Kjeldahl Practice Guide

From sample preparation to result calculation

www.buchi.com Quality in your hands

 The Kjeldahl Process
Introduction

At the time when Johan Kjeldahl published his method for the determination of nitrogen
in 1883 the electric lamp was just patented and the technical age in its childhood. Seldom in
human history has an invention remained basically unchanged for such a long time as Kjeldahl’s
method for nitrogen determination.

As in 1883 a Kjeldahl nitrogen determination starts with sample preparation, proceeds to the
digestion followed by separation using steam distillation and subsequent volumetric determina-
tion of the amount of ammonia formed in the process.

Kjeldahl’s visionary idea of providing a simple method for nitrogen and protein determinations,
which can also be carried out by non-academic lab personnel, has been put into practice by
BUCHI’s Kjeldahl systems since 1961.

With this short guide BUCHI wants to support you in your daily work not only by provi-
ding high quality instrumentation but also offering theoretical background information
and useful tables for your daily routine. The laminated tables can be taken out of the guide to
be placed at the locations in the lab where they are needed.

For more detailed information please refer to the BUCHI Kjeldahl Guide.
www.buchi.com/kjeldahl/en/applications/literature

In addition to this short guide you can find detailed information including our
application database at: www.buchi.com/kjeldahl/applications

And to download BUCHI’s practical tablet guide or Kjeldahl calculator for iOS,
Android and Windows Phone 7: www.buchi.com/kjeldahl/apps

2
The Kjeldahl Process
From sample preparation to result calculation

∙∙ Grinding Samples must be homoge-

∙∙ Sample tube size neous. The actual weight of a
Sample ∙∙ Weighing table sample depends on the nitro-
preparation ∙∙ Kjeldahl Tablets gen content as well as on the
inhomogeneity of the sample.

∙∙ Digestion: Organic matter is destroyed by

(CHNO) + H2SO4 → CO2 + SO2 + H2O + NH4+ boiling in concentrated sulfuric
∙∙ Digestion parameters acid. Kjeldahl Tablets raise the
Digestion boiling point and accelerate the
process.

∙∙ Neutralization/Alkalinization: The digestion mixture is alka-

H2SO4 + 2 NaOH → 2 Na+ + SO4 2- + 2 H2O lized with NaOH prior to distil-
∙∙ Distillation: lation to free up the ammonia.
Distillation NH4+ + OH– ⇌ NH3 (gas) + H2O The ammonia is steam distilled
into an acidic receiver solution.

∙∙ Receiver: The pH in the acidic receiver

B(OH)3+ NH3+ H2O ⇌ NH4+ + B(OH)4– solution rises upon addition of
∙∙ Titration: ammonia. The nitrogen and
Titration –
B(OH)4– + HX → X + B(OH)3 + H2O protein content is then deter-
mined by titration of the borate
complex.

∙∙ Calculation The nitrogen content is then

∙∙ LIMS calculated. To calculate the
∙∙ KjelLink protein content the nitrogen is
Result multiplied by a sample specific
protein factor.

3
 Sample preparation
Sample tube selection and weight

Micro
Sample weight < 0.2 g
Sample volume 2 – 3 mL
H2SO4 2 – 5 mL
Kjeldahl Tablets 1 (Micro)
Recommendation Homogeneous samples high in nitrogen/protein
Reduction of chemicals by 80 % compared to 300 mL
Benefit
tubes

300 mL
Sample weight 0.1 – 5 g
Sample volume < 200 mL
H2SO4 5 – 30 mL
Kjeldahl Tablets 2
Recommendation Standard tube for most applications
Benefit All-round sample tube

500 mL
Sample weight >4g
Sample volume < 400 mL
H2SO4 > 10 mL
Kjeldahl Tablets 2
Especially for high sample volumes or strongly foaming
Recommendation
samples
Benefit Problem-free digestion of strongly foaming samples

4
Sample preparation
Weighing

The actual weight depends on

∙∙ The protein or rather the nitrogen content of the sample

preparation
▶ the higher the N-content, the lower the weight can be

Sample
∙∙ The Homogeneity of the sample
▶ By increasing the sample amount the impact of the grain size is reduced
▶ Example: homogeneous samples < 1 g, inhomogeneous samples > 2 g

∙∙ The concentration of the titrant

▶ The consumption of the titrant should be in a range of 3 and 17 mL
(optimal accuracy of measurement when using a 20 mL burette)

5
 Sample preparation
Usage of the weighing table

Example for the usage of the weighing table

1 Expected %N of the sample must be selected (here 2 %)
2 Selection of the titrant concentration used (e.g. 0.05 mol/L)

3 Determination of the expected titrant consumption in mL ▶ here 3.6 and

14.3 mL

4 Result: For samples containing 2 % N and with titrant concentration of

0.05 mol/L, the expected consumption should be in a range of 3 – 17 mL.
Therefore the weight must be between 0.125 and 0.5 g.

Sample: weight [g] 4 Titrant conc.: [N]

5 2 1 0.5 0.125 0.01 0.05 0.1 0.5
N [mg] N [%] Titrant consumption for sample
per glas [mL]
0.5 0.01 0.03 0.05 0.10 0.40 3.6 2
2.0 0.04 0.10 0.20 0.40 1.60 14.3 2.9
2.5 0.05 0.13 0.25 0.50 2.00 3.6 1.8
1
3
7.0 0.14 0.35 0.70 1.40 5.60 10.0 5.0
10.0 0.20 0.50 1.00 2.00 8.00 1 14.3 7.1 1.4
3
50.0 1.00 2.50 5.00 10.00 40.00 7.1
100.0 2.00 5.00 10.00 20.00 80.00 X 14.3
3
1

The limit of quantification is 0.02 mg N per sample tube.

However, optimal would be nitrogen content of 1 – 200 mg per sample tube.

6
Sample preparation
Addition of chemicals and Kjeldahl Tablets

Addition of chemicals, rule-of-the-thumb:

∙∙ sulfuric acid: 2 mL H2SO4 per 1 g catalyst

preparation
∙∙ Kjeldahl Tablets: frequent practice 2 pieces per sample tube

Sample
The aim of the Kjeldahl Tablets is the acceleration of the digestion process
by means of:
∙∙ catalysis by metal salts
∙∙ raising the boiling point of the H2SO4 by sulfate salts (K 2SO4)

The selection of Kjeldahl Tablet depends on: For problem-free samples:

1. Safety aspects Ideal digestion conditions are

2. Digestion time ∙∙ Boiling point at 370 °C
3. Ecological aspects ∙∙ No nitrogen losses
4. Foam formation of the sample ∙∙ Minimal time needs

These are achieved with

▶ 2 mL H2SO4 to 1 g catalyst

For more demanding samples or samples that are high in fat or carbohydrate see
page 10.

7
Sample preparation
Weighing table for solid and liquid samples

Solid samples
Sample: weight [g] Titrant concentration [N]
5 2 1 0.5 0.125 0.01 0.05 0.1 0.5
N [mg] N [%] Titrant consumption sample [mL]
0.5 0.01 0.03 0.05 0.10 0.40 3.6
2.0 0.04 0.10 0.20 0.40 1.60 14.3 2.9
2.5 0.05 0.13 0.25 0.50 2.00 3.6 1.8
7.0 0.14 0.35 0.70 1.40 5.60 10.0 5.0
10.0 0.20 0.50 1.00 2.00 8.00 14.3 7.1 1.4
50.0 1.00 2.50 5.00 10.00 40.00 7.1
100.0 2.00 5.00 10.00 20.00 80.00 14.3

Procedure:
A: Select N % of sample
B: select titrant concentration
C: Choose weight in order that the titrant consumption can be expected
between 3 and 17 mL

Liquid samples
Sample Titrant
[mL] N [%] N mg/L Titrant [N]
4 0.1 – 0.6 0.10
6 0.06 – 0.4 0.10
10 100 – 200 0.01
25 50 – 100 0.01
50 20 – 50 0.01
100 10 – 20 0.01
250 5 – 10 0.01
400 <5 0.01

8
 Sample preparation
Kjeldahl Tablets overview

Article Composition Weight

Titanium 3.5 g K2SO4 / 0.105 g CuSO4 • 5 H2O 3.71 g
# 11057980 0.105 g TiO2
Benefit: Time saving
Recommen- Optimal compromise between environmental and performance
dation: priorities.
Titanium Micro 1.5 g K2SO4 / 0.045 g CuSO4 • 5 H2O 1.59 g
# 11057981 0.045 g TiO2
Benefit: Reduced chemical amount
Recommen- Same as Titanium (11057980) but for semi-micro & micro-
dation: Kjeldahl applications.
Missouri 4.98 g K2SO4 5g
# 11057982 0.02 g CuSO4 • 5 H2O
Benefit: Easy to use and universally applicable
Recommen- The digestion with Missouri is more eco-friendly
dation:
ECO 3.998 g K2SO4 4g
# 11057983 0.002 g CuSO4
Benefit: Eco-friendly
Recommen- Most environmentally friendly catalyst, due to the very low cop-
dation: per content
Antifoam 0.97 g Na2SO4 1g
# 11057984 0.03 g silicone antifoam
Benefit: Maximum foam reduction
Recommen- Used as general purpose foam suppressant. This tablet has to
dation: be combined with Titanium Micro (11057981) or Copper Micro
(11057985).
Copper Micro 1.5 g K2SO4 1.65 g
# 11057985 0.15 g CuSO4 • 5 H2O
Benefit: Reduced chemical amount
Recommen- Combo tablets for Antifoam or micro Kjeldahl applications.
dation:

9
 Sample preparation
Amount of sulfuric acid

The amount of H2SO4 is given by:

1. Conversion of K2SO4 to KHSO4 (K2SO4 is a component of Kjeldahl Tablets)

ca. 2 – 3 mL

2. Consumption by organic matter

Organic matter H2SO4/ g Example: e.g. for 1.5 g weight
[mL] Salami (weight ∙ org. matter):
Fat 9.7 27.3 % 1.5 ∙ 9.7 ∙ 27.3 = 3.97 mL
100
Protein 4.9 20.6 % 1.5 ∙ 4.9 ∙ 20.6 = 1.51 mL
100
Carbohydrates 4.0 0.0 % 1.5 ∙ 4.0 ∙ 0.0 = 0.0 mL
100

3. Losses due to evaporation

ca. 1 mL/h

4. Remaining volume
= amount of used Kjeldahl Tablet (e.g. 10 g Kjeldahl tablets = 10 mL H2SO4)

H2SO4 volume =
conversion + (total consumption by org. matter) + evaporation + remaining volume

3 mL + (3.97 + 1.51 + 0.00) mL + 1 mL + 10 mL = 18.48 mL ~18 mL

10
The digestion
Hints

Inlet for ambient air

Safety zone (≈ 5 cm)

Condensation zone

Boiling /digesting sample

Digestion
∙∙ Optimal conditions are achieved when the condensation zone remains 5 cm below the constric-
tion of the sample tube.

∙∙ For foaming samples one Kjeldahl Tablet «Antifoam» or stearic acid can be used.

∙∙ To reduce the digestion time H2O2 can be added.

∙∙ When samples crystalize despite optimal H2SO4/catalyst ratio ▶ the suction power of the
Scrubber should be reduced.

∙∙ The Scrubber must only be placed on the left of the digester.

∙∙ Minimal use of chemicals due to micro-Kjeldahl (only SpeedDigester).

∙∙ For liquid samples boiling rods prevent boiling delays.

∙∙ Boiling rods can in contrast to boiling stones, also be used for the following automated analysis
via the KjelSampler.

∙∙ Slowly increasing the digestion temperature reduces foam formation of problematic samples.

11
 Digestion
Digestion parameter: SpeedDigester (IR)

TKN using 500 mL sample tube

Digestion 1)
Step Temp. [°C] Level Time [min] Vol. [mL]
█ T nominal
First H2O has to █ T sample Preheating 380 7
evaporate before
the temperature Digestion 520 9 45 10
Temp. [°C]

can increase
50 25
60 50
80 100
135 250
Time [min] 185 400
Cooling 30
1)
The digestion time depends on the sample volume

The sample is heated to boil as fast Standard Kjeldahl using 300 mL sample tube
as possible.
Step Temp. [°C] Level Time [min]
The sample is kept
constantly boiling. Preheating 480 8.5
Digestion 480 8.5 10
Temp. [°C]

550 9.5 10
490 8.5 65
█ T nominal
█ T sample Cooling 30

Time [min]

The sample is heated to boil as fast Micro Kjeldahl using 100 mL sample tubes
as possible.
Due to the low volume used Step Temp. [°C] Level Time [min]
for micro-Kjeldahl lower
temperatures are required Preheating 480 8.5
(480 compared to 490°C) to
Digestion 480 8.5 10
Temp. [°C]

reach the boiling tempera-

ture and the digestion time 500 9.5 5
can significantly reduced.
480 8.5 45
█ T nominal
█ T sample Cooling 15

Time [min]
12
Digestion
Digestion parameter: KjelDigester (only 300 mL tubes)

Standard Kjeldahl digestion using Kjeldahl Tablets

To avoid foaming and over splashing of the

Step Temp. [°C] Time [min]
sample, it is heated up slowly (3 steps) Preheating 300 0
The sample is kept Digestion 340 15
constantly boiling
Temp. [°C]

420 105

█ T nominal
Cooling 35
█ T sample

Time [min]

Accelerated Kjeldahl digestion using Kjeldahl Tablets + H2O2

Step Temp. [°C] Time [min]

330 °C is used as preheat temperature to
avoid splashing, due to too fast heating of Preheating 330 0
the sample
The sample is kept Digestion 420 60
constantly boiling
Temp. [°C]

Cooling 25

█ T nominal
█ T sample

Time [min]

13
 Digestion
Highest sample throughput

Reduce heat-up / cool-down periods Reduce digestion period

█ Block: Tablets █ IR: Tablets █ Block: Tablets + H2O2 █ IR: H2O2 (operator’s presence required)

65 min
120

Time saving: 30 min Time saving: 100 min

Use optimized «Kjeldahl Throughput» solution to get the

highest sample throughput (batch size: 20 samples)

1. Sample preparation batch batch batch batch batch batch

per batch: 40 min #1 #2 #3 #4 #5 #6
Mixer B-400

2. Digestion batch #1 batch #2 batch #3 batch #4 batch #5 batch #6

per batch: 100 min
cooling

cooling

cooling

cooling

cooling

cooling
KjelDigester K-449

3. Distillation / Titration batch #6 of the batch #1 batch #2 batch #3 batch #4 batch #5

per batch: 100 min day before (unattended)
KjelMaster K-375,
KjelSampler K-376 / K-377

t [min] Start 8:00 am Finish 5:00 pm

14
Distillation
Overview

General overview

1. Dilution of the acidic digestion mixture ▶ H2O addition

2. Alkalinization to convert NH4+ in NH3 ▶ NaOH addition
3. Steam distillation to drive out the NH3 ▶ H2O steam addition
4. NH3 collection in acidic receiver ▶ acid (H3BO3 or H2SO4)

Step 4 can be varied as required for

∙∙ Boric acid titration
∙∙ Back titration

Distillation

15
Distillation
Parameter for boric acid titration

Step Why How much Rule-of-the-thumb

Step 1: Dilution
H2O dist. Dilution of the strongly acidic 25 – 90 mL 4 mL per mL used H2SO4,
solution, prevents violent 2.5 mL when a auto sampler
reactions is connected
Step 2: Alkalinization
NaOH 32 % Conversion of NH4+ in 15 – 90 mL 4.5 mL per mL used H2SO4
NH3 (gaseous)

Step 3: Preparation of the receiver

H3BO3 To collect the distilled NH3. 40 – 70 mL 2 % H3BO3 with KCl for low N
(pH 4.65) NH3 is bound as borate contents
complex (NH4B(OH4). 0.02 − 6.75 mg/sample tube

4 % H3BO3 for medium

and high N content
6.75 – 125 mg/sample tube
Step 4: Distillation
Water steam Separation of NH3 by boiling 180 – 300 s Distillation time:
(100 %) of the sample 180 s with KjelMaster
240 s with others
Step 5: Collection
NH3 In boric acid receiver of Condenser outlet tube
pH 4.65 and electrode must be
completely immersed

For the following analysis potentiometric as well as colorimetric titration can be chosen.

16
 Distillation
Parameter for back titration

Step Why How much Rule-of-the-thumb

Step 1: Dilution
H2O dist. Dilution of the strongly 25 – 90 mL 4 mL per mL used H2SO4,
acidic solution, prevents 2.5 mL when a auto sampler
violent reactions is connected
Step 2: Alkalinization
NaOH 32 % Conversion of NH4+ in 15 – 90 mL 4.5 mL per mL used H2SO4
NH3 (gaseous)

Step 3: Preparation of the receiver

H2SO4 To collect the distilled NH3. 10 – 20 mL Volume must be exactly
(0.25 mol/L) Surplus of H2SO4 is titrated dosed (usually 20 mL)
with NaOH.
Step 4: Distillation
Water steam Separation of NH3 by boiling 180 – 300 s Distillation time:
(100 %) of the sample 180 s with KjelMaster
240 s with others
Step 5: Collection
NH3 In sulfuric acid receiver Condenser outlet hose and
electrode must be immersed
completely.

For the following analysis only potentiometric titration can be done.

17
 Distillation
Hints

∙∙ After digestion the samples must cool-down to 50 – 100 °C before they can be further pro-
cessed.

∙∙ Optimal NaOH concentration is 32 %

∙∙ For very low N-contents 2 % H3BO3 with KCI (3 g/L) should be chosen as receiving solution,
to achieve lower detection limits.

∙∙ With KjelSampler samples can be processed overnight.

∙∙ With the «IntelliDist» function of the KjelMaster preheating of the instrument can be avoided.

∙∙ Distillation and titration can be synchronized by means of the «Online-Titration».

∙∙ Waste from the sample tube and the receiver can be collected separately with the
KjelMaster.

18
Titration
Overview of the different combination possibilities

Titration Titration Distillation Measuring Titration

type mode mode mode algorithm

Startpoint pH
Back titration

Endpoint pH

Setpoint mV
Fixed time
Boric acid

Standard

IntelliDist

Optimal
titration

Normal
Online
Potentiometric • • • • • • • • • •
Colorimetric • • • • • • •

Advantage Disadvantage
Potentiometric: direct pH measurement
Lower detection limit / back titration possible / calibration of sensor required / 6 – 12 months
„IntelliDist“ possible lifetime of electrode

Colorimetric: detection of the color change

No sensor calibration necessary / longer lifetime of „IntelliDist“ and back titration not possible /
the sensor / endpoint visible indicator required / protection mesh required /
daily setpoint determination required
Standard: the titration starts after the distillation is finished
Standardized process: Distillation → titration / Longer analysis time as for Online-Titration

Titration
easier monitoring

Online: the titration takes place whilst the distillation is still in progress
Time saving: Synchronizes distillation and titration Not useful for low titration volumes (> 9 mL)

Endpoint
Fixed and known pH pH of boric acid must be adjusted to 4.65

Startpoint
Exact adjustment of pH not needed More boric acid required (compared to end-
(pH must be between 4.4 and 5.0) point titration), to detect the startpoint
Start volume
Huge time saving, higher sample throughput Only for well-known titration volume

19
 Titration
Endpoint and Startpoint titration (potentiometric)

Endpoint titration

Parameter What Setting Rule-of-the-thumb

1. Algorithm Dosage steps Optimal „Optimal“ for Titrant < 0.5 N
for titration „Normal“ for Titrant ≥ 0.5 N
2. Start volume Titrant Only for well-known samples /
HCI / H2SO4 N-content and high titration volume
3. Titration Titrant Consumption should be between
3 and 17 mL
4. Endpoint pH-value pH 4.65 pH-value of the used H3BO3 must pre-
viously be adjusted to 4.65

Startpoint titration (only step 4 must be changed)

Parameter What Setting Rule-of-the-thumb

4. Startpoint pH is pH-value before Adjustment of the pH not necessary
detected NaOH addition

Back titration (potentiometric)

Endpoint titration

Parameter What Setting Rule-of-the-thumb

1. Algorithm Dosage steps Optimal „Optimal“ for Titrant < 0.5 N,
for titration „Normal“ for Titrant ≥ 0.5 N
2. Start volume Titrant NaOH Only for well-known samples /
N-content and high titration volume
3. Titration Titrant Consumption should be between
3 and 17 mL
4. Endpoint Titrant NaOH Back titration Endpoint neutral pH 7.00
pH-value pH 7.00

20
Titration
Setpoint titration (Colorimetric)

Setpoint titration

Parameter What Setting Rule-of-the-thumb

1. Algorithm Dosage steps Optimal When Sher indicator is used both
for titration algorithms „Optimal“ and „Normal“
are possible.
When bromocresol green / methyl red
indicator is used, it allows for algorithm
„Normal“ only.
2. Start volume Titrant Only for well-known samples /
HCI / H2SO4 N-content and high titration volume
3. Titration Titrant Consumption should be between
3 and 17 mL .
The titrant concentration must be
0.02 - 0.2N.
4. Setpoint Color change Sher Indicator in H3BO3 (2.5 mL/L) or
bromocresol green / methyl red
indicator in H3BO3 (17 mL/L)

21
 Titration
Hints

Endpoint Titration Boric Acid

B(OH)3 + 2 H2O ⇌ B(OH)4– + H3O+ (pKa = 9.24)

B(OH)3 + 2 H2O

Sher Indicator

Endpoint pH 4.65

B(OH)4– + H3O+

Acid consumption [mL]

∙∙ Depending on the regulation or method, either potentiometric or colorimetric titration can be

chosen.

∙∙ For endpoint and colorimetric titration the pH of the boric acid must be adjusted to 4.65.

∙∙ Back titration can be used when boric acid should be avoided (for potentiometric titration only).

∙∙ For well-known samples a start volume for titration might be used to accelerate the titration step.
(only useful for high titration volumes)

∙∙ The distillation and titration process can be synchronized, by means of online titration.

22
Result calculation
Hints

∙∙ The KjelMaster K-375 performs the calculation automatically.

∙∙ The PC software KjelLink facilitates error-free sample data editing. In addition, the results can be trans-
ferred to a PC for printing or if necessary for recalculation.

∙∙ To determine the optimal method parameters, the BUCHI App Kjeldahl Optimizer can be used as a
helpful tool.

[V(1) - V(Bl)] ∙ F ∙ c ∙ f ∙ M(N)

w(N) =————————————————
m ∙ 1000

% N = w(N) ∙ 100 %
% P = w(N) ∙ PF ∙ 100 %

(7.5mL – 1mL) ∙ 2 ∙ 0.25 ∙ 1 ∙ 14.007 ∙ 100 %

Example (Titrant is 0.25 M H2SO4,f=1.000): —————————————————————————— = 6.07 % N
0.750 g ∙ 1000
6.07 % N ∙ 6.25 = 37.94 % P

w(N): weight fraction of N

V(1): consumption of titrant, sample, [mL]
V(Bl): average consumption of titrant, blank, [mL]
F: molar reaction factor (1 = HCI, 2 = H2SO4)
c: concentration of titrant [mol /L]
f: factor of titrant
M(N): molecular weight of N (14,007 [g/mol])
m: sample weight [g]
1000: conversion factor (mL in L)
PF: Protein factor

Result
% N: % of weight of N
% P: % of weight of protein

23
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