable fraction of nitrite is decomposed and the method becomes less sensitive and more erratic .
(d) The determination is from now on the same as that for nitrite described in Part 11 .7. except that both sample volume and acidity are rather greater .
(e) The interference from urea and amino acids is less than in the original Richards and Kletsch method but may be up to 40 percent of the amino nitrogen of some compounds.
(j) Any nitrite initially present in the sample is unchanged by the analytical procedure, so a correction for its presence can be made in the manner shown . In this calculation allowance is made for the fact that the sample is diluted from 52 to 74 ml by reagents before extinction measurements are made and that only a fraction of the ammonia is converted to nitrite . This correction can be quite significant in water containing relatively large amounts of nitrite but little ammonia .
G. DETERMINATION OF BLAN K
1 . REAGENT BLANK .
Carry out the method exactly as described in Section F, paragraphs 1-3, using 50 ml of freshly de-ionized water and 1 ml of sodium bromide solution to act as a catalyst . A blank determination in triplicate must be carried out with each batch of samples being analysed . Blank extinctions using a 10-cm cell should not exceed 0 .15 .
2 . PRECAUTIONS TO REDUCE CONTAMINATION .
The greatest care is necessary to prevent the contamination of reagents and samples by ammonia (carried as gas or particles of ammonium salts) in the laboratory . Solutions should be kept in tightly stoppered bottles, except when in use, and samples should be stored in well-stoppered containers until the analysis commences . Under no circumstances must a bottle of ammonium hydroxide be opened in the laboratory, for however brief a period, whilst analyses for ammonia are being carried out . All glassware must be washed initially by dilute acid and rinsed very thoroughly with distilled water immediately prior to every use . Ordinary distilled water is satisfactory for this rinsing.
H . CALIBRATIO N
Carry out the calibration using filtered sea water .
AMMONIA PLUS AMINO ACIDS 85 1. STANDARD AMMONIA SOLUTION.
Dissolve 0.100 g of analytical reagent quality ammonium sulphate in 1000 ml of distilled water. Add 1 ml of chloroform and store sheltered from strong light. The solution is stable for many months if well stoppered.
1 ml 1.5 tg-at N
Pipette 1.00 ml of this solution into a 500-ml flask and complete with sea water to the mark. The resulting ammonium concentration is equivaknt to 3.0 tg-at N /liter.
2. PROŒDURE.
Measure out 50-ml portions of this dilute ammonia solution into each of four clean 125-ml Erlenmeyer flasks and take two 50 ml portions of the same sea water that was used to make the diluted standard solution into two additional clean 125-ml Erlenmeyer flasks. Carry out the determinations on all, exactly as described in Section F, paragraphs 1-3.
Evaluate the factor F from the expression:
F— 3.0 Es — Eb
where Es is the mean extinction of the standards and Eb is the mean extinction of the blanks. The value for F should be near 4.5.
11.9. DETERMINATION OF AMMONIA INTRODUCTION
The following method is specific for ammonia and, although not as sensitive as the procedure described in 11.8., should be used when an estimate of the concentra-tion of ammonia alone is desirable. The technique is a sensitive modificaconcentra-tion of the phenol-hypochlorite method used by many workers (e.g., Riley, Anal. Chim. Acta., 9:
575, 1953; Emmet, Naval Ship Res. and Devel. Cent. Rept. 2570, 1968) by L. Solorzano (Limnol. Oceanogr. 14: 799. 1969).
A. CAPABILITIES
METHOD
Range: 0.1-10 µg-at/liter 1. PRECISION AT THE 3 11G-AT /LITER LEVEL
The correct value lies in the range:
Mean of n determinations ± 0.15 In µg-at /liter.
2. PRECISION AT THE 1.0'.LG-AT/LITER LEVEL
The correct value lies in the range :
Mean of n determinations ± 0.10 In µg-at /liter.
3. LIMIT OF DETECTION
The smallest quantity of ammonia nitrogen that can be detected with certainty by a single determination is about 0.1 µg-at/liter.
Reject duplicate determinations if extinction values differ by:
more than 0.015 in the extinction range 0.1 to 0.2, more than 0.025 in the extinction range 0.2 to 0.5.
If duplicate extinction values differ by less than the above limits, take a mean value.
B. OUTLINE OF METHOD
The sea water is treated in an alkaline citrate medium with sodium hÿpochlorite and phenol in the presence of sodium nitroprusside which acts as a catalyzer. The blue indophenol formed with ammonia is measured using a 10-cm cell.
C. SPECIAL APPARATUS AND EQUIPMENT
125-ml Erlenmeyer flasks. These must be rinsed copiously with distilled water and drained immediately before use.
D. SAMPLING PROCEDURE AND SAMPLE STORAGE
Temporary storage of sea water prior to analysis appears to be satisfactory in either glass or polyethylene but the analysis should not be delayed for more than 1-2 hr at the most. If longer storage periods are necessary freeze the samples solid in a deep-freeze. There are indications that, even with refrigeration, losses or gains may be significant after more than a few days. Although more evidence is required it would seem desirable not to store samples.
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E. SPECIAL REAGENTS