Title: Determination of nitrogen (ammonia) using the manual phenate method

Objective:

Determination of concentration of NH4 –N/L samples provided from the plotted graph

(absorbance against concentration of ammonium mg/L) using the indophenol method.

Introduction:

In this experiment, the phenate method was used in order to develop the indophenol blue color in

the samples provided. In this particular method, ammonia combines with hypochlorite ions

(OCl-) to form mono chloramine (NH2Cl) which then reacts with phenate to form 5-

aminophenate.The 5-aminophenate is oxidized in the presence of a catalyst which is the sodium

nitroprusside, which results in the formation of indophenol (a blue-colored compound) which in

turn its absorbance was detected in a spectrometer and from a calibration curve obtained from

the ammonium standard solutions, the concentration of NH4-N/L found in the sample was

obtained.

Chloramination:

Phenate Reaction:

Color Formation:

Structure of indophenol:

Risk Assessment

Phenol is highly corrosive and a strong irritant. Use gloves when handling. Sodium hypochlorite

also known as a bleaching agent is corrosive. The former in liquid state and gas are harmful.

Hence it must be used in fume hood. Sodium hydroxide is hygroscopic and tri-sodium citrate as

well as ammonium chloride irritates eyes and the skin. Wear appropriate gloves, goggles and lab

coats.

Glassware: 5 volumetric flasks of 100 ml, 9 conical flasks of 50 ml, pipette, burette, measuring

cylinder and beakers.

Access to: Electronic balance, fume hood, spectrophotometer

Reagents: Liquid phenol, sodium nitroprusside solid, tri-sodium citrate solid, sodium hydroxide

pellets, sodium hypochlorite, ammonium chloride solution and ethanol solution.

Procedure:

1. Preparation of Reagents

A. Phenol solutions

11.1 ml liquefied phenol was dissolved in 95% ethanol in a 100 ml volumetric flask and made up

to the mark.

B. Sodium nitroprusside

0.5 g of sodium nitroprusside was dissolved in 100 ml deionized water in a volumetric flask.

C. Alkaline citrate

100 g tri-sodium citrate was dissolved with 5 g sodium hydroxide in 500 ml of deionized water.

D. Sodium hypochlorite

It was obtained by the lab technician. It is commercially available about 5%.

E. Oxidizing solution

100 ml of alkaline citrate solution was mixed with 25 ml of sodium hypochlorite and made up to

the mark in a 500 ml of volumetric flask.

F. Stock ammonium solution

3.819 g of anhydrous ammonium chloride was dissolved in 1000 ml of distilled water. This

solution was equivalent to 1000 mg NH4- N/L.

G. Standard ammonium solution

The stock solution was used to prepare a series of standard ammonium solutions in the next step.

2. Preparation of ammonium standards and to obtain color for absorbance

i. A series of standard solutions was prepared covering then concentrations of 1000,

100, 10, 1, 0.1 mg NH4-N/L by making appropriate dilutions with deionized water.

The standards were prepared in 100 ml of volumetric flask.

Standard Name Concentration / mgL-1 Volume of stock

ammonium solution/

cm3

Volume of deionized

water needed for

dilution/ cm3

A 1000 100 0

B 100 10 90

C 10 1 99

D 1 0.1 99.9

E 0.1

Table 1 shows the preparation of different standards with different concentrations.

Note: Sample number E was not carried out since the volume was too small to be measured with

a burette.

ii. To a 25 ml sample (standard ammonium solution) in a conical flask,

a. 1ml of phenol solution,

b. 1ml of sodium nitroprusside solution and

c. 2.5ml of oxidizing solution were added, was added with constant mixing.

The sample was then covered with Para film and the color was allowed to develop for 1 hour.

Observation

A blue color was developed ranging from dark blue to pale blue. When the absorbance of these

samples was measured at 640 nm, the spectrophotometer was unable to read for standard A,B,C

and D as they had the highest concentration of nitrogen as their color was the darkest.

Dilution of A, B, C and D.

From the conical flask, 5 ml of A was taken using a pipette and transferred to its respective 100

ml volumetric flask. Deionized water was added till the mark.

10 ml of B, C and D was diluted to 100 ml water up to the mark. Then the absorbances were

measured using deionized water as reference.

Sample Name Absorbance Corrected Absorbance Concentration of

NH4–N/L

Blank 2 0.174 0 0

A 0.607 0.433 50

B 0.433 0.259 10

Blank 2 0.164 0 0

C 0.216 0.052 1

D 0.222 0.058 0.1

River water 0.227 0.063

Tap water 0.252 0.088

Sea water 0.169 0.005

Table 2 shows different concentrations and its absorbances.

Calculations:

1. Concentration of NH 4-N/L in the standard ammonium solutions

Using m1V1 = m2V2

For example A: 1000 x V1 = 1000 x 100

V1 = (100 x 1000) / 1000 = 100 ml

2. Concentration of NH 4-N/L in samples after dilution and color has been developed

For A: 1000 ml of solution of A = 1000 x 10-3 mol

5 ml of solution of A = 5 x 10-3 mol

Thus in 100 ml of solution after dilution = 5 x 10-3 mol

1000 ml of solution = 0.05 mol = 50 mg/L

The above calculation was then repeated for B, C and D.

Example for B:

For B: 1000 ml of solution of A = 1000 x 10-3 mol

10 ml of solution of A = 10 x 10-3 mol

Thus in 100 ml of solution after dilution = 10 x 10-3 mol

1000 ml of solution = 0.10 mol = 10 mg/L

Analysis and Discussion:

1) Graph of corrected absorbance against concentration of NH4 –N/L

In this method adherence to Beer’s law was studied by measuring the absorbance values of

solutions varying nitrite concentration. A straight line graph was obtained by plotting absorbance

against concentration of nitrite. Beer’s law was said to be obeyed in the concentration range

NH4-NL-1 of ammonium. Adherence to Beer’s law graph for the determination of ammonium

using nitroprusside was presented below. The molar absorptivity of the method was found to be

approximately 0.00866 L mg-1 cm-2. The correlation coefficient, detection limit (DL=

0.926634791 σ/S), where σ is the standard deviation of the regent blank (n= Blank) and ‘S’ is the

slope of the calibration curve of the nitrite determination was found to be 15.69884949 μgmL-1

and 0.007817952 μgmL-1 respectively.

0 10 20 30 40 50 600

0.10.20.30.40.5

f(x) = 0.00934167880938558 xR² = 0.870871643331705

Corrected Absorbance against Concentration of NH4-N/L

Corrected AbsorbanceLinear (Corrected Absorbance)

Concentration of NH4-N/L

Corrected

Absorbance

Graph 1 shows a corrected absorbance against concentration of NH4-N/L

Correlation Factor 0.926634791Slope 0.007817952

Standard deviation 15.69884949Average 6.1902Median 0.1795Variance 246.4538753

0 10 20 30 40 50 600

0.10.20.30.40.5

f(x) = 0.00934167880938558 xR² = 0.870871643331705

Corrected Absorbance against Concentration of NH4-N/L

Corrected Absorbance

Linear (Corrected Absorbance)

Linear (Corrected Absorbance)

Concentration of NH4-N/L

Corrected

Absorbance

Graph 2 shows corrected absorbance against concentration of NH4-N/L with standard error bars

and forecasted calibration line (maroon color)

F- test 3.03953E-08

T -test0.13932525

7

The concentrations of the samples provided were calculated as follows:

Absorbance = 0.0093 x concentration + 0.000

Concentration = (Absorbance – 0.000) / 0.0093

Concentration mg/L AbsorbanceRiver water 6.77 0.063Tap water 9.46 0.088Sea water 0.54 0.005

The concentration of NH4-N/L of the following samples above was found to be in the range 5 –

50 mg/L except the concentration found in the sea water sample. In the samples having the

concentration of ammonium within the range, the nitrogen was said to be easily assimilable to

marine organisms and the concentration in the tap sample was said to be within tolerance limit

for consumption. (Tolerance limit of nitrogen in drinking water is below 10 mg/L).

If nitrogen level is greater than 10 mg/L, it is said to cause methemoglobinemia. The tolerance

level of nitrogen in sea water is below 1 mg/L. Reverse osmosis can be done to remove excess

nitrogen where pressure is applied to water to force it through a semi permeable membrane. As

water passes through, the membrane filters out the most of the impurities. It is said that 85-95%

of ammonium was removed from this process.

Effect of Divers Ions

The effect of various non-target species on the determination of nitrite and nitrate were

determined. The studies revealed that Mg (11) and Ca (II) can show severe interference which

was overcome by adding citrate which precipitates them at high pH. Other interferences can be

Glycine, urea, glutamic acid, cyanates, and acetamide hydrolyze very slowly in solution on

standing but, of these; only urea and cyanates will hydrolyze on distillation at pH of 9.5.

Hydrolysis amounts to about 7% at this pH for urea and about 5% for cyanates.

The proposed method was applied to the quantitative determinations of ammonium in different

water samples. Statistical analyses of the results by t- and F-tests show that, there was no

significant difference in accuracy and precision of the proposed and reported method. The

precision of the proposed method was evaluated by replicate analysis of samples containing

ammonium at five different concentrations. The reagents provide a simple and sensitive method

for the spectrophotometric determination of ammonium. The proposed method has been

successfully applied to the determination of trace amounts of ammonium in different water

samples. This method was said to be applied successfully to the determination of trace amounts

of ammonia in pharmaceutical preparations.

Various instrumental methods such as Nessler can be used in the determination of ammonium

where the Nessler reagent (K2HgI4) reacts with ammonia under strong alkaline conditions to

produce a yellow-colored species where the intensity of the color is directly proportional to the

ammonia concentration.

2KsHgI4 + NH3 + 3KOH → Hg2OINH2 + 7KI + 2H2O

Another method is the Ion Selective Electrode (ISE) method.

Conclusion

The reagents provide a simple and sensitive method for the spectrophotometric determination of

ammonium. The reagents have the advantage of high sensitivity and low absorbance of reagent

blank. The developed method does not involve any stringent reaction conditions and offers the

advantages of color stability for about more than 2 hours. The proposed method has been

successfully applied to the determination of trace amounts of ammonium in different water

samples.

References:

NICHOLS, M.S. & M.E. FOOTE. 1931. Distillation of free ammonia from buffered

solutions. Ind. Eng. Chem., Anal. Ed. 3:311.

TARAS, M.J. 1953. Effect of free residual chlorination of nitrogen compounds in water.

J.Amer. Water Works Assoc. 45:47.

GRASSHOFF, K., EHRHARDT, M. and KREMLING, K., Methods of Seawater

Analysis (Verlag Chemie, D-6940 Weinheirn, 1983), 363-365.

Manualfor Oceanographic Observation (Oceanographic Society of Japan, 1985).