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05
PT. WONOKOYO JAYA CORPORINDO
Revisi :1
DIBUAT DISETUJUI
Purpose: Standard Operation Procedure for Kjeldahl Nitrogen/ Crude Protein using Block
Digestion with Copper Catalysts and Steam Distillation into Boric Acid on basis of the global
standard AOAC 2001.11 /ISO 5983-2:2009.
Tujuan: Standard Operation Procedure untuk Kjeldahl Nitrogen/ Protein Kasar
menggunakan Digestion Block dengan katalis tembaga dan distilasi uap ke dalam larutan
asam borat yang mengacu pada standard AOAC 2001.11 /ISO 5983-2:2009.
01. Scope
The method is applicable from 0.5 - 50% nitrogen and to animal feed, forage (plant tissues),
grain & oilseeds, fish feed and pet food.
NOTE : This method does not measure oxidized forms of nitrogen and does not fully recover
heterocyclic nitrogen compounds. It should be used in conjunction with the instrument
manual and the Foss application note AN 300.
02. Principle
The method involves block digestion of the sample in sulphuric acid to convert the protein
nitrogen to ammonium sulphate. The boiling point is elevated by the addition of potassium
sulphate. The elevated boiling temperature is necessary to break the peptide bonds and convert
the amino groups in protein to ammonium ions. A copper catalyst is added to enhance the
reaction rate.
After the digestion, the digest-mix is diluted with water to avoid mixing concentrated alkali
with concentrated acid and to prevent the digest from solidifying. Water dilution can be done
manually or automatically in some distillation units. Ammonia is then liberated by addition of
alkali and steam distillation, trapped in a weak boric acid solution and titrated with a
standardized hydrochloric acid, using colorimetric end-point detection.
The amount of protein is calculated by multiplying the percentage nitrogen by 6.25.
03. Safety
The sulphuric acid and sodium hydroxide used are in concentrated forms and are highly
corrosive. Wear gloves and eye protection while handling. Do not mix concentrated acid and
sodium hydroxide. If splashed on the skin or in the eyes, flush with copious amounts of water.
Seek medical attention. The sulphur oxide fumes produced during digestion are hazardous to
breathe. An exhaust system should be connected to the digestion block.
04. Frequency
This analysis is performed at Wonokoyo on a daily basis.
04. Frekuensi
Analisis ini dilakukan setiap hari di Wonokoyo.
05. Reagents
05. Reagen
05.02. Salt and catalyst – Kjeltabs Cu (3.5g K2SO4 and 0.4g CuSO4 per tablet, ordering
no.15270018)
05.02. Kjeltabs Cu (3,5 g K2SO4 dan 0,4 g CuSO4 per tablet, cat. no.15270018)
05.04. Methyl red indicator solution – Dissolve 100 mg methyl red in 100 ml methanol.
05.04. Indikator Metil Red- Larutkan 100 mg metil merah dalam 100 ml metanol
05.05. Bromocresol indicator green solution – Dissolve 100 mg bromocresol green in 100 ml
methanol.
05.05. Indikator Bromocresol green- Larutkan 100 mg bromocresol green dalam 100 ml
metano
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06. Apparatus
06. Alat-alat
08. Procedure
08. Prosedur
08.01. Digestion
08.01.02. Weigh the samples, as indicated below, recording each sample weight (W) to
the nearest mg for weights >1 g and to the nearest 0.1 mg for weights <1.0 g.
Do not exceed 1.2 g. For samples in protein (< 80%), weigh approximately
0.5 g of the sample, (> 80%), weigh approximately 0.3 g.
Fold paper around the sample and drop into a numbered Kjeldahl tube.
08.01.02. Timbang sampel, catat setiap berat sampel (W) ke mg terdekat untuk berat >
1 g dan 0,1 mg terdekat untuk berat <1,0 g. Jangan melebihi 1,2 g.
Untuk sampel protein (< 80%), timbang sampel sekitar 0,5 g.
Untuk sampel protein tinggi (> 80%), timbang sampel sekitar 0,3 g.
Lipat kertas yang berisi sampel dan masukkan ke dalam tabung Kjeldahl.
08.01.03. Each run should contain a quality control sample and standards every day.
See section “Quality Control”.
08.01.03. Setiap menjalankan analisa harus disertakan juga sampel kontrol (kontrol
samples) dan standard setiap hari. Lihat bagian "Quality Control"
08.01.04. Each day run should contain a reagent blank tube containing a folded
nitrogen-free weighing paper.
08.01.04. Setiap hari saat menjalankan analisa harus disertakan juga tabung Kjeldahl
kosong berisi kertas timbang bebas nitrogen (blank).
08.01.07. Place the fume manifold tightly on the tubes and turn the Scrubber on
completely. Place the rack of tubes in the pre-heated block. The following
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steps 08.01.08-10 are done automatically with the digestion systems TD
2520.
08.01.07. Tempatkan manifold fume pada tabung dan putar Scrubber pada kecepatan
penuh. Tempatkan rak tabung dalam digestion block. Langkah-langkah
08.01.08-10 dapat dilakukan secara otomatis dengan digestion system TD
2520.
08.01.08. After 10 minutes, turn the H2O aspirator down until the acid fumes are just
contained within the exhaust hood. A condensation zone should be
maintained within the tube. After the bulk of the sulphur oxide fumes are
produced during the initial stages of digestion, the vacuum source must be
reduced to prevent loss of H2SO4.
08.01.08. Setelah 10 menit, putar aspirator H2O bawah sampai asap asam berada dalam
tabung scrubber. Tabung scrubber tersebut merupakan area kondensasi.
Setelah sebagian besar asap belerang oksida yang dihasilkan selama tahap
awal destruksi, kecepatan scrubber harus dikurangi untuk mencegah
hilangnya H2SO4.
08.01.10. Remove the rack of tubes with exhaust still in place and put in the stand to
cool for 30 minutes. Cooling can be increased by using a commercial air
blower or by placing in a hood with the hood sash pulled down to increase
airflow across tubes. When fuming has stopped, remove the manifold and
shut off aspirator.
08.01.10. Keluarkan rak tabung dengan tabung scrubber dan biarkan sampai dingin
hingga tabung scrubber mencapai suhu kamar (sekitar 30 menit).
Pendinginan dapat dipercepat dengan menggunakan blower udara. Setelah
asap habis, pindahkan scrubber dan matikan aspirator.
NOTE 1 : For manual pre-dilution wear gloves and eye protection! Carefully add a few ml of
deionised H2O to each tube. If spattering should occur, the tubes are still too hot.
Allow cooling for a few more minutes. Add H2O to each tube approximately 10 ml.
CATATAN 1: Pada penambahan akuades secara manual, gunakan sarung tangan dan pelindung
mata! Hati-hati dalam menambahkan akuades untuk setiap tabung. Jika terjadi
percikan, berarti tabung masih terlalu panas. Lakukan penambahan akuades
sekitar 10 ml setiap tabung.
NOTE 2 : The sample digest must contain residual sulphuric acid to prevent liberation of
ammonia.
CATATAN 2 : Sampel terdestruksi harus mengandung residu asam sulfat untuk mencegah
pembebasan amonia.
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NOTE 3 : If the sample should solidify, place the tube containing diluted digest in the block
digester and carefully warm with occasional swirling until salts dissolve.
CATATAN 3 : Jika terjadi kristalisasi setelah destruksi, masukkan kembali tabung ke digestion
block dan kocok sesekali sampai kristal larut.
08.02. Distillation
08.02. Distilasi
08.02.01. Place 40% NaOH in alkali tank of distillation unit. Adjust volume dispensed
to 60 ml. Select program and start analysis.
08.02.01. Masukkan NaOH 40% dalam tangki alkali unit distilasi. Set volume alkali ke
60 ml. Pilih program dan lakukan analisa.
09.02.01. Nitrogen loss. - Use 0,12 g ammonium sulphate and 0,7 g sucrose per flask.
Add all other reagents as stated in Sample Preparation. Digest and distil
under same conditions as for sample. Recovery shall be at least 99 %. Do this
check every time the control sample gives too low results.
09.02.01. Nitrogen loss – Timbang 0,12 g amonium sulfat dan 0,7 g sukrosa setiap
tabung. Tambahkan semua reagen lainnya sebagaimana tercantum dalam
Preparasi Sampel. Destruksi dan distilasi dalam kondisi yang sama seperti
untuk sampel. Recovery minimal adalah 99%. Lakukan pemeriksaan ini
setiap kali hasil analisa sampel kontrol terlalu rendah.
09.02.02. Digestion efficiency - Use 0,12 g acetanilide with about 0,7 g sucrose per
flask. Add all other reagents as stated in Sample Preparation. Digest and
distil under same conditions as for a sample. Recovery shall be at least 98%.
This check should be made at least once per week and be documented.
09.02.02. Digestion efficiency – Timbang 0,12 g Acetanilide dan 0,7 g sukrosa setiap
tabung. Tambahkan semua reagen lainnya sebagaimana tercantum dalam
Preparasi Sampel. Destruksi dan distilasi dalam kondisi yang sama seperti
untuk sampel. Recovery minimal adalah 98%. Pemeriksaan ini harus
dilakukan setidaknya sekali per minggu dan didokumentasikan.
10. Results
10. Hasil
10.02. Calculate the % crude protein by multiplying with the factor 6.25.
10.02. Hitung % protein kasar dengan mengalikan dengan faktor 6,25.
11. References
1. AOAC Official Method 2001.11, Crude Protein in Animal Feed, Forage, Grain, and
Oilseeds, Block Digestion Using Copper Catalyst, Steam Distillation into Boric Acid,
www.aoac.org
2. ISO 5983-2:2009 Animal feeding stuffs -- Determination of nitrogen content and calculation
of crude protein content -- Part 2: Block digestion/steam distillation method, www.iso.org
3. Foss Application Note AN 300, www.foss.dk
4. Handbook for Kjeldahl digestion, Foss, fourth edition, 2009, www.foss.dk
11. References
Prosedur Tanggal Terbit:
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1. AOAC Official Method 2001.11, Crude Protein in Animal Feed, Forage, Grain, and
Oilseeds, Block Digestion Using Copper Catalyst, Steam Distillation into Boric Acid,
www.aoac.org
2. ISO 5983-2:2009 Animal feeding stuffs -- Determination of nitrogen content and calculation
of crude protein content -- Part 2: Block digestion/steam distillation method, www.iso.org
3. Catatan aplikasi Foss AN 300, www.foss.dk
4. Buku pegangan untuk protein kjeldahl, Foss, edisi keempat, 2009, www.foss.dk
12. Annex
Checklist of Protein Analysis
Protein Logbook
Manual Instruction Operational Kjeltec 8400
12. Lampiran
Checklist Analisa Protein
Buku Protein
Panduan Praktis Operasional Kjeltec 8400
Revisi Tanggal Terbit Bagian Yang Berubah Pada Dokumen Yang Telah Diubah
0 10 Desember 2012 -
1 16 Oktober 2013 Halaman 7 Butir 08.01.10