Follow us on:

@büchi-labortechnik-ag

@buchilabequipment

@buchilabequipment

@buchi_labortechnik_ag

@buchilabeqpt
 Kjeldahl Process Overview

Step Process

Digestion
Organic matter is Digestion: (CHNO) + H2SO4
destroyed by boiling in  CO2 + SO2 + H2O + NH4+
concentrated sulfuric acid.

Distillation
Neutralization / Alkalization:
The mixture is alkalized
H2SO4 + 2 NaOH
with NaOH to free up
 2 Na+ + SO4 2- + 2 H2O
ammonia, which is steam
Distillation: NH4+ + OH– ৮ NH3 (gas)
distilled into an acidic
+ H2O
receiver solution.

Titration
The pH of the solution
Receiver:
increases as ammonia
B(OH)3+ NH3+ H2O ৭ NH4+ + B(OH)4
is added. The nitrogen
Titration:
content is determined
B(OH)4- + HX X- + B(OH)3 + H2O
by titration of the borate
complex.
 Sample Preparation – Overview

Sample amounts

Sample Sample Sample Kjeldahl Sulfuric

example amount tube tablet Acid

High 1 Tablet
2 – 5 mL of
protein Titanium Micro
1 – 2 mg 100 mL 98% sulfuric
pure of 1.59 g or
acid
proteins Copper Micro

Moderate
protein 2 Tablets
0.1 – 5 g 15 – 20 mL
most food Titanium of
(commonly 300 mL of 98%
samples 3.71 g or
0.5 – 1 g) sulfuric acid
(6 – 30% Missouri of 5 g
protein)

Low
protein Limit of Limit of
milk 10 g 500 mL
samples

General Tips
· Avoid boiling delays in the digestion step - use glass
digestion rods during the disgestion
· Avoid foam formation in the digestion step - add Kjeldahl
Tablet Antifoam
· Avoid foam formation in the distillation step - program steam
ramps with lower steam power in the beginning of the
distillation
 Sample Preparation

Mincing:
· Particle sizes should be smaller than 1 mm

Homogenization:
· Homogenize solid samples to maximize precision during nitrogen
determination
· Samples with protein content of 6 - 30% with relative standard
deviation (rsd) values > 1% indicate insufficient homogeneity or
nitrogen level close to the limit of detection (LOD)

Mixer B-400 for homogenization

Drying:
· If needed, dry samples or determine water content using Karl-
Fischer titration or gravimetric methods
 Digestion - Overview

Digestion initial – clearing

Sample mixture is fuming and carbonizing.
The black foam must not rise too high, as
they must be rinsed during the second
digestion step (completion) back into the
sample tube to avoid losses of nitrogen.

Safety zone Completion of digestion –

(= 5 cm)
total degradation
Maintaining the condensation zone at the
height of the BUCHI logo, 5 cm below the
Condensation constriction of the sample tube.
Zone

Digestion finished
Digestate is clear and green, blue-green or
colorless, depending on the catalyst used.
 Digestion – Carbonization

During digestion, the organically bonded nitrogen is converted into

ammonium ions.

Step 1: Carbonization
· Decomposition of carbonized material to carbon dioxide – reflected
by dark foam formation
· Appearance of clear liquid indicates completion of chemical reaction
– 30 minutes are usually added at this point to allow for complete
mineralization
· Optimal digestion conditions when condensation zone remains
5 cm below the constriction of the sample tube
· Addition of salts and catalysts or hydrogen peroxide results in
shorter digestion times

Glass funnels for hydrogen peroxide addition

 Comparison of infrared (IR)
vs block digestion

IR (SpeedDigester) Block (KjelDigester)

Automated for highest

Advantage Fast and flexible
sample throughput

Heating principle Radiation Contact

Preheat time
10 min 20 min
(400 °C)

Heat transfer to
Fast Slow
sample

Max. temperature 580 °C 450 °C

Programmable
Yes (K-439) Yes (K-449)
profiles

Lift No Yes (K-449)

Sample positions 6 / 12 20

Sample tube sizes

100 / 300 / 500 mL 300 mL
Kjeldahl

Reflux sample tubes

Other sample
(e.g. COD), 3rd party None
tubes
tubes
 Digestion – Acid mixtures

Application Acid

Kjeldahl applications 98% sulfuric acid

Mixtures of sulfuric and chromic

Modified Kjeldahl digestions
acid

Analysis of phosphates in waste Mixture of sulfuric and nitric

water acid*

Hydrochloric and nitric acid*

Analysis of heavy metals
(aqua regia)

* Be wary of production of very hazardous explosives

 Digestion – Acid consumption

The total required amount of sulfuric acid is given by:

1. Conversion of K2SO4 to KHSO4

· K2SO4 is a component of Kjeldahl Tablets, Consumption ca.
2 – 3 mL H2SO4

2. Consumption by organic matter, depends on sample type and

amount of added catalyst

3. Losses due to evaporation, of ca. 1 mL / hour

4. Required remaining volume, amount of used Kjeldahl Tablet

(ex. 10 g Kjeldahl Tablets = 10 mL H2SO4)

5. Example of calculation of required H2SO4 amount:

· H2SO4 volume required = conversion + (total consumption by
org. matter) + evaporation + remaining volume
· H2SO4 volume required = 3 mL + (3.97 + 1.51 + 0.00) mL +
1 mL + 10 mL = 18.48 mL ~ 18 mL
 Digestion - Catalysts

Kjeldahl Tablets usually consist of:

· Inert salt, which increases the boiling point of sulfuric acid:

· 354 °C with H2SO4 + potassium sulfate*
· 352 °C with H2SO4 + potassium and sodium sulfate
· 348 °C with H2SO4 + sodium sulfate
· 1-3% of one or several metal catalysts, which speed up the
chemical reaction
· Titanium – moderately fast, low toxicity;
· Copper – moderately fast, low toxicity;
· The shortest digestion times are reached when mixing titanium
dioxide and copper sulfate in a 1:1 ratio
· Optional: additives such as silicone to reduce foam formation

*Potassium sulfate achieves a higher boiling point than sodium sulfate and moreover
decreases the risk of crystallization
 Digestion – Kjeldahl Tablets

· With the addition of Kjeldahl Tablets, the boiling temperature of the

mixture in the sample tube is increased to a desired temperature of
350 to 370°C
· The ratio of sulfuric acid to sulfate salts is crucial for the boiling
temperature to be reached
· A good ratio is 1 g of Kjeldahl catalyst mixture to 3 mL of 98%
sulfuric acid
· At temperatures above 390°C, the formation of nitrogen gas (N2)
becomes a possibility, eventually leading to nitrogen results that are
too low
· Samples which exhibit final ratios close to the limits are more prone
to crystallization after cooling, which could impede sample transfer
 Distillation - Overview

· After digestion, the sample is cooled to room temperature

· Dilution: The acidic digestion mixture is diluted with distilled water
· Alkalization and distillation: In a chemical equilibrium, the
solvated ammonium ions (NH4+) are converted to ammonia gas
(NH3) by reacting with hydroxyl ions (OH-) of excess sodium
hydroxide
· Steam distillation: ammonia is separated from the sample and
condensed together with water in the receiving vessel
· Condensate collection in receiving flask – collect ammonia (NH3) in
the receiver containing typically boric acid B(OH)3 dissolved in water.
The ammonia is quantitatively captured by the boric acid solution
forming solvated ammonium ions
 Distillation – Typical parameters

Step How Rule of thumb

much

4 mL per mL used
H2SO4 / 2.5 mL when
1. Dilution H2O dist. 25 – 90 mL
an autosampler is
connected

NaOH 4.5 mL per mL used

2. Alkalization 15 – 90 mL
32 % H2SO4

2 % H3BO3 with KCl for

low N contents 0.02
− 6.75 mg N / sample
3. Preparation H3BO3
40 – 70 mL tube, 4 % H3BO3 for
of the receiver (pH 4.65)
medium and high N
content 6.75 – 125 mg
N / sample tube

Distillation time:
Water · 150 s with KjelMaster
150 – 300
4. Distillation steam · 180 s with
s
(100 %) KjelSampler
· 240 s with others

Condenser outlet tube

5. Collection NH3 and electrode must be
completely immersed
 Potentiometric vs colorimetric titration

· After steam distillation, titration is commonly used to quantify

ammonia
· Titration is carried out until endpoint of pH 4.65 is reached, either by
potentiometric or colorimetric titration

Advantage Disadvantage

· Lower detection · Daily calibration required

Potentiometric limit · 6 – 12 months lifetime of
direct pH · „IntelliDist“ and electrode
measurement back titration · Storage of pH electrode
possible in saturated KCl

· „IntelliDist“ and back

titration not possible
· No calibration · Indicator required (Sher
of the sensor or bromocresol green /
Colorimetric necessary methyl red indicator only!)
detection of the · Longer lifetime · protection mesh required
color change of the sensor to protect sensor from
(approx. 4 years) bubbles, to avoid titration
· Endpoint visible errors
· Daily determination of the
Setpoint necessary
 Titration – Method Parameters

Potentiometric Colorimetric

Boric acid · 2 % + KCl 3 g / L · 2 % + indicator

concentration · 4% · 4 % + indicator

· Standard titration · Standard titration

Boric acid
 50 mL  60 mL
volume in
· Online titration · Online titration
receiver
 60 mL  60 mL

· Sher
· Bromocresol green /
Indicator None
methyl red (according
to AOAC 2001.11)

pH electrode with KCl

Spectrosense with
Sensor storage solution and
protection mesh
calibration buffers

Titrant
0.01 – 0.5 N 0.02 – 0.2 N
concentration

Titrant
consumption 3 – 17 mL 3 – 17 mL
(optimum)
Titration – Boric acid vs back titration

Advantage Disadvantage

Boric acid titration Easy and

Receiver = H3BO3 approximate
Boric acid in use
Titrant = H2SO4 / HCl dosage of the
Endpoint = 4.65 receiver

Accurate dosage of
the receiving solution
Back titration by an additional dosing
Receiver = H2SO4 No boric acid unit required. More
Titrant = NaOH needed expensive, as an
Endpoint = 7.00 additional dosing unit and
more volumetric solutions
are required
 Blanks - Boric acid titration with water

pH vs Dilution of Boric Acid

5.9

5.7

5.5

5.3

pH
5.1

4.9

4.7

4.5

0 20 40 60 80 100 120 140

Added H2O [mL]

4% 2% 1%
 Calculation of nitrogen and
protein content

Nitrogen Content

Absolute amount of nitrogen [g]

(Vsample - Vblank) · M(N) · cacid · f · z

W(N) =
1000

Weight fraction of nitrogen [gN / g sample]

(Vsample - Vblank) · M(N) · cacid · f · z W(N)

w(N) = =
msample · 1000 msample

Weight percentage of nitrogen [%]

(Vblank - Vsample) · M(N) · cNaOH · fNaOH

%(N)wt = = 100 · w(N)
msample · 10

The parameter needed for the calculations is the empirical protein

factor (PF)

%(N) · 100%
%P = = %(N) · 6.25 = %(N) · fprotein
16%

· Empirical protein factors for individual protein containing foodstuffs

are determined based on average nitrogen content
· Protein factors are regulated by AOAC, ISO and others
 Determination of volatile acids

Relevant for samples: Wine, sparkling wine, juice, beer

Chemicals needed:
· Tartaric acid
· Sodium hydroxide solution
· Phenolphthalein solution
· Sulfuric acid: water solution (1:4)
· Iodine solution
· Starch solution

Determination procedure:
· Eliminate carbon dioxide present in sample prior to distillation
· Distill sulfur dioxide and sorbic acid; determine both to deduct
impact on volatile acid result
· Steam distillation and titration of the distillate with NaOH solution
 Alcohol

The determination of alcoholic strength includes the following steps:

· Distillation of the sample, using an EasyDist, BasicDist or

MultiDist
A known volume of the sample is distilled and collected in a
volumetric flask filled up close to the mark and finally adjusted to the
mark.

· Determination of the density

The density of the distillate is measured by means of a pycnometer.
Alternative measuring devices like an electronic densimeter or a
hydrostatic balance can also be used.

The alcoholic strength by mass is derived by means of a correlation

to the densities of aqueous alcoholic solutions as given in tables.