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Kjeldahl Practice Guide

From sample preparation to result calculation


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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 Kjeldahls
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 determination of the amount of ammonia formed in the process.
Kjeldahls 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
BUCHIs Kjeldahl systems since 1961.
With this short guide BUCHI wants to support you in your daily work not only by providing 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 BUCHIs practical tablet guide or Kjeldahl calculator for iOS,
Android and Windows Phone 7: www.buchi.com/kjeldahl/apps

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The Kjeldahl Process


From sample preparation to result calculation

Sample
preparation

Grinding
Sample tube size
Weighing table
Kjeldahl Tablets

Samples must be homogeneous. The actual weight of a


sample depends on the nitrogen content as well as on the
inhomogeneity of the sample.

Digestion:

(CHNO) + H2SO4 CO2 + SO2 + H2O + NH4+

Digestion

Digestion parameters

Neutralization/Alkalinization:

H2SO4 + 2 NaOH 2 Na+ + SO4 2- + 2 H2O

Distillation

Distillation:

NH4+ + OH NH3 (gas) + H2O

Receiver:

B(OH)3+ NH3+ H2O NH4+ + B(OH)4

Titration

Result

Titration:

B(OH)4 + HX X + B(OH)3 + H2O

Calculation
LIMS
KjelLink

Organic matter is destroyed by


boiling in concentrated sulfuric
acid. Kjeldahl Tablets raise the
boiling point and accelerate the
process.

The digestion mixture is alkalized with NaOH prior to distillation to free up the ammonia.
The ammonia is steam distilled
into an acidic receiver solution.

The pH in the acidic receiver


solution rises upon addition of
ammonia. The nitrogen and
protein content is then determined by titration of the borate
complex.
The nitrogen content is then
calculated. To calculate the
protein content the nitrogen is
multiplied by a sample specific
protein factor.

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Sample preparation
Sample tube selection and weight

Micro
Sample weight

< 0.2 g

Sample volume

23 mL

H2SO4

25 mL

Kjeldahl Tablets

1 (Micro)

Recommendation Homogeneous samples high in nitrogen/protein


Benefit

Reduction of chemicals by 80% compared to 300 mL


tubes

300 mL
Sample weight

0.1 5 g

Sample volume

< 200 mL

H2SO4

530 mL

Kjeldahl Tablets

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

Recommendation

Especially for high sample volumes or strongly foaming


samples

Benefit

Problem-free digestion of strongly foaming samples

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Sample preparation
Weighing
The actual weight depends on
Sample
preparation

The protein or rather the nitrogen content of the sample


the higher the N-content, the lower the weight can be
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)

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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%)

Selection of the titrant concentration used (e.g. 0.05 mol/L)

Determination of the expected titrant consumption in mL here 3.6 and


14.3 mL

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


0.05 mol/L, the expected consumption should be in a range of 317 mL.
Therefore the weight must be between 0.125 and 0.5 g.

Sample: weight [g]


5

4
0.5

0.125

Titrant conc.: [N]


0.01

N [mg]
per glas

N [%]

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

2.5

0.05

0.13

0.25

0.50

2.00

7.0

0.14

0.35

0.70

1.40

5.60

10.0

0.20

0.50

1.00

2.00

8.00

50.0

1.00

2.50

5.00 10.00 40.00

100.0

2.00

5.00 10.00 20.00 80.00

0.05

0.1

0.5

Titrant consumption for sample


[mL]

2
2.9
3.6
10.0

14.3

3
3

1.8
5.0
7.1

1.4
7.1
14.3

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


However, optimal would be nitrogen content of 1200 mg per sample tube.

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Sample preparation
Addition of chemicals and Kjeldahl Tablets

Sample
preparation

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


sulfuric acid: 2 mL H2SO4 per 1 g catalyst
Kjeldahl Tablets: frequent practice 2 pieces per sample tube
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.
2.
3.
4.

Ideal digestion conditions are


Boiling point at 370 C
No nitrogen losses
Minimal time needs

Safety aspects
Digestion time
Ecological aspects
Foam formation of the sample

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.

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Sample preparation
Weighing table for solid and liquid samples
Solid samples
Sample: weight [g]
5

Titrant concentration [N]


0.5

0.125

N [mg] N [%]

0.01

0.05

0.1

0.5

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

2.5

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

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

2.9

1.4

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
[mL]

Titrant
N [%]

N mg/L

Titrant [N]

0.10.6

0.10

0.060.4

0.10

10

100200

0.01

25

50100

0.01

50

2050

0.01

100

1020

0.01

250

510

0.01

400

<5

0.01
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Sample preparation
Kjeldahl Tablets overview
Article

Composition

Weight

Titanium
# 11057980

3.5 g K2SO4 / 0.105 g CuSO4 5 H2O


0.105 g TiO2

Benefit:
Recommendation:

Time saving
Optimal compromise between environmental and performance
priorities.

Titanium Micro 1.5 g K2SO4 / 0.045 g CuSO4 5 H2O


0.045 g TiO2
# 11057981

3.71 g

1.59 g

Benefit:
Recommendation:

Reduced chemical amount


Same as Titanium (11057980) but for semi-micro & microKjeldahl applications.

Missouri
# 11057982

4.98 g K2SO4
0.02 g CuSO4 5 H2O

Benefit:
Recommendation:

Easy to use and universally applicable


The digestion with Missouri is more eco-friendly

ECO
# 11057983

3.998 g K2SO4
0.002 g CuSO4

Benefit:
Recommendation:

Eco-friendly
Most environmentally friendly catalyst, due to the very low copper content

Antifoam
# 11057984

0.97 g Na2SO4
0.03 g Silikon Schaumhemmer

Benefit:
Recommendation:

Maximum foam reduction


Used as general purpose foam suppressant. This tablet has to
be combined with Titanium Micro (11057981) or Copper Micro
(11057985).

Copper Micro
# 11057985

1.5 g K2SO4
0.15 g CuSO4 5 H2O

Benefit:
Recommendation:

5g

4g

1g

1.65 g

Reduced chemical amount


Combo tablets for Antifoam or micro Kjeldahl applications.

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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. 23 mL
2. Consumption by organic matter
Organic matter

H2SO4/ g
[mL]

Example:
Salami

e.g. for 1.5 g weight


(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

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The digestion
Hints
Inlet for ambient air

Safety zone ( 5 cm)


Condensation zone
Digestion

Boiling /digesting sample

Optimal conditions are achieved when the condensation zone remains 5 cm below the constriction 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.

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Digestion
Digestion parameter: SpeedDigester (IR)
TKN using 500 mL sample tube
Digestion

Temp. [C]

T nominal
T sample

First H2O has to


evaporate before
the temperature
can increase

1)

Step

Temp. [C] Level Time [min] Vol. [mL]

Preheating

380

Digestion

520

Time [min]

Cooling
1)

The sample is heated to boil as fast


as possible.

Temp. [C]

The sample is kept


constantly boiling.

T nominal
T sample

45

10

50

25

60

50

80

100

135

250

185

400

30

The digestion time depends on the sample volume

Standard Kjeldahl using 300 mL sample tube


Step

Temp. [C] Level Time [min]

Preheating

480

8.5

Digestion

480
550
490

8.5
9.5
8.5

Cooling

10
10
65
30

Temp. [C]

Time [min]

The sample is heated to boil as fast


as possible.
Due to the low volume used
for micro-Kjeldahl lower
temperatures are required
(480 compared to 490C) to
reach the boiling temperature and the digestion time
can significantly reduced.
T nominal
T sample

Micro Kjeldahl using 100 mL sample tubes


Step

Temp. [C] Level Time [min]

Preheating

480

8.5

Digestion

480
500
480

8.5
9.5
8.5

Cooling

10
5
45
15

Time [min]

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Digestion
Digestion parameter: KjelDigester (only 300 mL tubes)
Standard Kjeldahl digestion using Kjeldahl Tablets
To avoid foaming and over splashing of the
sample, it is heated up slowly (3 steps)

Temp. [C]

The sample is kept


constantly boiling

T nominal

Step

Temp. [C]

Preheating

300

Digestion

340

15

420

105

Cooling

Time [min]

35

T sample

Time [min]

Accelerated Kjeldahl digestion using Kjeldahl Tablets + H2O2


330 C is used as preheat temperature to
avoid splashing, due to too fast heating of
the sample
Temp. [C]

The sample is kept


constantly boiling

Time [min]

Step

Temp. [C]

Preheating

330

Digestion

420

60

Cooling

25

T nominal
T sample

Time [min]

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Digestion
Highest sample throughput
Reduce heat-up / cool-down periods

Block: Tablets

IR: Tablets

Reduce digestion period

Block: Tablets + H2O2

IR: H2O2 (operators presence required)

65 min
120

Time saving: 100 min

Time saving: 30 min

Use optimized Kjeldahl Throughput solution to get the


highest sample throughput (batch size: 20 samples)

1. Sample preparation
per batch: 40 min

batch
#1

batch
#2

batch
#3

batch
#4

batch
#5

batch
#6

Mixer B-400

batch #6 of the batch #1


day before

batch #2

batch #3

batch #6

batch #4

cooling

batch #5
cooling

batch #4
cooling

batch #3
cooling

KjelDigester K-449

3. Distillation / Titration
per batch: 100 min

batch #2
cooling

batch #1
cooling

2. Digestion
per batch: 100 min

batch #5
(unattended)

KjelMaster K-375,
KjelSampler K-376 / K-377

t [min]

Start 8:00 am

Finish 5:00 pm

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Distillation
Overview
General overview
1. Dilution of the acidic digestion mixture
2. Alkalinization to convert NH4+ in NH3
3. Steam distillation to drive out the NH3
4. NH3 collection in acidic receiver

H2O addition
NaOH addition
H2O steam addition
acid (H3BO3 or H2SO4)

Distillation

Step 4 can be varied as required for


Boric acid titration
Back titration

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Distillation
Parameter for boric acid titration
Step

Why

How much

Rule-of-the-thumb

Step 1: Dilution
H2O dist.

Dilution of the strongly acidic 2590 mL


solution, prevents violent
reactions

4 mL per mL
used H2SO4

Step 2: Alkalinization
NaOH 32%

Conversion of NH4+ in
NH3 (gaseous)

1590 mL

4.5 mL per mL
used H2SO4

4070 mL

2% H3BO3 with KCl for


low N contents
0.026.75 mg/sample tube

Step 3: Preparation of the receiver


H3BO3
(pH 4.65)

To collect the distilled NH3.


NH3 is bound as borate
complex (NH4B(OH4).

4 % H3BO3 for medium


and high N content
6.75125 mg/sample tube
Step 4: Distillation
Water steam
(100%)

Separation of NH3 by boiling


of the sample

180300 s

Distillation time:
180 s with KjelMaster
240 s with others

Step 5: Collection
NH3

In boric acid receiver of


pH 4.65

Condenser outlet tube


and electrode must be
completely immersed

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

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Distillation
Parameter for back titration
Step

Why

How much

Rule-of-the-thumb

Dilution of the strongly


acidic solution, prevents
violent reactions

2590 mL

4 mL per mL
used H2SO4

1590 mL

4.5 mL per mL
used H2SO4

To collect the distilled NH3.


Surplus of H2SO4 is titrated
with NaOH.

1020 mL

Volume must be exactly


dosed (usually 20 mL)

Separation of NH3 by boiling


of the sample

180300 s

Distillation time:
180 s with KjelMaster
240 s with others

Step 1: Dilution
H2O dist.

Step 2: Alkalinization
NaOH 32%

Conversion of NH4+ in
NH3 (gaseous)

Step 3: Preparation of the receiver


H2SO4
(0.25 mol/L)
Step 4: Distillation
Water steam
(100%)
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.

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Distillation
Hints

After digestion the samples must cool-down to 50100 C before they can be further processed.
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.

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Titration
Overview
Boric acid titration
Potentiometric titration
Colorimetric titration

1. Standard

a) endpoint titration / b) startpoint titration

2. Online

a) endpoint titration / b) startpoint titration

1. Standard

a) endpoint titration

Start volume before titration can be used for all titration variants.
Back titration
Potentiometric Titration

1. Standard

Advantage

a) endpoint titration

Disadvantage

Potentiometric: direct pH measurement


Lower detection limit / online titration possible /
calibration required / 612 months lifetime
shorter analysis time / IntelliDist possible
of electrode
Colorimetric: detection of the color change
No calibration necessary / longer lifetime of the
IntelliDist not possible / indicator required /
probe / end point visible
more boric acid necessary (compared to pot.)

Titration

Standard: the titration starts after the distillation is finished


Standardized process: Distillation titration /
Longer analysis time as for Online-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)
Fixed and known pH
Adjustment of pH not needed

Endpoint
pH of boric acid must be adjusted to 4.65
Startpoint
More boric acid required (compared to end
point titration), to detect the startpoint

Start volume
Huge time saving, higher sample throughput
Only for well-known titration volume
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Titration
Endpoint and Startpoint titration (potentiometric)
Endpoint titration
Parameter

What

Setting

Rule-of-the-thumb

1. Algorithm

Dosage steps
for titration

Optimal

Optimal for Titrant < 0.5 N


Slow for Titrant 0.5 N

2. Start volume

Titrant
HCI / H2SO4

Only for well-known samples /


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 previously be adjusted to 4.65

Startpoint titration (only step 4 must be changed)


Parameter

What

Setting

Rule-of-the-thumb

4. Startpoint

pH is
detected

pH-value before
NaOH addition

Adjustment of the pH not necessary

Back titration (potentiometric)


Endpoint titration
Parameter

What

Setting

Rule-of-the-thumb

1. Algorithm

Dosage steps
for titration

Optimal

Optimal for Titrant < 0.5 N,


Slow 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
pH-value

Back titration
pH 7.00

Endpoint neutral pH 7.00

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Titration
Endpoint titration (Colorimetric)
Endpoint titration
Parameter

What

Setting

Rule-of-the-thumb

1. Algorithm

Dosage steps Optimal


for titration

Only Optimal, not Slow

2. Start volume

Titrant
HCI / H2SO4

Only for well-known samples /


N-content and high titration volume

3. Titration

Titrant

Consumption should be between


3 and 17 mL

4. Endpoint

Color change

Sher Indicator in H3BO3

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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 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.

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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 transferred to a PC for printing or if necessary for recalculation.
To determine the optimal method parameters, the BUCHI Kjeldahl-calculator 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%
Example (Titrant is 0.25 M H2SO4,f=1.000):

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


= 6.07% N
0.750 g 1000

6.07 % N 6.25 = 37.94% P

weight fraction of N
consumption of titrant, sample, [mL]
average consumption of titrant, blank, [mL]
molar reaction factor (1 = HCI, 2 = H2SO4)
concentration of titrant [mol /L]
factor of titrant
molecular weight of N (14,007 [g/mol])
sample weight [g]
conversion factor (mL in L)
Protein factor
% of weight of N
% of weight of protein

Result

w(N):
V(1):
V(Bl):
F:
c:
f:
M(N):
m:
1000:
PF:
% N:
% P:

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www.buchi.com/kjeldahl/applications

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BCHI Labortechnik AG
CH 9230 Flawil 1
T +41 71 394 63 63
F +41 71 394 65 65
buchi@buchi.com
www.buchi.com

BUCHI Italia s.r.l.


IT 20090 Assago (MI)
T +39 02 824 50 11
F +39 02 57 51 28 55
italia@buchi.com
www.buchi.it

BUCHI Hong Kong Ltd.


HK Central
T +852 2389 2772
F +852 2389 2774
china@buchi.com
www.buchi.com.cn

Nihon BUCHI K.K.


JP Tokyo 110-0008
T +81 3 3821 4777
F +81 3 3821 4555
nihon@buchi.com
www.nihon-buchi.jp

BUCHI Korea Inc


KR Seoul 153-782
T +82 2 6718 7500
F +82 2 6718 7599
korea@buchi.com
www.buchi.kr

BCHI Labortechnik GmbH


DE 45127 Essen
Freecall 0800 414 0 414
T +49 201 747 490
F +49 201 747 492 0
deutschland@buchi.com
www.buechigmbh.de

BCHI Labortechnik GmbH


Branch Office Benelux
NL 3342 GT
Hendrik-Ido-Ambacht
T +31 78 684 94 29
F +31 78 684 94 30
benelux@buchi.com
www.buchi.be

BUCHI Shanghai
CN 200052 Shanghai
T +86 21 6280 3366
F +86 21 5230 8821
china@buchi.com
www.buchi.com.cn

BUCHI India Private Ltd.


IN Mumbai 400 055
T +91 22 667 75400
F +91 22 667 18986
india@buchi.com
www.buchi.in

BUCHI Corporation
US New Castle,
Delaware 19720
Toll Free: +1 877 692 8244
T +1 302 652 3000
F +1 302 652 8777
us-sales@buchi.com
www.mybuchi.com

BUCHI Sarl
FR 94656 Rungis Cedex
T +33 1 56 70 62 50
F +33 1 46 86 00 31
france@buchi.com
www.buchi.fr

BUCHI UK Ltd.
GB Oldham OL9 9QL
T +44 161 633 1000
F +44 161 633 1007
uk@buchi.com
www.buchi.co.uk

BUCHI (Thailand) Ltd.


TH Bangkok 10600
T +66 2 862 08 51
F +66 2 862 08 54
bacc@buchi.com
www.buchi.co.th

PT. BUCHI Indonesia


ID Tangerang 15321
T +62 21 537 62 16
F +62 21 537 62 17
indonesia@buchi.com
www.buchi.co.id

BUCHI Russia/CIS
United Machinery AG
RU 127787 Moscow
T +7 495 981 05 22
F +7 495 981 05 20
russia@buchi.com
www.buchi.ru

11592548 en 1306 D/ Unangekndigte nderungen der technischen Daten vorbehalten /


Qualittssysteme ISO 9001 Die englischsprachige Ausgabe ist die originalsprachliche Ausgabe
und dient als Grundlage fr die bersetzung in alle anderen Sprachen.

Quality in your hands

We are represented by more than 100 distribution partners worldwide. Find your local representative at

www.buchi.com

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