METTLER TOLEDO Titrators

+
NH +
4 NH
4
+
NH
4

Nitrogen Determination by
Kjeldahl Digestion
Application brochure 13
Editorial

Dear Reader

Here is the answer to the Kjeldahl analysis. But where is the question?

If your question concerns the classical nitrogen determination according to Kjeldahl, consisting of the
digestion, water steam distillation and titration steps, please have a look at Applications M120 and
M121. If you ask for a more elegant procedure, the explanation is provided in Applications M125 and
M126. On the other hand, please check Applications M122 and M123 if you're looking for a time
saver.
All these Applications explain 3 different techniques. Whereas the digestion step is the same, the
determination steps differ. The principles are presented and summarized in the Introduction.
Since each of the 3 techniques offers its special advantages and also carries drawbacks, the choice of
the procedure remains with the user. In order to help with the decision, the Applications give comments
and remarks and a Comparison chapter has been added. The brochure ends with a Conclusion and a
Literature list.
The Applications in this brochure have been performed with titrators of the METTLER TOLEDO
DL70 Line. The flexibility provided by this titrator line's unique Methods concept has been applied for
automated and convenient analyses. However, the fundamentals of the Applications can be smoothly
transferred by the interested user to titrators of the DL 50 Line. A lesser degree of automation must be
taken into account, but quality and reliability of the results remains high.
We thank the author of this brochure, Cosimo A. De Caro, for the comprehensive presentation of the
Kjeldahl technique and the long working hours he spent "cooking" and titrating in our Market Support
Laboratory.

We wish you successful Kjeldahl determinations !

Georg Reutemann Rolf M. Rohner

Manager Market Support AnaChem Marketing Manager

Page 2/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
Contents

Introduction ......................................................................................................................................... 4

M120 Titration according to Kjeldahl:

Test with Ammonium Sulfate Solution .................................................................................... 6

M121 Titration according to Kjeldahl:

Dairy Products ......................................................................................................................... 9

M122 Ammonia Determination by ISE:

Test with Ammonium Sulfate Solution .................................................................................. 12

M123 Ammonia Determination by ISE:

Dairy Products ....................................................................................................................... 14

M124 Calibration of the Ammonia Selective Electrode ................................................................... 17

M125 Determination of Nitrogen after Urotropine Reaction:

Test with Ammonium Sulfate Solution .................................................................................. 19

M126 Protein Determination in Dairy Products

by Reaction to Urotropine...................................................................................................... 22

Comparison of three techniques ........................................................................................................ 26

Conclusion ......................................................................................................................................... 28

Literature ........................................................................................................................................... 29

Additional literature ........................................................................................................................... 30

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 3/36
Introduction

The Kjeldahl nitrogen analysis is a well established technique and has become the standard method
for the quantitative determination of organic-bound nitrogen (Classical Kjeldahl Analysis, CKA).
The CKA technique is the standard procedure for protein determination in food (Literature: 10). The
analysis is based on three steps:

1) Kjeldahl digestion: The sample is strongly heated in a Kjeldahl digestion flask containing
98% sulfuric acid, H2SO4 , during 2-3 hours. Nitrogen is released
from organic material and is present in the digested solution as
ammonium sulfate, (NH4)2SO4 .

2) Distillation of ammonia: To determine the nitrogen content in the digested solution,

concentrated sodium hydroxide solution (30% NaOH) is added.
Ammonia (NH3) is generated according to:

(NH4)2SO4 + 2 NaOH 2 NH3 + Na2SO4 + 2 H2O

Ammonia is distilled using water steam and collected in a 1-2%

boric acid solution, H3BO3 :
2 NH3 + 4 H3BO3 (NH4)2B4O7 + 5 H2O

3) Titration: Ammonium tetraborate, (NH4)2B4O7 , can be titrated with

hydrochloric acid (HCl):
(NH4)2B4O7 + 2 HCl + 5 H2O 2 NH4Cl + 4 H3BO3

Literature: 1, 2, 3, 4, and 5.

The ammonia distillation step represents an additional source of error and work and leads also to an
increase of the analysis time. This step can be avoided by direct determination of nitrogen using
the two following alternative determinations:
• Ammonia determination NaOH reacts with ammonium sulfate in the digested solution to gene-
by ISE (NH3-ISE): rate ammonia (NH3), sodium sulfate (Na2SO4) and water (see step 2).
Thus, ammonia can be directly determined by using an ammonia gas-
sensing electrode (NH3-ISE). Ammonia molecules diffuse through
the membrane of the NH3-ISE leading to a change of the pH-value of
the inner electrolyte. The change is detected by a glass pH-electrode
mounted inside the ammonia selective electrode.
(Literature: see ref. 6 and 4)
• Urotropine reaction Nitrogen is determined by first reacting ammonium with 37%
(HMTA- Method): formaldehyde to give urotropine ( (CH2)6N4 , Hexamethylene-
tetramine, HMTA) in the digested solution:
2 (NH4)2SO4 + 6 HCHO (CH2)6N4 + 2 H2SO4 + 6 H2O
The generated sulfuric acid is determined by titration with NaOH.
(Literature: see ref. 7, 8, 9 and 4).
Page 4/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
In this application brochure, the classical Kjeldahl analysis (CKA), the direct ammonia determination
(NH3-ISE), and the urotropine reaction (HMTA) have been compared with respect to 1) accuracy
(expressed as recovery, x) and 2) reproducibility (rel. standard deviation, RSD).

Overview of the techniques

A) CKA 1) Kjeldahl digestion

2) Ammonia destillation with water steam
3) Titration with hydrochloric acid
simplified scheme: (NH4)3BO3 + 3 HCl 3 NH4Cl + H3BO3

B) NH3-ISE 1) Kjeldahl digestion

2) Direct determination by NH3-ISE

C) HMTA-Method 1) Kjeldahl digestion

2) Direct titration of ammonium
This determination consists of 4 steps:
- Neutralization to pH 3.2
- Titration to the equivalence point
- Addition of 6 ml 37% formaldehyde previously neutralized
- Titration with NaOH to neutralize the produced sulfuric acid.

Based on these three analytical procedures, titration methods have been developed to perform automated
nitrogen determination. Priority was given to the accuracy and the precision achievable rather than to
a fast analysis procedure. If necessary, the analysis time can be shortened by modifying the
corresponding control parameters.

First, the methods were tested using ammonium sulfate in sulfuric acid. Then the methods were
compared by determining the nitrogen and protein content in different dairy products (milk, cheese,
yoghurt...). The protein content was calculated by multiplying the nitrogen content by a factor f which
depends on the sample, e.g., f = 6.38 for dairy products.

Applications for nitrogen determination by Kjeldahl digestion

A) CKA 3) Titration with HCl M120 Test with (NH4)2SO4

M121 Dairy products

B) NH3-ISE 2) Direct measurement M122 Test with (NH4)2SO4

M123 Dairy products
M124 NH3-ISE Calibration

C) HMTA-Method 2) Direct titration of ammonium M125 Test with (NH4)2SO4

M126 Dairy products

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 5/36
METTLER TOLEDO Titration Application No. M120

Titration according to Kjeldahl:

Test with Ammonium Sulfate Solution

Sample: Water solution of ammonium Instruments: METTLER DL67 DL70ES DL77

sulfate 0.06 … 0.07 g/50 mL METTLER ST20A Changer
METTLER TOLEDO AT261
Substance: Ammonium sulfate (NH4)2SO4 Printer HP DeskJet 510
M = 132.14 ; z = 2 Water steam distilling unit
Preparation: – max. 0.07 g in 50 mL flask; Accessories: Titration beaker ME-101974
– 20 mL deion. water; Peristaltic Pump ME-65241
– 15 mL sulfuric acid,H2SO4(98%). 1 Burette drive DV90
Titrant: Hydrochloric acid, HCl Temp. sensor DT120 ME-89624
c(HCl) = 0.1 mol/L Indication: DG111-SC at Sensor 1
Standard: Tris(hydroxymethyl)amino-
methane (THAM),
0.05 …0.1 g / 50 mL H2O.
Results: 1/5 Amm.Bor. Test 10 50.0 mL
R1 = 0.3266
g Fixed
mmol/L
volume U
(NH4)3BO3
R2 = 0.6864 mg N
R3 = 13.7289 mg N Total
1/6 Amm.Bor. Test 10 50.0 mL g Fixed volume U
R1 = 0.3273 mmol/L (NH4)3BO3
R2 = 0.6878 mg N
R3 = 13.7561 mg N Total
1/7 Amm.Bor. Test 10 50.0 mL g Fixed volume U
R1 = 0.3260 mmol/L (NH4)3BO3
R2 = 0.6851 mg N
R3 = 13.7027 mg N Total
1/8 Amm.Bor. Test 10 50.0 mL g Fixed volume U
R1 = 0.3266 mmol/L (NH4)3BO3
R2 = 0.6863 mg N
METTLER DL77 Titrator V3.1 Mettler-Toledo AG R3 = 13.7261 mg N Total
DL77/115 Application Laboratory 1/9 Amm.Bor. Test 10 50.0 mL g Fixed volume U
R1 = 0.3264 mmol/L (NH4)3BO3
Method KKA2 Kjeldahl / Titration 22-Mar-1995 15:17 R2 = 0.6860 mg N
User cdc R3 = 13.7197 mg N Total
Measured 22-Mar-1995 17:54 1/10 Amm.Bor. Test 10 50.0 mL g Fixed volume U
R1 = 0.3273 mmol/L (NH4)3BO3
RESULTS R2 = 0.6877 mg N
R3 = 13.7548 mg N Total
No ID1 ID2 Sample amount and results
STATISTICS
Number results R1 n = 10
1/1 Amm.Bor. Test 10 50.0 mL g Fixed volume U Mean value x = 0.3267 mmol/L (NH4)3BO3
R1 = 0.3269 mmol/L (NH4)3BO3 Standard deviation s = 0.000436 mmol/L (NH4)3BO3
R2 = 0.6870 mg N Rel. standard deviation srel = 0.133 %
R3 = 13.7408 mg N Total Outlier test: no outliers!
1/2 Amm.Bor. Test 10 50.0 mL g Fixed volume U
R1 = 0.3270 mmol/L (NH4)3BO3 STATISTICS
R2 = 0.6872 mg N Number results R2 n = 10
R3 = 13.7447 mg N Total Mean value x = 0.6867 mg N
1/3 Amm.Bor. Test 10 50.0 mL g Fixed volume U Standard deviation s = 0.000915 mg N
R1 = 0.3270 mmol/L (NH4)3BO3 Rel. standard deviation srel = 0.133 %
R2 = 0.6873 mg N Outlier test: no outliers!
R3 = 13.7457 mg N Total
1/4 Amm.Bor. Test 10 50.0 mL g Fixed volume U STATISTICS
R1 = 0.3262 mmol/L (NH4)3BO3 Number results R3 n = 10
R2 = 0.6855 mg N Mean value x = 13.7330 mg N Total
R3 = 13.7108 mg N Total Standard deviation s = 0.018309 mg N Total
Rel. standard deviation srel = 0.133 %
Outlier test: no outliers!

Page 6/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
Method Sample preparation
Method KKA2 Kjeldahl / Titration
Version 22-Mar-1995 15:17
1) Ammonium sulfate stock solution:
Title
Method ID . . . . . . . . .
Title . . . . . . . . . . .
. . . . KKA2
. . . . Kjeldahl / Titration
a) Prepare a stock solution of ca. 0.7 g am-
Date/time . . . . . . . . . . . . . 22-Mar-1995 15:17 monium sulfate in a 500 mL flask and fill with
Sample
Number samples . . . . . . . . . . . 10
200 mL deionized water;
Titration stand . . . . . . . . . . ST20A 1
Entry type . . . . . . . . . . . . . Fixed volume U b) Place the 500 mL flask in a cold water bath
Volume [mL] . . . . . . .
ID1 . . . . . . . . . . . .
.
.
.
.
.
.
.
.
50.0
Amm.Bor.
and add slowly 150 mL 98% sulfuric acid
Molar mass M . . . . . . . . . . . . 112.96 (Merck, for nitrogen determination, Nitrogen
Equivalent number z . . . . . . . . 3
Temperature sensor . . . . . . . . . TEMP A max. 0.0005%); (CAUTION: hot !)
Titration
Titrant . . . . . . . . . . . . . . HCl c) Allow the solution to cool down to room
Concentration [mol/L] . . .
Sensor . . . . . . . . . . .
.
.
.
.
.
.
.
.
0.1
DG111-SC
temperature and fill to the mark with deion.
Unit of meas. . . . . . . . . . . . As installed water.
Titration mode . . . . . . . . . . . EQP
Titrant addition . . . . . . . . DYN
∆E(set) [mV] . . . . .
Limits ∆V . . . . . .
.
.
.
.
.
.
.
.
8.0
Absolute 2) Distillation of ammonia:
∆V(min) [mL] . . . . . . . 0.02
∆V(max) [mL] . . .
Measure mode . . . . . .
.
.
.
.
.
.
.
.
0.3
EQU
To distill ammonia it is necessary to use an
∆E [mV] . . . . . . . . . . . 1.0 appropriate distilling unit with a water steam
∆t [s] . . . . . . . . . . . . 2.0
t(min) [s] . . . . . . . . . . 5.0 generator, e.g. Kjeltec 1002 -Tecator- or
t(max) [s] . . . . . .
Threshold . . . . . . . .
.
.
.
.
.
.
.
.
30.0
100.0
Büchi 316 Distilling Unit.
Maximum volume [mL] . . .
Termination after n EQPs
.
.
.
.
.
.
.
.
30.0
Yes a) Place 50 mL 1.5% boric acid solution (H3BO3)
n = . . . . . . . . .
Evaluation procedure . .
.
.
.
.
.
.
.
.
1
Standard
in a flask at the outlet of the distilling unit and
Calculation add 3 drops of mixed indicator 5 (Merck). Be
Result name . . . . . . . .
Formula . . . . . . . . . .
.
.
.
.
.
.
.
.
(NH4)3BO3
R=Q*C/U
sure that the delivery tube of the distilling unit
Constant . . . . . . . . . . . . . . C=1000/z is dipped into the boric acid solution;
Result unit . . . . . . . . . . . . mmol/z
Decimal places . . . . . . .
Calculation
. . . . 4 b) Pipette 50 mL of the ammonium sulfate
Result name . . . . . . . . . . . . N solution into the appropriate glass tube for
Formula . . . . . . . . . .
Constant . . . . . . . . . .
.
.
.
.
.
.
.
.
R2=R1*(U/1000)*C2
C2=z*14.01
distillation;
Result unit . . . . . . . . . . . . mg
Decimal places . . . . . . . . . . . 4 c) Add ca. 50 mL water and fix the glass tube to
Calculation
Result name . . . . . . . . . . . . N Total
the inlet of the distilling unit;
Formula . . . . . . . . . . . . . . R3=R2*20
Constant . . . . . . . . . . . . . . d) Add ca. 60-80 mL 30% sodium hydroxide
Result unit . . . . . . . .
Decimal places . . . . . . .
.
.
.
.
.
.
.
.
mg
4
solution (NaOH) and immediately start the
Record distillation by opening the valve of the water
Output unit . . . . . . . .
Raw results last sample . .
.
.
.
.
.
.
.
.
Printer
Yes
steam generator;
Table of values . . . . . . . . . . Yes
E - V curve . . . . . . . . . . . . Yes e) When the indicator colour has changed from
Rinse
Auxiliary reagent . . . . . . . . . H2O
violet to green, wait ca. 1 minute and then stop
Volume [mL] . . . . . . . . . . . . 20.0 the distillation.
Statistics
Ri (i=index) . . . . . . . .
Standard deviation s . . . .
.
.
.
.
.
.
.
.
R1
Yes
Literature: see ref. 1, 2, 3.
Rel. standard deviation srel . . . . Yes
Outlier test . . . . . . . . . . . . Yes
Statistics 3) Titration:
Ri (i=index) . . . . . . . . . . . . R2
Standard deviation s . . . .
Rel. standard deviation srel
.
.
.
.
.
.
.
.
Yes
Yes
a) Pour the distilled sample into a 250 mL flask
Outlier test . . . . . . . .
Statistics
. . . . Yes (or 500 mL or 1000 mL) and fill up to the
Ri (i=index) . . . . . . . . . . . . R3 mark with deionized water;
Standard deviation s . . . . . . . . Yes
Rel. standard deviation srel . . . . Yes b) Pipette 50 mL into a titration beaker and start
Outlier test . . . . . . . . . . . . Yes
Record the method KKA2 (automated titration with
Output unit . . . . . . . .
All results . . . . . . . .
. . . . Printer
. . . . Yes
0.1 mol/L HCl).

Author: C. De Caro

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 7/36
Comments to Method
1) To check wether the 30% sodium hydroxide solution added is enough for the ammonia distillation,
it is very helpful to put 1-3 drops of mixed indicator 5 in the sample (colour change from violet to
green);
2) The neutralization of sulfuric acid with concentrated alkaline solution leads to a strong heating of
the sample. To avoid this, deionized water is added before starting the distillation step (see sample
preparation, 2c).
3) The distilled sample was diluted with deionized water in a 1000 mL flask. Thus, the corresponding
factor (20) was taken into account in the calculation R3.

General Remarks
1) A blank determination did not give any results.
2) The analysis time can be decreased by predispensing (see function "TITRATION")

Waste disposal
After titration, the weakly acidic samples should be neutralized before final disposal.

Titration Curve Table of Measured Values

|Volume |Increment| Signal | Change |1st deriv. | Time
| mL | mL | mV | mV | mV/mL | min:s
C
ET1 | 0.0000| | -29.3 | | | 0:11
| 0.0200| 0.0200 | -27.7 | 1.6 | 80.5 | 0:16
| 0.0400| 0.0200 | -26.2 | 1.5 | 73.5 | 0:21
| 0.0800| 0.0400 | -23.7 | 2.5 | 63.0 | 0:26
| 0.1600| 0.0800 | -17.7 | 6.0 | 75.2 | 0:34
| 0.2510| 0.0910 | -8.6 | 9.1 | 100.0 | 0:43
| 0.3150| 0.0640 | 0.4 | 9.0 | 141.1 | 0:52
| 0.3580| 0.0430 | 9.0 | 8.5 | 198.6 | 1:01
| 0.3890| 0.0310 | 18.0 | 9.0 | 291.3 | 1:10
| 0.4090| 0.0200 | 25.3 | 7.3 | 364.0 | 1:19
| 0.4290| 0.0200 | 36.6 | 11.3 | 567.0 | 1:29
| 0.4490| 0.0200 | 52.4 | 15.8 | 791.0 | 1:41
| 0.4690| 0.0200 | 75.2 | 22.8 | 1141.0 | 1:50
| 0.4890| 0.0200 | 116.1 | 40.8 | 2040.5 | 2:03
EQP1 | 0.5090| 0.0200 | 158.1 | 42.1 | 2103.5 | 2:17
| 0.5290| 0.0200 | 173.7 | 15.5 | 777.0 | 2:29
| 0.5490| 0.0200 | 181.6 | 7.9 | 395.5 | 2:38

Results of Series (Titration)

Range of Sample
Concentration n Recovery Reproducibility
( mg N / 50 mL ) % %

2–3 4 98.90 0.036

4 99.39 0.080
4 99.39 0.106
4 99.31 0.123

1–2 5 99.11 0.113

4 98.92 0.085

0.5 – 1 10 98.60 0.226

8 98.53 0.075
10 98.29 0.133
9 97.99 0.136
9 99.02 0.220

Page 8/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
METTLER TOLEDO Titration Application No. M121

Titration according to Kjeldahl:

Dairy Products

Sample: Dairy products Instruments: METTLER DL67 DL70ES DL77

0.25…3 g / 250 mL Kjeldahl flask METTLER ST20A Changer
METTLER TOLEDO AT261
Substance: Ammonium sulfate (NH4)2SO4 Printer HP DeskJet 510
M = 132.14; z = 2
Accessories: Titration beaker ME-101974
Preparation: - 15…20 mL 98% sulfuric acid Peristaltic pump ME-65241
- 1 tablet Missouri catalyst (Merck) 1 Burette drive DV90
Titrant: Hydrochloric acid, HCl Temp. sensor DT120 ME-89624
c(HCl) = 0.1 mol/L Indication: DG111-SC at Sensor 1
Standard: Tris(hydroxymethyl)aminomethane
(THAM), 0.05…0.1 g / 50 mL H2O
Results:
METTLER DL77 Titrator V3.1 Mettler-Toledo AG
DL77/115 Application Laboratory

Method KKA2 Kjeldahl / Titration 10-Mar-1995 14:10

User cdc
Measured 10-Mar-1995 15:38

RESULTS

No ID1 ID2 Sample amount and results

1/1 Amm.Bor. Sbrinz 2 50.0 mL g Fixed volume U

R1 = 0.6439 mmol/L (NH4)3BO3
R2 = 1.3532 mg N
R3 = 13.5320 mg N Total
1/2 Amm.Bor. Sbrinz 2 50.0 mL g Fixed volume U
R1 = 0.6432 mmol/L (NH4)3BO3
R2 = 1.3516 mg N
R3 = 13.5159 mg N Total
1/3 Amm.Bor. Sbrinz 2 50.0 mL g Fixed volume U
R1 = 0.6446 mmol/L (NH4)3BO3
R2 = 1.3547 mg N
R3 = 13.5472 mg N Total
1/4 Amm.Bor. Sbrinz 2 50.0 mL g Fixed volume U
R1 = 0.6439 mmol/L (NH4)3BO3
R2 = 1.3532 mg N
R3 = 13.5321 mg N Total
1/5 Amm.Bor. Sbrinz 2 50.0 mL g Fixed volume U
R1 = 0.6432 mmol/L (NH4)3BO3
R2 = 1.3518 mg N
R3 = 13.5177 mg N Total

STATISTICS
Number results R1 n = 5
Mean value x = 0.6438 mmol/L (NH4)3BO3
Standard deviation s = 0.000606 mmol/L (NH4)3BO3
Rel. standard deviation srel = 0.094 %
Outlier test: no outliers!

STATISTICS
Number results R2 n = 5
Mean value x = 1.3529 mg N
Standard deviation s = 0.001274 mg N
Rel. standard deviation srel = 0.094 %
Outlier test: no outliers!

STATISTICS
Number results R3 n = 5
Mean value x = 13.5290 mg N Total
Standard deviation s = 0.012738 mg N Total
Rel. standard deviation srel = 0.094 %
Outlier test: no outliers!

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 9/36
Method
Method KKA2 Kjeldahl / Titration Calculation
Version 10-Mar-1995 14:10 Result name . . . . . . . . . . . (NH4)3BO3
Formula . . . . . . . . . . . . . R=Q*C/U
Title Constant . . . . . . . . . . . . . C=1000/z
Method ID . . . . . . . . . . . . KKA2 Result unit . . . . . . . . . . . mmol/z
Title . . . . . . . . . . . . . . Kjeldahl / Titration Decimal places . . . . . . . . . . 4
Date/time . . . . . . . . . . . . 10-Mar-1995 14:10 Calculation
Sample Result name . . . . . . . . . . . N
Number samples . . . . . . . . . . 5 Formula . . . . . . . . . . . . . R2=R1*(U/1000)*C2
Titration stand . . . . . . . . . ST20A 1 Constant . . . . . . . . . . . . . C2=z*14.01
Entry type . . . . . . . . . . . . Fixed volume U Result unit . . . . . . . . . . . mg
Volume [mL] . . . . . . . . . . 50.0 Decimal places . . . . . . . . . . 4
ID1 . . . . . . . . . . . . . . . Amm.Bor. Calculation
Molar mass M . . . . . . . . . . . 112.96 Result name . . . . . . . . . . . N Total
Equivalent number z . . . . . . . 3 Formula . . . . . . . . . . . . . R3=R2*10
Temperature sensor . . . . . . . . TEMP A Constant . . . . . . . . . . . . .
Titration Result unit . . . . . . . . . . . mg
Titrant . . . . . . . . . . . . . HCl Decimal places . . . . . . . . . . 4
Concentration [mol/L] . . . . . . 0.1 Record
Sensor . . . . . . . . . . . . . . DG111-SC Output unit . . . . . . . . . . . Printer
Unit of meas. . . . . . . . . . . As installed Raw results last sample . . . . . Yes
Titration mode . . . . . . . . . . EQP Table of values . . . . . . . . . Yes
Titrant addition . . . . . . . DYN E - V curve . . . . . . . . . . . Yes
∆E(set) [mV] . . . . . . . . 8.0 Rinse
Limits ∆V . . . . . . . . . Absolute Auxiliary reagent . . . . . . . . H2O
∆V(min) [mL] . . . . . . 0.02 Volume [mL] . . . . . . . . . . . 20.0
∆V(max) [mL] . . . . . . 0.3 Statistics
Measure mode . . . . . . . . . EQU Ri (i=index) . . . . . . . . . . . R1
∆E [mV] . . . . . . . . . . 1.0 Standard deviation s . . . . . . . Yes
∆t [s] . . . . . . . . . . . 2.0 Rel. standard deviation srel . . . Yes
t(min) [s] . . . . . . . . . 5.0 Outlier test . . . . . . . . . . . Yes
t(max) [s] . . . . . . . . . 30.0 Statistics
Threshold . . . . . . . . . . . 100.0 Ri (i=index) . . . . . . . . . . . R2
Maximum volume [mL] . . . . . . 30.0 Standard deviation s . . . . . . . Yes
Termination after n EQPs . . . Yes Rel. standard deviation srel . . . Yes
n = . . . . . . . . . . . . 1 Outlier test . . . . . . . . . . . Yes
Evaluation procedure . . . . . Standard Statistics
Ri (i=index) . . . . . . . . . . . R3
Standard deviation s . . . . . . . Yes
Rel. standard deviation srel . . . Yes
Outlier test . . . . . . . . . . . Yes
Author: C. De Caro Record
Output unit . . . . . . . . . . . Printer

All results . . . . . . . . . . . Yes

Sample preparation
1) Kjeldahl digestion:
a) The substance is weighed and carefully placed into the Kjeldahl digestion flask;
b) A tablet of Missouri catalyst (Merck) is added;
c) 15-20 mL sulfuric acid (Merck, for nitrogen determination) are slowly added;
d) The digestion flask is heated until the solution becomes colourless and transparent;
e) Keep heating the solution for additional 60 minutes.
2) Distillation of ammonia:
To distill ammonia, it is necessary to use an appropriate distilling unit with a water steam generator,
e.g., Kjeltec 1002 -Tecator- or Büchi 316 Distilling Unit.
a) Place 50 mL 1.5% boric acid solution (H3BO3) in a flask at the outlet of the distilling unit
and add 3 drops of mixed indicator 5 (Merck). Be sure that the delivery tube of the distilling
unit is dipped into the boric acid solution;
b) Pipette 50 mL of the ammonium sulfate solution into the appropriate glass tube for distillation;
c) Add ca. 50 mL water and fix the glass tube to the inlet of the distilling unit;
d) Add ca. 60-80 mL 30% sodium hydroxide solution (NaOH) and immediately start the
distillation by opening the valve of the water steam generator;
e) When the indicator colour has changed from violet to green, wait ca. 1 minute and then stop
the distillation.
3) Titration:
a) Pour the distilled sample into a 250 mL (or 500 or 1000 mL) glass flask and fill with deionized
water up to the mark.
b) Pipette 50 mL into a titration beaker and start method KKA2.
Literature: see ref. 1, 2, 3, 5 and 10.

Page 10/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
Results
Sample Nitrogen and protein content in digested solution Nominal Content
n Nitrogen Protein RSD Protein Protein
(mg) (mg) (%) (%) (%)

Milk powder 5 13.2 84.22 0.18 34.31 34 (10x dilution)

(ca. 250 mg) 5 13.2 84.22 0.35 34.31

«Sbrinz» 5 13.4 85.49 0.10 33.86 34 (10x dilution)

grated cheese, hard 5 13.5 86.13 0.09 33.50
(ca. 250 mg) 5 12.8 81.75 0.60 33.70 (5x dilution)
4 13.7 87.41 0.09 33.44

Yoghurt (Nature) 5 19.5 124.74 0.18 3.90 3.5 (10x dilution)

(ca. 3 g) 10 19.5 124.74 0.23 3.91
5 15.1 96.34 0.08 3.46 (10x dilution)
4 16.4 104.63 0.08 3.62 (5x dilution)
4 17.0 108.46 0.09 3.60

Milk (UHT) 5 17.3 110.37 0.05 3.12 3.12 (10x dilution)

low fat 5 17.9 114.20 0.04 3.13
(ca. 3 g) 4 17.9 114.2 0.08 3.13
4 17.5 111.65 0.08 3.11 (10x dilution)

Titration Curve Table of Measured Values

|Volume |Increment| Signal | Change |1st deriv. | Time
| mL | mL | mV | mV | mV/mL | min:s

ET1 | 0.0000| | -30.4 | | | 0:11

| 0.0200| 0.0200 | -29.2 | 1.3 | 63.0 | 0:17
| 0.0400| 0.0200 | -28.4 | 0.8 | 38.5 | 0:22
| 0.0800| 0.0400 | -27.2 | 1.3 | 31.5 | 0:27
| 0.1600| 0.0800 | -24.6 | 2.5 | 31.5 | 0:33
| 0.3200| 0.1600 | -18.3 | 6.3 | 39.4 | 0:41
| 0.4890| 0.1690 | -9.7 | 8.6 | 50.9 | 0:50
| 0.6190| 0.1270 | -0.1 | 9.7 | 76.1 | 1:00
| 0.6950| 0.0790 | 7.6 | 7.7 | 97.5 | 1:09
| 0.7620| 0.0670 | 17.0 | 9.4 | 140.0 | 1:19
| 0.8050| 0.0430 | 25.9 | 8.9 | 206.7 | 1:29
| 0.8340| 0.0290 | 33.7 | 7.8 | 270.3 | 1:38
| 0.8570| 0.0230 | 41.6 | 7.8 | 340.9 | 1:47
| 0.8770| 0.0200 | 49.8 | 8.2 | 409.5 | 1:56
| 0.8970| 0.0200 | 60.3 | 10.6 | 528.5 | 2:05
| 0.9170| 0.0200 | 75.2 | 14.8 | 742.0 | 2:14
| 0.9370| 0.0200 | 91.7 | 16.5 | 826.0 | 2:23
| 0.9570| 0.0200 | 122.4 | 30.7 | 1533.0 | 2:36
EQP1 | 0.9770| 0.0200 | 159.2 | 36.8 | 1841.0 | 2:51
| 0.9970| 0.0200 | 172.1 | 12.9 | 644.0 | 3:01
| 1.0170| 0.0200 | 180.7 | 8.7 | 434.0 | 3:10

Comments to Method
1) The protein content is determined by multiplying the nitrogen amount with a factor f. In the case of dairy
products, f is equal to 6.38 (Literature: see ref. 2)
2) Add 1-3 drops of mixed indicator 5 into the sample to check if the ammonia liberation is complete (colour
change from violet to green);
3) Deionized water is added before starting the distillation step to avoid a too strong heating of the sample due
to the neutralization of sulfuric acid by 30% NaOH.
4) The distilled sample was diluted with water in a 500 mL flask (dil. factor 10 in R3).

General Remarks
1) A blank determination did not give any results.
2) The analysis time can be shortened by predispensing (see function "TITRATION")

Waste disposal
After titration, the weakly acidic samples should be neutralized before final disposal.

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 11/36
METTLER TOLEDO Titration Application No. M122

Ammonia Determination by ISE:

Test with Ammonium Sulfate Solution

Sample: Ammonium sulfate solutions Instruments: METTLER DL67 DL70ES DL77

0.06…0.07 g / 50 mL water METTLER ST20A Changer
METTLER TOLEDO AT261
Substance: Ammonium sulfate (NH4)2SO4 Printer HP DeskJet 510
M = 132.14; z = 2 Accessories: Titration beaker ME-101974
Preparation: 15 mL sulfuric acid, H2SO4 (98%) Burette drive DV90
Temp. sensor DT120 ME-89624
Burette: EDTA / NaOH solution
c(EDTA) = 0.1 mol/L Indication: NH3 selective electrode,
c(NaOH) = 1 mol/L e.g. DX217 at sensor 1

Results:
METTLER DL77 Titrator V3.1 Mettler-Toledo AG
DL77/115 Application Laboratory

Method N002 NH3 ISE-Sensor 01-Feb-1995 15:58

User cdc
Measured 01-Feb-1995 16:38

RESULTS

No ID1 ID2 Sample amount and results

1/1 NH3 0.5359 g Weight m

R1 = 1.19296 pX-NH3 pX
R2 = 53.4386 mmol/L NH3
R3 = 54.5967 mg NH3
R4 = 545.9666 mg NH3 tot.
R5 = 101.878 % NH3 tot. %
1/2 NH3 0.5359 g Weight m
R1 = 1.19100 pX-NH3 pX
R2 = 53.6807 mmol/L NH3
R3 = 54.8441 mg NH3
R4 = 548.4407 mg NH3 tot.
R5 = 102.340 % NH3 tot. %
1/3 NH3 0.5359 g Weight m
R1 = 1.18705 pX-NH3 pX
R2 = 54.1707 mmol/L NH3
R3 = 55.3447 mg NH3
R4 = 553.4467 mg NH3 tot.
R5 = 103.274 % NH3 tot. %
1/4 NH3 0.5359 g Weight m
R1 = 1.18866 pX-NH3 pX
R2 = 53.9705 mmol/L NH3
R3 = 55.1402 mg NH3
R4 = 551.4018 mg NH3 tot.
R5 = 102.893 % NH3 tot. %
1/5 NH3 0.5359 g Weight m
R1 = 1.19052 pX-NH3 pX
R2 = 53.7402 mmol/L NH3
R3 = 54.9049 mg NH3
R4 = 549.0488 mg NH3 tot.
R5 = 102.454 % NH3 tot. %
1/6 NH3 0.5359 g Weight m
R1 = 1.19114 pX-NH3 pX
R2 = 53.6629 mmol/L NH3
R3 = 54.8259 mg NH3
R4 = 548.2586 mg NH3 tot.
R5 = 102.306 % NH3 tot. %

STATISTICS
Number results R1 n = 6
Mean value x = 102.524 % NH3 tot. %
Standard deviation s = 0.490255 % NH3 tot. %
Rel. standard deviation srel = 0.478 %
Outlier test: no outliers!

Page 12/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
Method Sample preparation
Method N002 NH3 ISE-Sensor
Version 01-Feb-1995 15:58 1) Prepare a stock solution of ca. 0.7 g ammo-
Title nium sulfate in a 500 mL flask and fill with
Method ID . . . . . . . . .
Title . . . . . . . . . . .
. N002
. NH3 ISE-Sensor
200 mL deionized water;
Date/time . . . . . . . . . . 01-Feb-1995 15:58
Sample 2) Place the flask in a cold water bath and add
Number samples . . . . . . . . 6
Titration stand . . . . . . . ST20 1 slowly 150 mL 98% sulfuric acid (Merck, for
Entry type . . . . . . . . .
Lower limit [g] . . . . .
.
.
Weight m
0.003
nitrogen determination, Nitrogen max.
Upper limit [g] . . . . . . 0.3 0.0005%);
ID1 . . . . . . . . . . . . . NH3
Molar mass M . . . . . . . .
Equivalent number z . . . .
.
.
17.03
1
3) Allow the solution to cool down;
Temperature sensor . . . . . . TEMP A
Dispense 4) Fill with deionized water to the mark;
Titrant . . . . . . . . . . . EDTA/NaOH (1M)
Concentration [mol/L] . . . . 0.1
Volume [mL] . . . . . . . . . 10 5) Pipette 50 mL of the solution into a titration
Stir
Speed [%] . . . . . . . . . . 60
beaker;
Time [s] . . . . . . . . . . . 360
Measure 6) Start titration method N002.
Sensor . . . . . . . . . . . . NH3-Sensor
Unit of meas.. . . . . . . . . mV
∆E [mV] . . . . . . . . . . . 0.5
∆t [s] . . . . . . . . . . . . 2.0 Comments to Method
t(min) mode . . . . . . . . . Fix
t(min) [s] . . . . . . .
t(max) [s] . . . . . . . . .
.
.
30.0
60.0
- Calibration and measurements always must
Calculation be performed at the same temperature, the
Result name . . . . . . . .
Formula . . . . . . . . . .
.
.
pX
R=E
same stirring speed and by keeping the same
Constant . . . . . . . . . . . distance between the NH3-electrode and the
Result unit . . . . . . . . . pX-NH3
Decimal places . . . . . . . . 5 stirrer.
Calculation
Result name . . . . . . . . . NH3 - After each measurement, the electrode is
Formula . . . . . . . . . .
Constant . . . . . . . . . .
.
.
R2=pw(-R1)*50/60*1000 immersed in 0.1 mol/L NaOH solution
Result unit . . . . . . . . . mmol/L (conditioning beakers).
Decimal places . . . . . . . . 4
Calculation - Keep the electrode in 0.1 mol/L ammonium
Result name . . . . . . . . . NH3
Formula . . . . . . . . . . . R3=R2*M*(60/1000)-C3 chloride solution overnight. If the results are
Constant . . . . . . . . . .
Result unit . . . . . . . .
.
.
C3=0.0069
mg
not reproducible, it is recommended to keep
Decimal places . . . . . . . . 4 the electrode in distilled water overnight.
Calculation
Result name . . . . . . . . . NH3 tot. - A blank value (C3) was determined before
Formula . . . . . . . . . .
Constant . . . . . . . . . .
.
.
R4=R3*10
starting the analysis and it was taken into
Result unit . . . . . . . . . mg account in calculation R3 (see Method).
Decimal places . . . . . . . . 4
Calculation
Result name . . . . . . . .
Formula . . . . . . . . . .
.
.
NH3 tot.
R5=R4/(10*m)
Waste disposal
Constant . . . . . . . . . . .
Result unit . . . . . . . .
Decimal places . . . . . . .
.
.
%
3
It is necessary to neutralize the strong alkaline
Statistics
Ri (i=index) . . . . . . . . . R5
solutions before final disposal.
Standard deviation s . . . . . Yes
Rel. standard deviation srel . Yes
Outlier test . . . . . . . . . R5 Author: C. De Caro
Record
Output unit . . . . . . . . . Printer
All results . . . . . . . . . Yes
Literature: see ref. 4,6.
Results of Series
Range of Sample
Concentration n Recovery Reproducibility
mg N / 50 mL % %

20 6 102.52 0.478

5-6 6 103.50 0.749

4 102.69 0.784

1-2 6 99.56 0.711

4 103.31 0.098
4 103.38 0.470

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 13/36
METTLER TOLEDO Titration Application No. M123

Ammonia Determination by ISE:

Dairy Products

Sample: Dairy products, Instruments: METTLER DL67 DL70ES DL77

0.25-3 g / 250 mL Kjeldahl flask METTLER ST20A Changer
METTLER TOLEDO AT261
Substance: Ammonium sulfate (NH4)2SO4
Printer HP DeskJet 510
M = 132.14; z = 2
Accessories: Titration beaker ME-101974
Preparation: See "Sample preparation", p.15
Burette drive DV90
Burette: EDTA / NaOH solution Temp. sensor DT120 ME-89624
c(EDTA) = 0.1 mol/L
Indication: NH3 selective electrode,
c(NaOH) = 2 mol/L
e.g. DX217 at sensor 1
Results:
METTLER DL77 Titrator V3.1 Mettler-Toledo AG
DL77/115 Application Laboratory

Method N003 NH3 ISE-Sensor 28-Mar-1995 10:17

User cdc
Measured 28-Mar-1995 14:53

RESULTS

No ID1 ID2 Sample amount and results

1/1 NH3 Sbrinz 12 50.0 mL Fixed volume U

R1 = 3.54253 pX-NH3 pX
R2 = 0.2389 mmol/L NH3
R3 = 0.2394 mg NH3
R4 = 0.1970 mg N
1/2 NH3 Sbrinz 12 50.0 mL Fixed volume U
R1 = 3.54005 pX-NH3 pX
R2 = 0.2403 mmol/L NH3
R3 = 0.2408 mg NH3
R4 = 0.1981 mg N
1/3 NH3 Sbrinz 12 50.0 mL Fixed volume U
R1 = 3.54666 pX-NH3 pX
R2 = 0.2367 mmol/L NH3
R3 = 0.2371 mg NH3
R4 = 0.1951 mg N
1/4 NH3 Sbrinz 12 50.0 mL Fixed volume U
R1 = 3.54399 pX-NH3 pX
R2 = 0.2381 mmol/L NH3
R3 = 0.2386 mg NH3
R4 = 0.1963 mg N
1/5 NH3 Sbrinz 12 50.0 mL Fixed volume U
R1 = 3.54569 pX-NH3 pX
R2 = 0.2372 mmol/L NH3
R3 = 0.2377 mg NH3
R4 = 0.1955 mg N

STATISTICS
Number results R1 n = 5
Mean value x = 3.54378 pX-NH3 pX
Standard deviation s = 0.002620 pX-NH3 pX
Rel. standard deviation srel = 0.074 %
Outlier test: no outliers!

STATISTICS
Number results R4 n = 5
Mean value x = 0.1964 mg N
Standard deviation s = 0.001210 mg N
Rel. standard deviation srel = 0.616 %
Outlier test: no outliers!

Page 14/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
Method Sample preparation
Method N003 NH3 ISE-Sensor
Version 28-Mar-1995 10:17 1) Weigh substance and place carefully into the
Title Kjeldahl digestion flask;
Method ID . . . . . . . . . . . N00
Title . . . . . . . . . . . . . NH3 ISE-Sensor
Date/time . . . . . . . . . . . 28-Mar-1995 10:17 2) Add a tablet of Missouri catalyst (Merck);
Sample
Number samples . . . . . . .
Titration stand . . . . . .
.
.
.
.
5
ST20 1
3) Carefully add 15-20 mL sulfuric acid;
Entry type . . . . . . . . . . . Fixed volume U
Volume [mg] . . . . . . . . . 50.0 4) Heat the sample until it becomes transparent
ID1 . . . . . . . . . . . . . . NH3
Molar mass M . . . . . . . . . . 17.03 and almost colourless;
Equivalent number z . . . . . . 1
Temperature sensor . . . . .
Dispense
. . TEMP A 5) Heat for additional 60 min;
Titrant . . . . . . . . . . . . EDTA/NaOH (2M)
Concentration [mol/L] . . . . . 0.1 6) Let the Kjeldahl flask to cool down to room
Volume [mL] . . . . . . . . . . 10
Stir temperature;
Speed [%] . . . . . . . . . . . 60
Time [s] . . . . . . . . . .
Measure
. . 180 7) Cool a 250, 500 or 1000 mL flask in a cold
Sensor . . . . . . . . . . . . . NH3-Sensor water bath;
Unit of meas.. . . . . . . . . . As installed
∆E [mV] . . . . . . . . . . . . 0.2
∆t [s] . . . . . . . . . . . . . 5.0 8) Carefully pour the digested solution into the
t(min) mode . . . . . . . .
t(min) [s] . . . . . . .
.
.
.
.
Fix
30.0 250 mL flask;
t(max) [s] . . . . . . . . . . . 600.0
Calculation
Result name . . . . . . . . . . pX
8) Slowly fill the flask up to the mark with
Formula . . . . . . . . . . . . R=E deionized water (CAUTION: heat ! );
Constant . . . . . . . . . . . .
Result unit . . . . . . . . . . pX-NH3
Decimal places . . . . . . . . . 5 9) Transfer 50 mL of the diluted solution into
Calculation
Result name . . . . . . . . . . NH3
the titration beaker with a 50 ml pipette;
Formula . . . . . . . . . . . . R2=pw(-R1)*50/60*1000
Constant . . . . . . . . . . . . 10) Start titration method N003.
Result unit . . . . . . . . . . mmol/L
Decimal places . . . . . . . . . 4
Calculation
Result name . . . . . . . . . . NH3
Formula . . . . . . . . . . . . R3=R2*M*(60/1000)-C3
Constant . . . . . . . . . . . . C3=0.0047
Result unit . . . . . . . . . . mg
Decimal places . . . . . . . . . 4
Calculation
Result name . . . . . . . . . . N
Formula . . . . . . . . . . . . R4=R3*(14.01/17.03)
Constant . . . . . . . . . . . .
Result unit . . . . . . . . . . mg
Decimal places . . . . . . . . . 4
Statistics
Ri (i=index) . . . . . . . . . . R1
Standard deviation s . . . . . . Yes
Rel. standard deviation srel . . Yes
Outlier test . . . . . . . . . . Yes
Statistics
Ri (i=index) . . . . . . . . . . R4
Standard deviation s . . . . . . Yes
Rel. standard deviation srel . . Yes Author: C. De Caro
Outlier test . . . . . . . . . . Yes
Record
Output unit . . . . . . . . . . Printer
All results . . . . . . . . . . Yes

Comments to Method
1) Calibration and measurements always must be performed at the same temperature, the same
stirring speed and by keeping the same distance between the NH3-electrode and the stirrer.
2) After each measurement the electrode is immersed in 0.1 mol/L NaOH solution (conditioning
beakers).
3) Keep the electrode in 0.1 mol/L ammonium chloride solution (NH4Cl) overnight. If the results are
not reproducible, it is recommended to keep the electrode in distilled water overnight.
4) A blamk value (C3) is taken into account in calculation R3.

Waste disposal
It is necessary to neutralize the strong alkaline solutions before final disposal.

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 15/36
Results of Series:

Sample Nitrogen and protein content in digested solution Nominal Content

(Producer)
n Nitrogen Protein RSD Protein Protein
(mg) (mg) (%) (%) (%)

Milk powder 5 11.6 74 2.39 30.46 34

(ca. 250 mg) 5 10.9 69.5 1.86 28.61
5 11 70.2 3.04 27.2
5 10.7 68.3 2.31 26.47

«Sbrinz» 5 10.9 69.5 1.88 26.22 34

grated cheese, hard 5 10.9 69.5 1.46 26.22
(ca. 250 mg) 3 13 82.9 2.93 31.27

Yoghurt (Nature) 5 11.7 74.6 0.89 2.39 3.5

(ca. 3 g) 5 12.2 77.8 1.17 2.27
5 12.2 77.8 1.80 2.27

Milk (UHT) 4 12.1 77.2 1.40 2.26 3.12

low fat 5 19.8 126.3 3.71 16.76
(ca. 3 g)

«Gruyère» 5 5.2 33.2 1.33 23.43 26

grated cheese, hard 5 4.6 29.3 1.88 20.68
(ca. 250 mg)

Yoghurt (Chocolate) 4 14.0 89.3 1.10 2.94 3.7

(ca. 3 g) 5 13.4 85.5 1.08 2.82

«Certosino» 5 7.3 46.6 0.68 12.93 17

fresh cheese 5 7.0 44.7 0.97 12.40
(ca. 700 mg)

Literature: see references 4, 5, 6 and 10.

Page 16/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
METTLER TOLEDO Titration Application No. M124

Calibration of the Ammonia Selective Electrode

Sample: 50 mL ammonium chloride solu- Instruments: METTLER DL67 DL70ES DL77

tion 0.1 mol/L, 0.01 mol/L, METTLER ST20A Changer
0.001 mol/L Printer HP DeskJet 510
Substance: Ammonium chloride NH4Cl Accessories: Titration beaker ME-101974
(Suprapur, Merck) Burette drive DV90
M = 53.491, Z = 1 Temp. sensor DT120 ME-89624
Preparation: deion. water Indication: NH3 selective electrode,
e.g. DX217 at sensor 1
Titrant: –––
Burette: EDTA / NaOH solution
c(EDTA) = 0.1 mol/L
c(NaOH) = 1 mol/L
Results:

METTLER DL77 Titrator V3.1 Mettler-Toledo AG

DL77/115 Applications Laboratory

Method N001 NH3 ISE-Sensor 31-Jan-1995 13:34

User cdc
Measured 02-Feb-1995 14:18

RESULTS

Nr ID1 ID2 Sample amount and results

1/1 50.0 mL mL pH,pM,pX 3.000

R1 = -108.008 mV Potential
1/2 50.0 mL mL pH,pM,pX 2.000
R1 = -164.427 mV Potential
1/3 50.0 mL mL pH,pM,pX 1.000
R1 = -222.386 mV Potential

CALIBRATION
Sensor NH3-Sensor
Buffer type pH,pM,pX
Zero point 4.884 pX0
Slope 57.19 mV/pX
Calibration temperature 22.4 °C

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 17/36
Method Remarks
Method N001 NH3 ISE-Sensor
Version 31-Jan-1995 13:34 Preparation of the electrode:
Title
Method ID . . . . . . . . . . . . . N001
a) The electrode is mounted and filled with the
Title . . . . . . . . . . . . . . . NH3 ISE-Sensor inner electrolyte solution according to the
Date/time . . . . . .
Sample
. . . . . . . 31-Jan-1995 13:34
instructions;
Number samples . . . . . . . . . . . 3
Titration stand . . . . . . . . . . ST20A 1 b) Before starting the measurements, the
Entry type . . . . . .
Volume [mL] . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Fixed volume U
50.0
electrode is kept in 0.1 mol/L ammonium
ID1 . . . . . . . . . . . . . . . . chloride solution for at least 30 min.
Molar mass M . . . . . . . . . . . . 0
Equivalent number z . . . . . . . . 1 c) After each sample, the electrode is condi-
Temperature sensor . .
Dispense
. . . . . . . TEMP A
tioned in a titration beaker containing 0.1
Titrant . . . . . . . . . . . . . . EDTA/NaOH (1M) mol/L NaOH solution (see method).
Concentration [mol/L] . . . . . . . 0.1
Volume [mL] . . . . . . . . . . . . 10
Stir
Speed [%] . . . . . . . . . . . . . 60
Time [s] . . . . . . . . . . . . . . 360 Calibration:
Measure
Sensor . . . . . . . . . . . . . . . NH3-Sensor
Unit of meas.. . . . . . . . . . . . mV a) To calibrate the ammonia selective electrode,
∆E [mV] . . . . . . .
∆t [s] . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
0.5
2.0
a dilution series is prepared from a stock
t(min) mode . . . . . . . . . . . . Fix solution of 5.35 g ammonium chloride
t(min) [s] . . . .
t(max) [s] . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
30.0
60.0
(Suprapur, Merck) in a 1000 mL glass flask
Conditioning (0.1 mol/L).
Interval . . . . . . . . . . . . . . 1
Time [s] . . . . . . . . . . . . . . 30 b) 100 mL and 10 mL of the 0.1 mol/L stock
Calculation
Result name . . . . . . . . . . . . Potential
solution are diluted in 1000 mL flasks to
Formula . . . . . . . . . . . . . . R=E obtain 0.01 mol/L and 0.001 mol/L,
Constant . . . . . . .
Result unit . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
. mV respectively.
Decimal places . . . . . . . . . . . 3
Calibration c) Addition of 10 mL EDTA/NaOH solution
Sensor . . . . . . . .
Buffer type . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
NH3-Sensor
pH,pM,pX
generates ammonia (NH3) , which diffuses
First buffer . . . . . . . . . . 3.0 through the membrane of the ISE-electrode.
Second buffer . . .
Third buffer . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
2.0
1.0
EDTA is added in order to avoid interference
Fourth buffer . . . . . . . . . . 0.0 by metal ions.
Fifth buffer . . . . . . . . . . 0.0
Sixth buffer . . . . . . . . . . 0.0 d) Start the calibration procedure with the most
Seventh buffer . . . . . . . . . 0.0
Eight buffer . . . . . . . . . . 0.0 diluted standard solution to avoid membrane
Ri (i=index) . . . . .
Minimal slope . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
R1
-55.0
saturation due to higher concentration, i.e.
Maximal slope . . . . . . . . . . . 60.0 0.001 mol/L (pX=3) , 0.01 mol/L (pX=2) and
Record
Output unit . . . . . . . . . . . . Printer
0.1 mol/L (pX=1).

Measurement conditions:
a) Calibration and measurements always must be performed at the same temperature, the same
stirring speed and keeping the same distance between the NH3-electrode and the stirrer.
b) Up to 8 standard solutions can be used for the calibration. Choose the appropriate standard solutions
according to the nitrogen content of the sample. For instance, for a sample containing 0.005-
0.008 mol/L nitrogen use 0.1 mol/ L, 0.01 mol/L and 0.001 mol/L standard solutions.
c) Keep the electrode in 0.1 mol/L ammonium chloride solution (NH4Cl) overnight. If the electrode
does not lead to reproducible results, it is recommended to keep it in distilled water overnight.

Waste disposal
The strong alkaline solution (pH = 10-11) has to be neutralized before final disposal.

Literature: see ref. 4 and 6

Author: C. De Caro

Page 18/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
METTLER TOLEDO Titration Application No. M125

Determination of Nitrogen after Urotropine Reaction:

Test with Ammonium Sulfate Solution

Sample: Water solution of ammonium Instruments: METTLER DL67 DL70ES DL77

sulfate 0.06 … 0.07 g/50 mL METTLER ST20A Changer
METTLER TOLEDO AT261
Substance: Ammonium sulfate (NH4)2SO4
Printer HP DeskJet 510
M = 132.14 ; z = 2
Accessories: Titration beaker ME-101974
Preparation: – max. 0.07 g in 50 mL flask;
Peristaltic Pump ME-65241
– 20 mL deion. water;
3 (2) Burette drives DV90
– 15 mL sulfuric acid,
Temp. sensor DT120 ME-89624
H2SO4 (98%);
– 30 mL 30 % sodium hydroxide Indication: DG111-SC at Sensor 1
Titrant 1: Sodium hydroxide
c(NaOH) = 3.0 mol/L
Titrant 2: Sodium hydroxide
c(NaOH) = 0.1 mol/L
Burette 3: Formaldehyde solution
HCHO > 37 %; neutralized.
Standard: Potassium hydrogen phthalate 1/3 H2SO4 0.033482 g Weight m
R1 = 22.246 mL NaOH 3M
(KHP), R2
R3
=
=
5.882
4.879
mL
mL
NaOH 0.1M
NaOH 0.1M
0.07 …0.12 g / 50 mL H2O. R4
R5
=
=
4.928
32.5620
mL
mg
Tot NaOH 0.1M
(NH4)2SO4
R6 = 97.2522 %
1/4 H2SO4 0.033597 g Weight m
Results: R1
R2
=
=
19.836
6.174
mL
mL
NaOH 3M
NaOH 0.1M
R3 = 4.905 mL NaOH 0.1M
R4 = 4.945 mL Tot NaOH 0.1M
R5 = 32.6704 mg (NH4)2SO4
R6 = 97.2419 %
1/5 H2SO4 0.034225 g Weight m
METTLER DL77 Titrator V3.1 Mettler-Toledo AG
R1 = 17.752 mL NaOH 3M
DL77/115 Application Laboratory
R2 = 5.277 mL NaOH 0.1M
R3 = 4.971 mL NaOH 0.1M
Method r002 (NH4)2SO4 / HMTA 06-Jan-1995 17:01
R4 = 5.016 mL Tot NaOH 0.1M
User cdc
R5 = 33.1400 mg (NH4)2SO4
Measured 06-Jan-1995 18:41
R6 = 96.8298 %
1/6 H2SO4 0.034075 g Weight m
RESULTS
R1 = 15.758 mL NaOH 3M
R2 = 5.400 mL NaOH 0.1M
No ID1 ID2 Sample amount and results
R3 = 4.977 mL NaOH 0.1M
R4 = 5.020 mL Tot NaOH 0.1M
1/1 H2SO4 0.042192 g Weight m
R5 = 33.1658 mg (NH4)2SO4
R1 = 15.636 mL NaOH 3M
R6 = 97.3316 %
R2 = 4.607 mL NaOH 0.1M
R3 = 6.218 mL NaOH 0.1M
STATISTICS
R4 = 6.271 mL Tot NaOH 0.1M
Number results R6 n = 6
R5 = 41.4353 mg (NH4)2SO4
Mean value x = 97.2986 %
R6 = 98.2065 %
Standard deviation s = 0.487168 %
1/2 H2SO4 0.030855 g Weight m
Rel. standard deviation srel = 0.501 %
R1 = 12.162 mL NaOH 3M
R2 = 4.632 mL NaOH 0.1M
Outlier test : sample No. 1/1
R3 = 4.479 mL NaOH 0.1M
Number results R6 n = 5
R4 = 4.527 mL Tot NaOH 0.1M
Mean value x = 97.1170 %
R5 = 29.9077 mg (NH4)2SO4
Standard deviation s = 0.222204 %
R6 = 96.9297 %
Rel. standard deviation srel = 0.229 %

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 19/36
 Method r002 (NH4)2SO4 / HMTA Calculation
Version 06-Jan-1995 17:01 Result name . . . . . . . . . . . . NaOH 0.1M
Formula . . . . . . . . . . . . . . R3=VEQ[3]
Title Constant . . . . . . . . . . . . . .
Method ID . . . . . . . . . . . . . r002 Result unit . . . . . . . . . . . . mL
Title . . . . . . . . . . . . . . . (NH4)2SO4 / HMTA Decimal places . . . . . . . . . . . 3
Date/time . . . . . . . . . . . . . 06-Jan-1995 17:01 Calculation
Sample Result name . . . . . . . . . . . . Tot NaOH 0.1M
Number samples . . . . . . . . . . . 6 Formula . . . . . . . . . . . . . . R4=R3+VEX[2]
Titration stand . . . . . . . . . . ST20A 1 Constant . . . . . . . . . . . . . .
Entry type . . . . . . . . . . . . . Weight m Result unit . . . . . . . . . . . . mL
Lower limit [g] . . . . . . . . . 0.025 Decimal places . . . . . . . . . . . 3
Upper limit [g] . . . . . . . . . 0.035 Calculation
ID1 . . . . . . . . . . . . . . . . H2SO4 Result name . . . . . . . . . . . . (NH4)2SO4
Molar mass M . . . . . . . . . . . . 98.02 Formula . . . . . . . . . . . . . . R5=(R4/C)*132.14
Equivalent number z . . . . . . . . 2 Constant . . . . . . . . . . . . . . C=10*z
Temperature sensor . . . . . . . . . TEMP A Result unit . . . . . . . . . . . . mg
Stir Decimal places . . . . . . . . . . . 4
Speed [%] . . . . . . . . . . . . . 60 Calculation
Time [s] . . . . . . . . . . . . . . 45 Result name . . . . . . . . . . . .
Titration Formula . . . . . . . . . . . . . . R6=R5/(10*m)
Titrant . . . . . . . . . . . . . . NaOH Constant . . . . . . . . . . . . . .
Concentration [mol/L] . . . . . . . 3.0 Result unit . . . . . . . . . . . . %
Sensor . . . . . . . . . . . . . . . DG111-SC Decimal places . . . . . . . . . . . 4
Unit of meas. . . . . . . . . . . . As installed Record
Titration mode . . . . . . . . . . . DOS Output unit . . . . . . . . . . . . Printer
Dose 2 . . . . . . . . . . . . . To potential Table of values . . . . . . . . . . Yes
Potential [mV, pH, ...] . . . 3.2 E - V curve . . . . . . . . . . . . Yes
Maximum volume [mL] . . . . . 30.0 Rinse
Record Auxiliary reagent . . . . . . . . . H2O
Output unit . . . . . . . . . . . . Printer Volume [mL] . . . . . . . . . . . . 20.0
Table of values . . . . . . . . . . Yes Conditioning
E - V curve . . . . . . . . . . . . Yes Interval . . . . . . . . . . . . . . 1
Titration Time [s] . . . . . . . . . . . . . . 30.0
Titrant . . . . . . . . . . . . . . NaOH Statistics
Concentration [mol/L] . . . . . . . 0.1 Ri (i=index) . . . . . . . . . . . . R6
Sensor . . . . . . . . . . . . . . . DG111-SC Standard deviation s . . . . . . . . Yes
Unit of meas. . . . . . . . . . . . As installed Rel. standard deviation srel . . . . Yes
Titration mode . . . . . . . . . . . EQP Outlier test . . . . . . . . . . . . Yes
Titrant addition . . . . . . . . DYN Record
∆E(set) [mV] . . . . . . . . . 8.0 Output unit . . . . . . . . . . . . Printer
Limits ∆V . . . . . . . . . . Absolute All results . . . . . . . . . . . . Yes
∆V(min) [mL] . . . . . . . 0.02
∆V(max) [mL] . . . . . . . 0.2
Measure mode . . . . . . . . . . EQU
∆E [mV] . . . . . . . .
∆t [s] . . . . . . . . .
.
.
.
.
.
.
0.5
1.0
Sample preparation
t(min) [s] . . . . . . . . . . 3.0
t(max) [s] . . . . . . . . . . 30.0 1) Dissolve 70 mg (NH4)2SO4 (Merck, p.a. ) in
Threshold . . . . . . . . .
Maximum volume [mL] . . . .
.
.
.
.
.
.
7.0
20.0
a 50 mL flask with 20 mL deionized water.
Termination after n EQPs .
n = . . . . . . . . . .
.
.
.
.
.
.
Yes
1
2) Place the 50 mL flask in a cold water bath
Evaluation procedure . . . . . . Standard and slowly add 15 mL 98% sulfuric acid
Record
Output unit . . . . . . . . . . . . Printer (Merck, for nitrogen determination). Be
Table of values . . . . . . .
E - V curve . . . . . . . . .
. . . Yes
. . . Yes
careful: the solution becomes hot!
Dispense
Titrant . . . . . . . . . . . . . . HCHO
3) Allow the solution to cool down and fill to
Concentration [mol/L] . . . . . . . 1.23 the mark with deionized water.
Volume [mL] . . . . . . . . . . . . 6
Stir 4) Transfer 25 mL of the (NH4)2SO4 solution with
Speed [%] . . . . . . . . . .
Time [s] . . . . . . . . . . .
. . . 70
. . . 150
a 25 mL pipette into the titration beaker.
Titration
Titrant . . . . . . . . . . . . . . NaOH
5) Place the titration beaker in a cold water bath
Concentration [mol/L] . . . . . . . 0.1 and slowly add 30 mL 30% NaOH .
Sensor . . . . . . . . . . . . . . . DG111-SC
Unit of meas. . . . . . . . . . . . As installed Be careful: the solution becomes hot!
Titration mode . . . . . . . . . . . EQP
Titrant addition . . . . . . . . DYN 6) Allow the solution to cool down to room
∆E(set) [mV] . . . . . .
Limits ∆V . . . . . . .
.
.
.
.
.
.
3.0
Absolute
temperature before starting the titration.
∆V(min) [mL] . . . . . . . 0.04
∆V(max) [mL] . . . . . . . 0.2
Measure mode . . . . . . .
∆E [mV] . . . . . . . .
.
.
.
.
.
.
EQU
0.5
Comments to method
∆t [s] . . . . . . . . . . . . 1.0
t(min) [s] . . . . . . . . . . 3.0 • The calculations are based on the titrant
t(max) [s] . . . . . . .
Threshold . . . . . . . . .
.
.
.
.
.
.
30.0
1.8
consumption in mL; thus, the result R5 must
Maximum volume [mL] . . . . . . . 10.0 be corrected by the titer. You can avoid this
Termination after n EQPs .
n = . . . . . . . . . .
.
.
.
.
.
.
Yes
1
step by defining the consumption in mmol.
Evaluation procedure . . .
Calculation
. . . Standard • The recovery of ammonium sulfate (in %) is
Result name . . . . . . . . . . . . NaOH 3M determined in calculation R6 .
Formula . . . . . . . . . . . . . . R1=VEQ[1]
Constant . . . . . . . . . . . . . . • The analysis procedure can be shortened by
Result unit . . . . . . . . .
Decimal places . . . . . . . .
.
.
.
.
.
.
mL
3
use of predispensing.
Calculation
Result name . . . . . . . . . . . . NaOH 0.1M
• When using the DL67 titrator, you have to
Formula . . . . . . . . . . . . . . R2=VEQ[2] change the titrant after the second titration.
Constant . . . . . . . . . . . . . .
Result unit . . . . . . . . . . . . mL
Decimal places . . . . . . . . . . . 3

Page 20/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
Waste disposal
1) Excess formaldehyde is neutralized with hydrogen peroxide (formation of formic acid).
2) The samples are weakly basic and are easily neutralized before final disposal.
Titration Curve (3rd Titration) Table of Measured Values (3rd Titration)
|Volume |Increment| Signal | Change |1st deriv. | Time
| mL | mL | pH | pH | pH/mL | min:s

ET1 | 0.0000| | 5.503 | | | 0:03

| 0.0400| 0.0400 | 5.531 | 0.028 | 0.698 | 0:06
| 0.0800| 0.0400 | 5.552 | 0.021 | 0.524 | 0:10
| 0.1600| 0.0800 | 5.605 | 0.054 | 0.669 | 0:14
| 0.2210| 0.0610 | 5.641 | 0.036 | 0.591 | 0:18
| 0.3180| 0.0970 | 5.693 | 0.051 | 0.528 | 0:21

| 4.5565| 0.0425 | 7.521 | 0.049 | 1.150 | 2:46

| 4.5965| 0.0400 | 7.572 | 0.051 | 1.280 | 2:50
| 4.6365| 0.0400 | 7.628 | 0.056 | 1.396 | 2:55
| 4.6765| 0.0400 | 7.694 | 0.066 | 1.658 | 3:00
| 4.7165| 0.0400 | 7.760 | 0.066 | 1.658 | 3:04
| 4.7565| 0.0400 | 7.839 | 0.079 | 1.978 | 3:09
| 4.7965| 0.0400 | 7.922 | 0.083 | 2.065 | 3:14
| 4.8365| 0.0400 | 8.006 | 0.084 | 2.095 | 3:19
| 4.8765| 0.0400 | 8.097 | 0.091 | 2.269 | 3:25
EQP1 | 4.9165| 0.0400 | 8.187 | 0.091 | 2.269 | 3:30
| 4.9565| 0.0400 | 8.275 | 0.087 | 2.182 | 3:35
| 4.9965| 0.0400 | 8.355 | 0.080 | 2.007 | 3:40

General Remarks
1) CAUTION: Formaldehyde is hazardous. Avoid inhalation and contact,wear appropriate
protective clothes (e.g. gloves) and work in a well ventilated laboratory (ev. in a fume hood).
2) It was not necessary to subtract a blank value from result R5 since blank measurements (i.e.,
without ammonium sulfate) did not give a result. This is an indication of the very low nitrogen
content in the chemicals used for the titration method.
3) The results obtained with neutralized formaldehyde were compared to the ones with formaldehyde
at pH 9 (see below). The results show a very good agreement.
In addition, measurements have been performed using formaldehyde without neutralization. The
acidic pH value (pH ca. 3) has been taken into account by determining the blank value.
Results of Series

Sample n x (%) RSD (%) xcorr (%) Remarks

1 stock solution 4 97.132 0.639 97.247 Formaldehyde solution neutralized

1 stock solution 6 96.871 0.644 96.986 Formaldehyde solution neutralized
1 stock solution 6 97.619 0.733 97.734 Formaldehyde solution neutralized
5 solutions 5 98.100 0.682 98.216 Formaldehyde solution neutralized
5 solutions 5 96.731 0.617 96.845 Formaldehyde solution neutralized

6 solutions 4 97.866 0.410 97.014 Formaldehyde solution pH=9

1 stock solution 4 96.608 0.133 95.748 Formaldehyde solution pH=9
1 stock solution 6 96.892 0.247 96.029 Formaldehyde solution pH=9

4 solutions 4 96.200 0.775 98.153 Formaldehyde NOT neutralized

4 solutions 4 96.163 0.733 98.115 Formaldehyde NOT neutralized
7 solutions 7 97.200 0.715 99.173 Formaldehyde NOT neutralized
1 stock solution 5 96.464 0.188 98.422 Formaldehyde NOT neutralized
Author: C. De Caro
Literature: see ref. 4, 7, 8 and 9.

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 21/36
METTLER TOLEDO Titration Application No. M126

Protein Determination in Dairy Products

by Reaction to Urotropine

Sample: Dairy products Instruments: METTLER DL67 DL70ES DL77

0.25 …3 g / 250 mL Kjeldahl flask METTLER ST20A Changer
Printer HP DeskJet 510
Substance: Ammonium sulfate (NH4)2SO4
M = 132.14 ; z = 2 Accessories: Titration beaker ME-23829
Peristaltic Pump ME-65241
Preparation: – 0.25…3 g in a 250 mL Kjeldahl
3 (2) Burette drives DV90
digestion flask;
Temp. sensor DT120 ME-89624
– 15…20 mL sulfuric acid,
H2SO4(98%); Indication: DG111-SC at Sensor 1
– 1 tablet Missouri catalyst.
Titrant 1: Sodium hydroxide, NaOH
c(NaOH) = 3.0 mol/L Results:

Titrant 2: Sodium hydroxide, NaOH METTLER DL77 Titrator V3.1 Mettler-Toledo AG

DL77/115 Application Laboratory
c(NaOH) = 0.1 mol/L
Method r005 Kjeldahl / HMTA 23-Jan-1995 17:46
User cdc
Burette 3: Formaldehyde solution Measured 23-Jan-1995 18:54

HCHO > 37 %; neutralized. RESULTS

No ID1 ID2 Sample amount and results

Standard: Potassium hydrogen phthalate
1/1 H2SO4 50.0 mL Fixed volume
(KHP), R1 = 17.554 mL NaOH 3M
R2 = 0.574 mL NaOH 0.1M
0.07 …0.12 g / 50 mL H2O. R3 = 0.896 mL NaOH 0.1M
R4 = 0.946 mL Tot NaOH 0.1M
R5 = 1.3247 mg N
1/2 H2SO4 50.0 mL Fixed volume
R1 = 17.728 mL NaOH 3M
R2 = 2.204 mL NaOH 0.1M
R3 = 0.880 mL NaOH 0.1M
R4 = 0.923 mL Tot NaOH 0.1M
R5 = 1.2933 mg N
1/3 H2SO4 50.0 mL Fixed volume
R1 = 17.208 mL NaOH 3M
R2 = 1.599 mL NaOH 0.1M
R3 = 0.856 mL NaOH 0.1M
R4 = 0.917 mL Tot NaOH 0.1M
R5 = 1.2849 mg N
1/4 H2SO4 50.0 mL Fixed volume
R1 = 17.216 mL NaOH 3M
R2 = 1.849 mL NaOH 0.1M
R3 = 0.872 mL NaOH 0.1M
R4 = 0.923 mL Tot NaOH 0.1M
R5 = 1.2928 mg N
1/5 H2SO4 50.0 mL Fixed volume
R1 = 17.250 mL NaOH 3M
R2 = 2.074 mL NaOH 0.1M
R3 = 0.867 mL NaOH 0.1M
R4 = 0.921 mL Tot NaOH 0.1M
R5 = 1.2903 mg N

STATISTICS
Number results R5 n = 5
Mean value x = 1.2972 mg N
Standard deviation s = 0.015717 mg N
Rel. standard deviation srel = 1.212 %

Outlier test : sample No. 1/1

Number results R5 n = 4
Mean value x = 1.2903 mg N
Standard deviation s = 0.003823 mg N
Rel. standard deviation srel = 0.296 %

Page 22/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
 Method r005 Kjeldahl / HMTA Calculation
Version 23-Jan-1995 17:46 Result name . . . . . . . . . . . . NaOH 0.1M
Formula . . . . . . . . . . . . . . R3=VEQ[3]
Title Constant . . . . . . . . . . . . . .
Method ID . . . . . . . . . . . . . r005 Result unit . . . . . . . . . . . . mL
Title . . . . . . . . . . . . . . . Kjeldahl / HMTA Decimal places . . . . . . . . . . . 3
Date/time . . . . . . . . . . . . . 23-Jan-1995 17:46 Calculation
Sample Result name . . . . . . . . . . . . Tot NaOH 0.1M
Number samples . . . . . . . . . . . 5 Formula . . . . . . . . . . . . . . R4=R3+VEX[2]
Titration stand . . . . . . . . . . ST20A 1 Constant . . . . . . . . . . . . . .
Entry type . . . . . . . . . . . . . Fixed volume U Result unit . . . . . . . . . . . . mL
Volume [mL] . . . . . . . . . . . 50.0 Decimal places . . . . . . . . . . . 3
ID1 . . . . . . . . . . . . . . . . H2SO4 Calculation
Molar mass M . . . . . . . . . . . . 98.02 Result name . . . . . . . . . . . . N
Equivalent number z . . . . . . . . 2 Formula . . . . . . . . . . . . . . R5=(R4/C)*14.01*2
Temperature sensor . . . . . . . . . TEMP A Constant . . . . . . . . . . . . . . C=10*z
Stir Result unit . . . . . . . . . . . . mg
Speed [%] . . . . . . . . . . . . . 60 Decimal places . . . . . . . . . . . 4
Time [s] . . . . . . . . . . . . . . 45 Record
Titration Output unit . . . . . . . . . . . . Printer
Titrant . . . . . . . . . . . . . . NaOH Table of values . . . . . . . . . . Yes
Concentration [mol/L] . . . . . . . 3.0 E - V curve . . . . . . . . . . . . Yes
Sensor . . . . . . . . . . . . . . . DG111-SC Rinse
Unit of meas. . . . . . . . . . . . As installed Auxiliary reagent . . . . . . . . . H2O
Titration mode . . . . . . . . . . . DOS Volume [mL] . . . . . . . . . . . . 20.0
Dose 2 . . . . . . . . . . . . . To potential Conditioning
Potential [mV, pH, ...] . . . 3.2 Interval . . . . . . . . . . . . . . 1
Maximum volume [mL] . . . . . 30.0 Time [s] . . . . . . . . . . . . . . 30.0
Record Statistics
Output unit . . . . . . . . . . . . Printer Ri (i=index) . . . . . . . . . . . . R5
Table of values . . . . . . . . . . Yes Standard deviation s . . . . . . . . Yes
E - V curve . . . . . . . . . . . . Yes Rel. standard deviation srel . . . . Yes
Titration Outlier test . . . . . . . . . . . . Yes
Titrant . . . . . . . . . . . . . . NaOH Record
Concentration [mol/L] . . . . . . . 0.1 Output unit . . . . . . . . . . . . Printer
Sensor . . . . . . . . . . . . . . . DG111-SC All results . . . . . . . . . . . . Yes
Unit of meas. . . . . . . . . . . . As installed
Titration mode . . . . . . . . . . . EQP
Titrant addition . . . . . . . . DYN
∆E(set) [mV] . . . . . . . . . 8.0
Limits ∆V . . . . . . . . . . Absolute
∆V(min) [mL] . . . . . . . 0.02
∆V(max) [mL] . . . . . . . 0.2
Measure mode . . . . . . . . . . EQU
∆E [mV] . . . . . . . . . . . 0.5
∆t [s] . . . . . . . . .
t(min) [s] . . . . . . .
.
.
.
.
.
.
1.0
3.0
Sample preparation
t(max) [s] . . . . . . . . . . 30.0
Threshold . . . . . . . . . . . . 6.5
Maximum volume [mL] . . . . . . . 20.0 1) Kjeldahl digestion:
Termination after n EQPs . . . . Yes
n = . . . . . . . . . . . . . 1 • Weigh the substance and carefully
Record
Evaluation procedure . . . . . . Standard
place it into the Kjeldahl digestion
Output unit . . . . . . . . . . . . Printer flask;
Table of values . . . . . . . . . . Yes
E - V curve . . . . . . . . . . . . Yes • Add a tablet of Missouri catalyst
Dispense
Titrant . . . . . . . . . . . . . . HCHO
(Merck);
Concentration [mol/L] . . . .
Volume [mL] . . . . . . . . .
. . . 1.23
. . . 6
• Slowly add 15-20 mL sulfuric acid
Stir (Merck, for nitrogen determination);
Speed [%] . . . . . . . . . . . . . 70
Time [s] . . . . . . . . . . . . . . 150 • The digestion flasks are heated for
Titration
Titrant . . . . . . . . . . . . . . NaOH
90–120 minutes.
Concentration [mol/L] . . . .
Sensor . . . . . . . . . . . .
.
.
.
.
.
.
0.1
DG111-SC
Literature: see ref. 1
Unit of meas. . . . . . . . . . . . As installed
Titration mode . . . . . . . . . . . EQP
Titrant addition . . . . .
∆E(set) [mV] . . . . . .
.
.
.
.
.
.
DYN
3.0
2) Titration (HMTA-Method):
Limits ∆V . . . . . . .
∆V(min) [mL] . . . .
.
.
.
.
.
.
Absolute
0.04
• Allow the digested solution to cool
∆V(max) [mL] . . . . . . . 0.2 down;
Measure mode . . . . . . . . . . EQU
∆E [mV] . . . . . . . . . . . 0.5 • Cool a 100 mL flask in a cold water
∆t [s] . . . . . . . . .
t(min) [s] . . . . . . .
.
.
.
.
.
.
1.0
3.0
bath and carefully pour the digested
t(max) [s] . . . . . . . . . . 30.0 solution into it;
Threshold . . . . . . . . . . . . 1.65
Maximum volume [mL] . . . . . . . 10.0 • Slowly fill the flask up to the mark
Termination after n EQPs .
n = . . . . . . . . . .
.
.
.
.
.
.
Yes
1
with deionized water (heat ! );
Evaluation procedure . . .
Calculation
. . . Standard • Transfer 10 mL of the diluted solution
Result name . . . . . . . . . . . . NaOH 3M into the titration beaker with a 10 mL
Formula . . . . . . . . . . .
Constant . . . . . . . . . . .
.
.
.
.
.
.
R1=VEQ[1] pipette;
Result unit . . . . . . . . .
Decimal places . . . . . . . .
.
.
.
.
.
.
mL
3
• Dilute the solution to 50 mL with deio-
Calculation nized water;
Result name . . . . . . . . . . . . NaOH 0.1M
Formula . . . . . . . . . . . . . . R2=VEQ[2] • Start titration method r005.
Constant . . . . . . . . . . . . . .
Result unit . . . . . . . . . . . . mL
Decimal places . . . . . . . . . . . 3

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 23/36
Results

Sample Nitrogen and protein content in digested solution Nominal Content

(corrected for the titer) (Producer)
n Nitrogen Protein RSD Protein Protein
(mg) (mg) (%) (%) (%)

Milk powder 4 12.8 81.7 0.30 33.76 34

(ca. 250 mg) 4 14.1 90.0 0.62 33.72
5 12.8 81.7 0.65 33.67

«Quark» 4 21.3 135.9 0.18 11.88 12

fresh cheese, 0% fat 4 21.3 135.9 0.40 11.92
(ca. 700 mg)

«Parmesan» 3 14.5 92.5 0.33 35.82 32

grated cheese, hard 4 14.7 93.8 5.19 36.30
(ca. 250 mg)

«Certosino» 4 20.6 131.4 0.58 17.51 17

fresh cheese 5 19.8 126.3 3.71 16.76
(ca. 700 mg)

«Gruyère» 4 10.8 68.9 2.21 26.22 26

grated cheese, hard 5 11.5 73.4 6.64 27.74
(ca. 250 mg)

Milk 4 15.2 97.0 2.52 3.05 3.12

low fat 5 15.3 97.6 3.01 3.07
(ca. 3 g)

Yoghurt (Chocolate) 4 17.6 112.3 0.44 3.57 3.7

(ca. 3 g) 5 17.0 108.5 3.58 3.45

«Sbrinz» 5 12.9 82.3 0.43 32.37 34

grated cheese, hard 4 12.9 82.3 0.21 32.37
(ca. 250 mg) 7 13.0 82.9 0.40 32.30

Yoghurt (Nature) 9 16.1 102.7 0.53 3.33 3.5

(ca. 3 g) 10 17.7 112.9 0.48 3.55

Milk UHT 6 16.4 104.6 0.23 3.00 3.12

low fat 7 15.3 97.6 0.47 2.97
(ca. 3 g) 7 14.4 91.9 0.34 2.89

Page 24/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
 Comments to Method
1) The calculations are based on the titrant consumption in mL, thus result R5 must be corrected by
the titer of the titrant. You can avoid this by defining the consumption in mmol.
2) Blank measurements did not give any results. This is an indication of the very low nitrogen
content in the chemicals used for the titration.
3) The analysis time can be shortened by predispensing.

Waste disposal
Excess formaldehyde (caution: hazardous) is neutralized with hydrogen peroxide (formation of formic
acid). The samples are weakly basic and are easily neutralized before final disposal.

Titration Curve Table of Measured Values

|Volume |Increment| Signal | Change |1st deriv. | Time
| mL | mL | pH | pH | pH/mL | min:s

ET1 | 0.0000| | 5.649 | | | 0:03

| 0.0400| 0.0400 | 5.687 | 0.038 | 0.954 | 0:07
| 0.0800| 0.0400 | 5.731 | 0.044 | 1.098 | 0:11

| 0.8190| 0.0520 | 6.427 | 0.050 | 0.956 | 1:09

| 0.8685| 0.0495 | 6.471 | 0.044 | 0.887 | 1:12
| 0.9285| 0.0600 | 6.535 | 0.064 | 1.059 | 1:17
| 0.9685| 0.0400 | 6.578 | 0.044 | 1.098 | 1:21
| 1.0120| 0.0435 | 6.627 | 0.049 | 1.116 | 1:25
| 1.0555| 0.0435 | 6.677 | 0.050 | 1.143 | 1:30
| 1.0975| 0.0420 | 6.733 | 0.057 | 1.348 | 1:35
| 1.1375| 0.0400 | 6.787 | 0.053 | 1.329 | 1:39
| 1.1775| 0.0400 | 6.848 | 0.061 | 1.531 | 1:44
| 1.2175| 0.0400 | 6.916 | 0.068 | 1.705 | 1:49
| 1.2575| 0.0400 | 6.995 | 0.079 | 1.965 | 1:54
| 1.2975| 0.0400 | 7.077 | 0.082 | 2.052 | 2:00
| 1.3375| 0.0400 | 7.183 | 0.106 | 2.658 | 2:06
| 1.3775| 0.0400 | 7.311 | 0.128 | 3.207 | 2:12
| 1.4175| 0.0400 | 7.475 | 0.164 | 4.103 | 2:19
| 1.4575| 0.0400 | 7.693 | 0.217 | 5.432 | 2:28
EQP1 | 1.4975| 0.0400 | 7.932 | 0.239 | 5.981 | 2:36
| 1.5375| 0.0400 | 8.164 | 0.232 | 5.808 | 2:44
| 1.5775| 0.0400 | 8.341 | 0.177 | 4.421 | 2:51

General Remarks
CAUTION: Formaldehyde is hazardous. Avoid inhalation and contact, wear appropriate protective
clothes (e.g. gloves) and work in a fume hood or a well ventilated laboratory.

Author: C. De Caro

Literature: see ref. 4, 5, 7, 8, 9 and 10

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 25/36
Comparison of three techniques

Test with ammonium sulfate solution

In order to check the accuracy and the reproducibility, the methods for nitrogen determination by
Kjeldahl digestion were first tested using aqueous solutions of ammonium sulfate in sulfuric acid
(Tab.1).
The classical Kjeldahl analysis (CKA) yields very good accuracy and reproducibility. The recovery is
99 % and the reproducibility better than 0.1 % . This proves to be a considerably reliable method.
The results obtained with the ammonia selective electrode are clearly less reliable. Specifically, the
determined recovery x (theoretical value: 100 %) is always 2-3 % higher than the expected value. The
reproducibility is good. Thus, the results are systematically shifted to higher values.
In the case of the HMTA-method, the recovery varies between 97 and 98 %, which represents a more
realistic value and a good result. The reproducibility is in the order of 0.6-0.7 %.

Tab.1: Comparison by ammonium sulfate

CKA NH3-ISE HMTA

n RSD (%) x (%) n RSD (%) x (%) n RSD (%) x (%)

(NH4)2SO4 4 0.036 98.90 6 0.478 102.52 4 0.639 97.247

4 0.080 99.39 6 0.749 103.50 6 0.644 96.986

4 0.106 99.39 4 0.784 102.69 6 0.733 97.734

4 0.123 99.31 6 0.711 99.56 5 0.682 98.216

5 0.113 99.11 4 0.098 103.31 5 0.617 96.847

4 0.085 98.92 4 0.470 103.38

Dairy products
Next, the three methods were compared by determining the nitrogen content in different dairy products
(Tab.2). Once again, the classical Kjeldahl analysis is the method which leads to very good results
(recovery: 99-100% , reproducibility: 0.1-0.3%).
The direct determination by ammonia ISE may not be the appropriate method for nitrogen determination
in these samples. A typical recovery rate of 103% is found.
On the other side, the HMTA method shows promising results. If we exclude some outliers, recovery
and reproducibility are both good. This method therefore represents an interesting alternative to the
CKA.

Page 26/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
Tab.2: Comparison by dairy products

CKA NH3-ISE HMTA

n RSD (%) x (%) n RSD (%) x (%) n RSD (%) x (%)

Milk 5 0.18 100.91 5 2.39 89.59 4 0.30 99.29

powder 5 0.35 100.91 5 1.86 84.15 4 0.62 99.18
5 0.65 99.03

Sbrinz 5 0.10 99.59 5 1.88 77.12 5 0.43 95.21

5 0.09 98.53 5 1.46 77.12 4 0.21 95.21
5 0.60 99.12 3 2.93 91.97 7 0.40 99.03
4 0.09 98.35

Yoghurt 5 0.08 98.86 5 0.89 68.29 9 0.53 95.14

(nature) 4 0.08 103.42 5 0.37 67.71 10 0.48 101.43
4 0.09 102.86 5 0.55 67.14

Milk 5 0.05 100.00 4 1.40 72.44 6 0.23 96.15

(UHT) 5 0.04 100.32 5 1.17 72.76 7 0.47 95.19
4 0.08 100.32 5 1..80 72.76 7 0.34 92.63
4 0.08 99.68

Literature: see ref. 11

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 27/36
Conclusion

The classical Kjeldahl analysis CKA is the technique yielding the best results in terms of reproducibility
(RSD) and recovery rate. This is valid both for the ammonium test solutions and the dairy samples.
The ammonia ISE technique deviates the most. A rather low reproducibility was attained with the test
solutions as well as with the samples. The recovery rate is sometimes as low as 67%. The easy method
of ammonia determination by ISE is possible but may require a correction factor.
The direct determination of nitrogen by reaction of ammonium with formaldehyde to urotropine (HMTA)
offers a good alternative to the CKA considering both RSD and recovery rate. Furthermore, no water
steam distillation unit is necessary. However, formaldehyde is very hazardous and requires appropriate
working conditions and tools (i.e., fume hood, ventilation, gloves, safety goggles, etc...). Also, waste
containing formaldehyde must be treated accordingly due to ecological reasons.
The methods given in the Applications may also be applied to other samples, for example by following
information given in the additional literature. And, of course, the method parameters may be modified
as you wish, e.g., to obtain faster titrations or reduce reagent consumption.

Page 28/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
Literature

[1] H.P. Latscha, H. A. Klein,

«Analytische Chemie, Chemie–Basiswissen III»,
2nd Edition, Springer Verlag, 1990, pp. 266-270.

[2] «Laborpraxis», Band 4: Analytische Methoden,

Birkhäuser Verlag,1983, pp. 19, 26-32.

[3] «Nitrogen according to Kjeldahl»,

Mettler Application No. 148 MemoTitrator DL40RC.

[4] G. Pignatelli,
«Stickstoffbestimmung nach Kjeldahl: Vergleich verschiedener Methoden zur Bestimmung
von Ammoniak», Diplomarbeit Ingenieurschule beider Basel, HTL Muttenz,17.10.1994.

[5] Schweizerisches Lebensmittelbuch, 5. Auflage, 1. Band,

Eidg. Drucksachen- und Materialzentrale Bern, 1964,
Kap. 6.2 «Stickstoffverbindungen»,
Paragraph 4, «Bestimmung des Stickstoffs», pp. 519-528.

[6] «Guide to ion selective measurement»,

Mettler Toledo, Brochure No. 51300075, 1994.

[7] J. F. Walker,
«Formaldehyde», Reinhold Publishing Corporation, 3. Auflage,
New York, 1964, pp. 490-491 and references therein.

[8] H.P. Latscha, H. A. Klein

«Analytische Chemie, Chemie–Basiswissen III»,
2nd Edition, Springer Verlag, 1990, p 264.

[9] Handbuch der Lebensmittelchemie, Band 2 / 2. Teil,

«Analytik der Lebensmittel: Nachweis und Bestimmung von Lebensmittel-Inhaltstoffen»,
Springer-Verlag, Berlin-Heidelberg-New York, 1967, pp.172-174.

[10] Schweizerisches Lebensmittelbuch, 5. Auflage, 2. Band,

Eidg. Drucksachen- und Materialzentrale Bern, 1964,
Kap. 4 «Milchdauerwaren»,
Paragraph 4, «Bestimmung des Stickstoffs nach Kjeldahl», pp. 1-3, 1993.
Kap. 22 «Diätetische Lebensmittel und Speziallebensmittel» ,
Paragraph 3.2 «Berechnung des Rohproteins», S.1 (1982).
Paragraph 4.1 «Bestimmung des Stickstoffs nach Kjeldahl», S. 1-3, (1991).
Paragraph 4.2 «Berechnung des Rohproteins», S. 1-2, (1991).

[11] C. A. De Caro,
"Nitrogen determination according to Kjeldahl: a comparison of three different techniques"
Chimia 49 (7/8), 1995, p.251 (NSCG 1995 Herbsttagung, University of Berne, Switzerland).

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 29/36
Additional literature

• S. Jacobs,
«The determination of nitrogen in biological materials»,
CRC Crit. Rev, Anal. Chem. 7, 1978, pp. 297-322;

• A.L. Lakin,
«Determination of nitrogen and estimation of protein in foods»,
in „Developments in food analysis techniques», 1, ed. R.D. King,
Applied Science Publishers Ltd, 1978, pp. 43-74;

• K.L. Watkins, T.L. Veum, G.F. Krause,

«Total nitrogen determination of various sample types: a comparison of the Hach,
Kjeltec, and Kjeldahl Methods»,
J. Assoc. Off. Anal. Chem. 70 (3), 1987, pp. 410-412;

• H. W. Harvey,
«Micro-Determination of Nitrogen in Organic Matter without Distillation»,
Analyst 76, 1951, pp. 657-660.

• T. Dévény, J. Gergely,
«Analytische Methoden zur Untersuchung von Aminosäuren, Peptiden und Proteinen,
in «Methoden der Analyse in der Chemie», Band 10,
Akademische Verlagsgesellschaft, Frankfurt am Main, 1968, pp. 254-257.

• F. Ehrenberger,
«Quantitative organische Elementaranalyse»,
Verlag Chemie, New York Berlin Basel Cambridge, 1991.

• R. Matissek, F-M. Schnepel, G. Steiner,

«Lebensmittelanalytik»,
Springer Lehrbuch, 1989.

• R.Lange, R. Friebe, F. Linow,

«Zur Anwendung der Methodenkombination Kjeldahl-Nassaufschluss / Berthelot-Reaktion
bei der Stickstofbestimmung in biologischen Materialien»,
1. Mitt. Stand der Kenntnisse Teil I., Die Nahrung 23(3), 1979, pp. 255-261;
ibid., Teil 2, , Die Nahrung 23(4), 1979, pp. 431-439.

• R. Bock,
«Aufschlussmethoden der anorganischen und organischen Chemie»
Verlag Chemie, 1972, pp. 142-145.

Page 30/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
• Literaturliste Kjeldahl-Methodik Chemie, Riedel-de Häen.

• Schweizerisches Lebensmittelbuch, 5. Auflage, 2. Band, EDMZ Bern, 1964,

Kap. 1 «Milch» ,
Kap. 5 «Käse»,
Kap. 9 «Speiseeis».

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 31/36
Notes

Page 32/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
 This application bulletin represents selected, possible application examples. These have been tested with all
possible care in our lab with the analytical instrument mentioned in the bulletin. The experiments were conducted
and the resulting data evaluated based on our current state of knowledge.
However, the application bulletin does not absolve you from personally testing its suitability for your intended
methods, instruments and purposes. As the use and transfer of an application example are beyond our control,
we cannot accept responsibility therefore.
When chemicals and solvents are used, the general safety rules and the directions of the producer must be
observed.

Nitrogen Determination by Kjeldahl Digestion METTLER TOLEDO DL67 DL70ES DL77 Page 33/36
Page 34/36 METTLER TOLEDO DL67 DL70ES DL77 Nitrogen Determination by Kjeldahl Digestion
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