11592548 Kjeldahl Practice Guide 210x210 en D Low

Kjeldahl Practice GuideFrom sample preparation to result calculation

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11592548_Mini_KjeldahlGuide_210x210_multilanguage_D_2.indd 1 10.07.2013 12:48:18

The Kjeldahl ProcessIntroduction

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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


Sample preparation

Digestion

Distillation

Titration

Result

∙ Grinding ∙ Sample tube size ∙ Weighing table ∙ Kjeldahl Tablets

Samples must be homoge-neous. The actual weight of a sample depends on the nitro-gen content as well as on the inhomogeneity of the sample.

∙ Digestion: (CHNO) + H2SO4 → CO2 + SO2 + H2O + NH4

+

∙ Digestion parameters

Organic matter is destroyed by boiling in concentrated sulfuric acid. Kjeldahl Tablets raise the boiling point and accelerate the process.

∙ Neutralization/Alkalinization: H2SO4 + 2 NaOH → 2 Na+ + SO4

2- + 2 H2O ∙ Distillation:

NH4+ + OH– ⇌ NH3 (gas) + H2O

The digestion mixture is alka-lized with NaOH prior to distil-lation to free up the ammonia. The ammonia is steam distilled into an acidic receiver solution.

∙ Receiver: B(OH)3+ NH3+ H2O ⇌ NH4

+ + B(OH)4–

∙ Titration: B(OH)4

– + HX → X– + B(OH)3 + H2O

The pH in the acidic receiver solution rises upon addition of ammonia. The nitrogen and protein content is then deter-mined by titration of the borate complex.

∙ Calculation ∙ LIMS ∙ KjelLink

The nitrogen content is then calculated. To calculate the protein content the nitrogen is multiplied by a sample specific protein factor.

The Kjeldahl ProcessFrom sample preparation to result calculation

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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

BenefitReduction of chemicals by 80 % compared to 300 mL tubes

500 mL

Sample weight > 4 g

Sample volume < 400 mL

H2SO4 > 10 mL

Kjeldahl Tablets 2

RecommendationEspecially for high sample volumes or strongly foaming samples

Benefit Problem-free digestion of strongly foaming samples

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

Sample preparationSample tube selection and weight

4


The actual weight depends on

∙ 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)

Sample preparation Weighing

Sam

ple

prep

arat

ion

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

5 2 1 0.5 0.125 0.01 0.05 0.1 0.5

N [mg] per glas

N [%] Titrant consumption for 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 X 14.3

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.

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2

4

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Example for the usage of the weighing table

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 3 – 17 mL. Therefore the weight must be between 0.125 and 0.5 g.

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Sample preparation Usage of the weighing table

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2

3

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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 (K2SO4)

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

1. Safety aspects2. Digestion time3. Ecological aspects4. Foam formation of the sample

Ideal digestion conditions are ∙ Boiling point at 370 °C ∙ No nitrogen losses ∙ 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.

Sample preparationAddition of chemicals and Kjeldahl Tablets

7

Sam

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prep

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Sample preparationWeighing 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 sampleB: select titrant concentrationC: 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

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9

Article Composition Weight

Titanium# 11057980

3.5 g K2SO4 / 0.105 g CuSO4 • 5 H2O

0.105 g TiO2

3.71 g

Benefit: Recommen-dation:

Time savingOptimal compromise between environmental and performance priorities.

Titanium Micro# 11057981

1.5 g K2SO4 / 0.045 g CuSO4 • 5 H2O

0.045 g TiO2

1.59 g


Reduced chemical amountSame as Titanium (11057980) but for semi-micro & micro-Kjeldahl applications.

Missouri# 11057982

4.98 g K2SO4

0.02 g CuSO4 • 5 H2O

5 g


Easy to use and universally applicableThe digestion with Missouri is more eco-friendly

ECO# 11057983

3.998 g K2SO4

0.002 g CuSO4

4 g


Eco-friendlyMost environmentally friendly catalyst, due to the very low cop-per content

Antifoam# 11057984

0.97 g Na2SO4

0.03 g Silikon Schaumhemmer1 g


Maximum foam reductionUsed 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 H2O1.65 g


Reduced chemical amountCombo tablets for Antifoam or micro Kjeldahl applications.

Sample preparationKjeldahl Tablets overview


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 [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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∙ 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.

The digestion Hints

Inlet for ambient air

Safety zone (≈ 5 cm)

Condensation zone

Boiling /digesting sample

Dig

estio

n

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█ █

█ █

█ █

TKN using 500 mL sample tube

Step Temp. [°C] Level Time1)

[min] Vol. [mL]

Preheating 380 7

Digestion 520 9 45 10

50 25

60 50

80 100

135 250

185 400

Cooling 301) 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 8.5 10

550 9.5 10

490 8.5 65

Cooling 30

Micro Kjeldahl using 100 mL sample tubes

Step Temp. [°C] Level Time [min]

Preheating 480 8.5

Digestion 480 8.5 10

500 9.5 5

480 8.5 45

Cooling 15

DigestionDigestion parameter: SpeedDigester (IR)

12

T nominalT sample

T nominalT sample

T nominalT sample

First H2O has to evaporate before the temperature can increase

Digestion

The sample is kept constantly boiling.

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 490°C) to reach the boiling tempera-ture and the digestion time can significantly reduced.

The sample is heated to boil as fast as possible.

Tem

p. [°

C]

Tem

p. [°

C]

Tem

p. [°

C]

Time [min]

Time [min]

Time [min]


█ █

█ █

█ █

█ █

Standard Kjeldahl digestion using Kjeldahl Tablets

Step Temp. [°C] Time [min]

Preheating 300 0

Digestion 340 15

420 105

Cooling 35

Accelerated Kjeldahl digestion using Kjeldahl Tablets + H2O2

Step Temp. [°C] Time [min]

Preheating 330 0

Digestion 420 60

Cooling 25

DigestionDigestion parameter: KjelDigester (only 300 mL tubes)

13

To avoid foaming and over splashing of the sample, it is heated up slowly (3 steps)

The sample is kept constantly boiling

330 °C is used as preheat temperature to avoid splashing, due to too fast heating of the sample

The sample is kept constantly boiling

T nominalT sample

T nominalT sample

Time [min]

Time [min]

Tem

p. [°

C]

Tem

p. [°

C]


█ █ ██

120

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Digestion Highest sample throughput

Use optimized «Kjeldahl Throughput» solution to get the highest sample throughput (batch size: 20 samples)

1. Sample preparation per batch: 40 min Mixer B-400

batch #1

batch #2

batch #3

batch #4

batch #5

batch #6

2. Digestion per batch: 100 min KjelDigester K-449

batch #1

cool

ing

batch #2

cool

ing

batch #3

cool

ing

batch #4

cool

ing

batch #5

cool

ing

batch #6co

olin

g

3. Distillation / Titrationper batch: 100 minKjelMaster K-375, KjelSampler K-376 / K-377

batch #6 of the day before

batch #1 batch #2 batch #3 batch #4 batch #5(unattended)

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

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

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

Time saving: 100 minTime saving: 30 min

65 min


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 ▶

Step 4 can be varied as required for ∙ Boric acid titration ∙ Back titration

H2O additionNaOH additionH2O steam additionacid (H3BO3 or H2SO4)

DistillationOverview

Dis

tilla

tion

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Step Why How much Rule-of-the-thumb

Step 1: Dilution

H2O dist. Dilution of the strongly acidicsolution, prevents violent reactions

25 – 90 mL 4 mL per mL used H2SO4

Step 2: Alkalinization

NaOH 32 % Conversion of NH4+ in

NH3 (gaseous)15 – 90 mL 4.5 mL per mL

used H2SO4

Step 3: Preparation of the receiver

H3BO3 (pH 4.65)

To collect the distilled NH3. NH3 is bound as borate complex (NH4B(OH4).

40 – 70 mL 2 % H3BO3 with KCl for low N contents 0.02 − 6.75 mg/sample tube

4 % H3BO3 for medium and high N content6.75 – 125 mg/sample tube

Step 4: Distillation

Water steam (100 %)

Separation of NH3 by boiling of the sample

180 – 300 s Distillation time:180 s with KjelMaster240 s with others

Step 5: Collection

NH3 In boric acid receiver of pH 4.65

Condenser outlet tubeand electrode must be completely immersed

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

DistillationParameter for boric acid titration

16


DistillationParameter for back titration

17

Step Why How much Rule-of-the-thumb

Step 1: Dilution

H2O dist. Dilution of the strongly acidic solution, prevents violent reactions

25 – 90 mL 4 mL per mL used H2SO4

Step 2: Alkalinization

NaOH 32 % Conversion of NH4+ in

NH3 (gaseous)15 – 90 mL 4.5 mL per mL

used H2SO4

Step 3: Preparation of the receiver

H2SO4 (0.25 mol/L)

To collect the distilled NH3.

Surplus of H2SO4 is titrated with NaOH.

10 – 20 mL Volume must be exactly dosed (usually 20 mL)

Step 4: Distillation

Water steam(100 %)

Separation of NH3 by boiling of the sample

180 – 300 s Distillation time:180 s with KjelMaster240 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.


∙ 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.

DistillationHints

18


Boric acid titration

Potentiometric titration 1. Standard a) endpoint titration / b) startpoint titration

2. Online a) endpoint titration / b) startpoint titration

Colorimetric titration 1. Standard a) endpoint titration

Start volume before titration can be used for all titration variants.

Back titration

Potentiometric Titration 1. Standard a) endpoint titration

Advantage Disadvantage

Potentiometric: direct pH measurementLower detection limit / online titration possible / shorter analysis time / „IntelliDist“ possible

calibration required / 6 – 12 months lifetimeof electrode

Colorimetric: detection of the color changeNo calibration necessary / longer lifetime of the probe / end point visible

„IntelliDist“ not possible / indicator required / more boric acid necessary (compared to pot.)

Standard: the titration starts after the distillation is finishedStandardized process: Distillation → titration / easier monitoring

Longer analysis time as for Online-Titration

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)

EndpointFixed and known pH pH of boric acid must be adjusted to 4.65

StartpointAdjustment of pH not needed More boric acid required (compared to end

point titration), to detect the startpoint

Start volumeHuge time saving, higher sample throughput Only for well-known titration volume

TitrationOverview

Titr

atio

n

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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 pre-viously be adjusted to 4.65

Startpoint titration (only step 4 must be changed)


4. Startpoint pH is detected

pH-value before NaOH addition

Adjustment of the pH not necessary

TitrationEndpoint and Startpoint titration (potentiometric)

Endpoint 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


4. Endpoint Titrant NaOHpH-value

Back titration pH 7.00

Endpoint neutral pH 7.00

Back titration (potentiometric)

20


Endpoint titration



Optimal Only „Optimal“, not „Slow“

2. Start volume Titrant HCI / H2SO4

Only for well-known samples / N-content and high titration volume


4. Endpoint Color change Sher Indicator in H3BO3

TitrationEndpoint titration (Colorimetric)

21


∙ 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.

TitrationHints

22

Endpoint Titration Boric Acid B(OH)3 + 2 H2O ⇌ B(OH)4

– + H3O+ (pKa = 9.24)

Sher Indicator

Endpoint pH 4.65

B(OH)3 + 2 H2O

B(OH)4– + H3O

+

Acid consumption [mL]


∙ 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 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):

6.07 % N ∙ 6.25 = 37.94 % P

(7.5mL – 1mL) ∙ 2 ∙ 0.25 ∙ 1 ∙ 14.007 ∙ 100 % —————————————————————————— = 6.07 % N 0.750 g ∙ 1000

Result calculationHints

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 % N: % of weight of N% P: % of weight of protein R

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www.buchi.com/kjeldahl/applications

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11592548 Kjeldahl Practice Guide 210x210 en D Low