a. kjeldahl method

ANALISIS PROTEIN
--------------------------------------------Disampaikan oleh
Sri Naruki
Fakultas Teknologi Pertanian UGM
PROTEIN
• Protein are an abundant component in all
cells (Nielsen, 2004)
• Food protein : very complex; MW ranging
from 5000 to more than a million Daltons
• Function : growth & maintenance of
tissue, formation of essential body
compounds, transportation of nutrients,
etc. (Guthrie, 1982)
• Protein consist of polypeptides, extremely
long chains of many amino acid units
• All protein in all species, regardless of
their function or biological activity, are
built from the same basic set of 20
standard amino acids, which by
themselves HAVE NO intrincsic biological
activity
• General structure of amino acids :
COOH
H2NCH
R
R = side chain or R group
• The simples amino acid : glycine  R = H
• In all amino acids, except glycine, the 
carbon atom has 4 different substituent
groups and is thus asymetric or chiral
carbon (Lehninger, p. 96)
The Peptide Bond
• Peptide bond : how the amino acids are
linked together to make a protein
H
H
H2NCCOOH + H2NCCOOH
R
R’
H O H
H2N-C-C-N-C-COOH + H2O
R H R’
• Proteins are composed of H, C, N, O, and
S (Nielsen, 2004)
• Composition of elements in proteins :
- C : 50 – 55 %
- O : 20 – 25 %
- N : 13.4 – 19.1 %
- H:5–7%
- S : 0.4 – 2.5 %
- P, Fe, Cu : trace
• C (karbon) : merupakan penyusun terbesar
• Bila C digunakan sebagai dasar analisis
protein
• Analisis protein berdasarkan C mempunyai
beberapa keuntungan :
a. Digesti lebih mudah bila dibandingkan
dengan digesti pada metoda analisis
berdasarkan N
b. Kadar C protein = tinggi sehingga
- mengurangi error
- faktor konversi relatif konstan
• TAPI INGAT : ada beberapa senyawa lain
yang juga mengandung C  karbohidrat
dan lipida
• Oleh karena itu, C nonprotein tersebut
harus dihilangkan terlebih dahulu
• Pemisahan C nonprotein tersebut  sulit
Oleh karena itu, yang lazim dilakukan
adalah : penentuan kadar protein berdasarkan kadar nitrogen
• Nitrogen is the most distinguishing
element present in proteins
• N content in various food protein ranges
from 13.4% to 19.1%  an average =
16.0%
• Therefore, protein content (%)
= 100/16x % N
= 6.25 x % N
conversion factor/CF (faktor konversi/FK)
Conversion Factors
• N content in various food protein ranges
from 13.4% to 19.1%  conversion
factors for various food are varied :
- Egg or meat
 CF = 6.25
- Dairy products
6.38
- Wheat
5.70
- Other cereal grains & oilseeds
6.25
- Almonds
5.18
- Peanuts & Brazil nuts
5.46
- Other tree nuts & coconut
5.30
ANALYSIS OF PROTEIN
• Analysis of protein is very complicated
since some food components posses
similar physicochemical properties
• Nonprotein nitrogen could come from :
- Free amino acids
- Some vitamins
- Small peptide
- Alkaloids
- Nucleic acids
- Uric acid
- Porphyrin
- Urea
- Ammonium ions
- Etc
Aspartame is a small peptide composed of Asp, Phe,
and methanol
Nucleic acids are polymer formed from linking of
various nucleotides
Struktur vitamin B1 (tiamin)  juga mengandung N
Caffeine alkaloid
Chemical structure of uric acid
Chemical structure of urea
• The total organic nitrogen in foods would
represent nitrogen :
- Primarily from protein
- A lesser extent from all organic nitrogencontaining nonprotein substances
• Therefore, % Protein = CF x % N
is called : crude protein (protein kasar)
• Other major food components, including
carbohydrate and lipid, may interfere
physically with analysis of food proteins
Importance of Analysis
1. Nutrition labeling
2. Functional property investigation
Proteins of food have unique functional
properties. Example : gliadin & glutenin
in wheat flour for bread making
3. Biological activity determination, including
enzymes & enzyme inhibitor. Enzyme
activity is expressed in terms of specific
activity : unit of enzyme/mg of protein
• Protein analysis is required if you want to
know :
1. Total protein content
2. Content of particular protein in mixture
3. Protein content during isolation and
purification of protein
4. Nonprotein nitrogen (ex. TMA content)
5. Amino acid composition (profil asam amino)
6. Nutritive value of protein (ex. PER, BV)
7. Activity (of enzyme)
Protein Content of Selected Foods
Food item
% Protein
(wet weight basis)
CEREALS & PASTA
Rice, brown, raw
Wheat flour
Corn flour, yellow
Spaghetti, dry, enriched
Cornstarch
7.9
13.7
6.9
12.8
0.3
DAIRY PRODUCTS
Milk, whole, fluid
Cheese, cheddar
3.3
24.9
FRUITS AND VEGETABLE
Apple, raw, with skin
Potato, whole, flesh and skin
0.2
2.0
LEGUMES
Soybean, mature seeds, raw
36.5
MEAT, POULTRY. FISH
Egg, raw, whole
Ham, sliced, regular
12.5
17.6
The Basic Principles of Some
Methods to Measure Protein Content
• Determination of nitrogen, peptide bonds,
aromatic amino acids
• Based on the dye-binding capacity of
protein
• Based on the UV absorptivity of protein
• Based on the light scattering properties of
protein
The Basic Principles of Some Methods
to Measure Protein Content
I. Determination of nitrogen,
peptide bonds, aromatic amino
acids
A. Kjeldahl method
B. Biuret method
C. Lowry method
D. Titrasi formol
E. Dumas method
F. BCA method
A. KJELDAHL METHOD
• On March 7, 1883, Johann Kjeldahl presented
his method of N analysis
• Today, the Kjeldahl method for the
determination of organic nitrogen is the
worldwide standard for the purpose of
calculating the protein content in both
human food and animal feed
• The Kjeldahl method has been adapted as
a standard method of N analysis in
fertilizer, fossil fuel, waste water, etc.
PRINCIPLE
• Proteins & other organic food components
in a sample are digested with sulfuric acid
in the presence of catalysts
• The total organic nitrogen is converted to
ammonium sulfate
• The digest is neutralized with alkali and
distilled into a boric acid solution
• The borate anions formed are titrated with
standardized acid, which is converted to N
in the sample
• The result of analysis represents the crude
protein content of the food since N also
comes from nonprotein components
• The Kjeldahl method for nitrogen analysis
is composed of three distinct steps :
1. Digestion (destruksi)
2. Neutralization and Distillation
3. Titration
IMPROVEMENTS
1. Katalis logam seperti Hg, Cu, dan Se ditambahkan ke asam sulfat untuk menyempurnakan oksidasi.
- Yang terbaik : Hg
- Selenium dioksida dan Cu-sulfat (3:1) juga
efektif untuk digesti
- Copper and titanium dioxide also have been
used as a mixed catalyst for digestion  the
use of them poses less safety concern than
Hg in the post-analysis disposal of the waste
• Bila digunakan Hg, selama digesti akan
terbentuk senyawa kompleks Hg-amonia
• Hg harus diendapkan (sebagai HgS) dengan
penambahan Na-tiosulfat (Na2S2O3) 
presipitan Na2S2O3 dicampur dengan
NaOH yang digunakan untuk membebaskan NH3 (saat tahap netralisasi sebelum
distilasi)
• Na-tiosulfat juga ditambahkan untuk
membantu membebaskan N dari ikatannya dengan Hg (lihat butir 3)
• Di lapangan, campuran (NaOH + Na2S2O3)
dikenal dengan istilah NaOH-thio
• Katalis Se (selenium) :
- efek lebih hebat daripada Hg
- Tidak perlu step tambahan (yaitu penam-
bahan Na-tiosulfat /Na2S2O3)
- TAPI : bila jumlah Se berlebihan maka
suhu destruksi/digesti tidak terkontrol
sehingga ada kemungkinan kehilangan N
(karena menguap)
2. Penambahan potasium sulfat dapat dila-
kukan untuk menaikkan titik didih asam
sulfat sehingga meningkatkan digesti
Suhu digesti yang ideal : 370 – 410 oC.
TAPI bila K-sulfat berlebihan maka akan
terjadi kehilangan N
3. Sulfide or sodium thiosulfate are added to
the diluted digest to help release nitrogen
from mercury, which tends to bind to
ammonium
4. The ammonia is distilled directly into a
boric acid solution, followed by titration
with standard acid
5. Colorimetry Nesslerization, or ion chromatography to measure ammonia, is used to
determine nitrogen content after digestion
Beberapa Modifikasi
1. Cara makro Kjeldahl
- Berat sampel : 1 g
- Katalis : K2SO4-HgO
- Distilasi : memapak lempeng Zn dengan
tujuan agar supaya
* tidak terjadi superheating
* tidak terjadi percikan cairan
* tidak terbentuk gelembung gas yang
besar
2. Cara mikro Kjeldahl
- Berat sampel : 10 – 30 mg
- Katalis : Na2SO4 –HgO
3. Cara Gunning
- Berat sampel : 0,7 – 5,5 g
- Katalis : K2S atau Na2SO4 dan CuSO4
GENERAL PROCEDURE & REACTIONS
• As stated before, the Kjeldahl method for
nitrogen analysis is composed of three
distinct steps. Sample preparation should
be done prior to these three steps.
1.
2.
3.
4.
Sample preparation
Digestion (destruksi)
Neutralization and Distillation
Titration
1. Sample Preparation
• Solid food are ground to pass a 20 mesh
screen
• Samples for analysis should be
homogeneous
• No other special preparation are required
2. Digestion Step
• The purpose of digestion : to break the
intricate structure and chemical bonds that
hold a chemical substances (piece of
meat, cup of flour, or quart of oil) down
into simple chemicals and ionic structures
• Specifically, proteins and other forms of
nitrogen are broken down and converted
to ammonia
• To digest the sample, 1 – 2 g of the
sample are placed on a digestion tube
with 12 – 15 mL of concentrated H2SO4.
Seven grams of K2SO4 and a metallic
catalyst, usually Cu, are the added.
• The digestion tube is heated to the boiling
temperature of the mixture
• Digestion was done to complete oxidation
and conversion of N to ammonium sulfate
• The digestion is usually completed after
one hour at 370 – 400 oC
a. Procedure of Digestion
• Place sample (accurately weighed) in a
Kjeldahl flask
• Add sulfuric acid and catalyst
UNTUK MENGOKSIDASI
MEMERLUKAN H2SO4
1 g Protein
+ 9,0 g
1 g Lemak
+ 17,8 g
1 g Karbohidrat
+ 7,3 g
• Digest until CLEAR to get complete breakdown
of all organic matter
• Digesti diakhiri bila larutan menjadi jernih
dan tak berwarna
b. Reactions During Digestion
• Nonvolatile ammonium sulfate is formed
from nitrogen and sulfuric acid :
Sulfuric acid
Protein
(NH4)2SO4
Heat, catalyst
• During digestion :
- protein nitrogen is liberated to form
ammonium ions
- Sulfuric acid oxidized organic matter &
combines with ammonium formed
- C is converted to CO2 (volatile/menguap)
- H is converted to H2O (volatile/menguap)
• Lama digesti bervariasi, tergantung dari
jenis sampel yang dianalisis
- Protein kaya akan asam amino histidin
(His) dan triptofan (Trp) : digesti lama
(perlu katalis lebih banyak)
- Sampel kaya lemak : digesti lama (perlu
H2SO4 lebih banyak)
c. Reaksi lengkap
HgO + H2SO4  HgSO4 + H2O
HgSO4  Hg2SO4 + SO2 + On
(CHON) + On + H2SO4  CO2 + H2O +
(NH4)2SO4
protein
3. Neutralization & Distillation Step
• Pada tahap ini (NH4)2SO4 dipecah menjadi
NH3 dengan penambahan NaOH (sampai
dica-pai kondisi alkalis) dan pemanasan
(distilasi)
• Selama distilasi dapat ditambahkan lempeng
Zn
• NH3 yang dibebaskan ditangkap oleh larutan
asam standar berlebihan. Asam dapat berupa
- Asam borat 4%
- HCl 0,1N
• Ujung alat distilasi harus tercelup larutan
asam
• Distilasi diakhiri bila destilat yang dihasilkan tidak lagi bereaksi basa. Caranya ????
• Reaksi yang terjadi saat netralisasi dan
distilasi :
(NH4)2SO4 + 2NaOH  2NH3 + Na2SO4 +
2H2O
• Selanjutnya NH3 yang dibebaskan akan
bereaksi dengan larutan penampung
sebagai berikut :
• Bila penampungnya asam borat :
NH3 + H3BO3 (boric acid)  NH4 +
H2BO3– (borate ion)
• Bila penampungnya HCl :
2NH3 + 2HCl (berlebihan)  2NH4Cl +
HCl (sisa)
Procedure of Netralization & Distillation
• The digest is diluted with water
• Alkali-containing sodium thiosulfate is
added to neutralize the sulfuric acid
• The ammonia formed is distilled into a
boric acid solution containing the indicator
methylene blue and methyl red
4. Titration
a. Bila digunakan penampung asam
borat (METODA TITRASI LANGSUNG)
- Borate anion (proportional to the amount
of nitrogen) is titrated with standardized
HCl (anion borat dalam penampung
dititrasi dengan HCl standar 0,1N )
- Reaction : H2BO3– + H+  H3BO3
- Calculation
Moles of HCl = moles of NH3 = moles N
in the sample
• A reagent blank should be run to subtract
reagent nitrogen from the sample nitrogen
• %N=
N HCl x (corrected acid volume/g of sample)
x (14g N/mol) x 100
Where :
- N HCl = normality of HCl, in moles/1000ml
- Corrected acid volume =
(ml std acid for sample) – (ml std acid
for blank)
• Atau :
% N = (ml HCl sampel – ml HCl blanko) :
berat sampel (g) x 1000 x NHCl x
14,008 x 100%
• Untuk menghitung kadar protein, maka %
N dikalikan faktor konversi (CF atau FK)

CF beberapa jenis bahan makanan : lihat
slide sebelumnya
b. Bila digunakan penampung HCl
(METODA KONVENSIONAL / BACK
TITRATION / TITRASI BALIK)
• Bila digunakan penampung HCl, maka HCl
sisa dititrasi dengan NaOH standar (0,1N)
• Reaksi
- Saat distilasi :
2NH3 + 2HCl (berlebihan)  2NH4Cl +
HCl (sisa)
- Saat titrasi :
HCl (sisa) + NaOH  NaCl + H2O
Perhitungan
• Pada metoda konvensional, perlu dua (2)
macam larutan standar yaitu HCl standar
dan NaOH standar
• meq NH3 hasil distilasi = (meq asam mulamula – meq asam setelah distilasi)
• meq NH3 = (ml asam x N asam) –
(ml NaOH x N NaOH)
• Berat N (g) = meq N x 0,014007
• Kadar N (%) =
(ml HCl x N HCl) – (ml NaOH x N NaOH)x 1,4
g sampel
• Atau :
%N =(ml NaOH blanko – ml NaOH sampel)
: berat sampel (g) x 1000 x NNaOH
x 14,008 x 100%
• A factor is used to convert % N to %
crude protein
• Most proteins contain 16% N, so the
conversion factor (CF) is 6.25
% Protein = % N x 6.25
PROSEDUR LENGKAP METODA
KJELDAHL
0,7 – 2.2 g sampel (dalam labu Kjeldahl)
0,7g HgO (atau 0,65g logam Hg)
15g K2SO4 (atau Na2SO4 anhidrat)
25ml H2SO4 pekat
DESTRUKSI/DIGESTI (didihkan) pelan-pelan,
tambahkan sedikit parafin
Setelah jernih, didihkan lagi + 30 menit
PENDINGINAN (next slide)
PENDINGINAN
+ 200 ml aquades dingin
Butiran Zn
25ml larutan Na2S2O3 8%
+ 15g NaOH
PEMASANGAN RANGKAIAN DISTILASI
PEMASANGAN ERLENMEYER PENAMPUNG
(15ml HCl 0,02N + 6 tetes MR-BCG)
DISTILASI (Diakhiri bila distilat mencapai > 150ml)
(Cuci ujung kondenser)
TITRASI HCl sisa dalam Erlenmeyer dengan NaOH
LAKUKAN PENENTUAN BLANKO !!
PROSEDUR LENGKAP METODA
MIKRO KJELDAHL
+ 30mg Sampel (dalam labu Kjeldahl)
2g K2SO4; 40mg HgO
2ml H2SO4 pekat
Batu didih
DESTRUKSI/DIGESTI
Bila larutan sudah jernih, tambahkan aquades
dan didihkan lagi + 1,5 jam
PENDINGINAN & PEMINDAHAN KE LABU DISTILASI
PENDINGINAN
8 – 10ml NaOH-Na2S2O3
DISTILASI
- Tampung distilat dalam Erlenmeyer berisi :
5ml H3BO3 4% + 4 tetes indikator
(0,2% MR + 0,2% BCG)
- Distilasi diakhiri saat distilat sudah tidak
lagi alkalis
TITRASI
- Distilat dititrasi dengan HCl 0,02N
LAKUKAN PENENTUAN BLANKO !!
ALTERNATE PROCEDURES
• In place of distillation & titration with acid,
ammonia or nitrogen can be quantitated
by :
1. NESSLERIZATION :
4NH4OH + 2HgI2 + 4KI + 3KOH 
mercuric iodide
NH2Hg2IO + 7KI + 2H2O
ammonium dimercuric iodide
• NH2Hg2IO (red-orange) can be determined
spectrophotometrically at 440nm
• This method is rapid & sensitive, but the
ammonium dimercuric iodide is colloidal and
color is not stable
OH–
2. NH3 + phenol + hypochlorite
indophenol (blue, 630nm)
3. pH measurement after distillation into known
volume of boric acid
4. Direct measurement of ammonia, using ion
chromatographic method
ADVANTAGES
1. Applicable to all types of foods
2. Inexpensive(if not usingan automated
system)
3. Accurate; an official method for crude
protein content)
4. Has been modified (micro Kjeldahl
method) to measure microgram
quantities of protein)
DISADVANTAGES
1. Measures total organic nitrogen, not just
protein nitrogen
2. Time consuming (at least 2 hr to
complete)
3. Poorer precision than the Biuret method
4. Corrosive reagent
CONTOH SOAL
• Pada analisa kadar protein dengan metoda
Kjeldahl, diperoleh hasil kadar protein
susu bubuk skim adalah 40%. Penampung
distilat yang digunakan adalah HCl
berlebihan. Bila berat sampel = 0,25g dan
titrasi blanko membutuhkan NaOH 0,1N
sebanyak 45ml, hitunglah volume NaOH
untuk titrasi sampel. Faktor konversi N ke
protein skim = 6,38.
B. BIURET METHOD
1. PRINCIPLE
• A violet-purple color is produced when
cupric ions are complexes with > 2 peptide bonds, under alkaline conditions)
• The absorbance of the color produced is
read at 540 nm
• The color intensity (absorbance) is
proportional to the protein content of the
sample
2. REACTIONS
H
O H
HOO–C–N–C –C –NH2 + CuSO4 + NaOH 
R H
R
R H H R
2HN–C–C–N–C–COOH
HO
H
Cu
+ Na2SO4 + H2O
H
O
HOO–C–N–C–C–NH2
R H H R (senyawa kompleks ungu)
• Hasil yang diperoleh dapat dipengaruhi
oleh adanya lipida dan komponen lain
yang dapat mengubah warna ataupun
memberikan respons yang sama dengan
ikatan peptida.
• Contoh gugus yang memberi respons yang
sama dengan ikatan peptida :
–CSNH2
–C(NH)NH2
–CH2NH2
–CRHNH2
–CHNH2CH2OH
–CHNH2CHOH
–CHOHCH2NH2
3. PROCEDURE
1. A 5-ml biuret reagent is mixed with a
1-ml portion of protein solution (1 to
10mg protein/ml). The reagent include
copper sulfate, NaOH, and K-Na-tartrate,
which is used to stabilize the cupric ion in
the alkaline solution
2. After the reaction mix is allowed to stand
at room temperature for 15 or 30 min,
the absorbance is read at 540 nm against
a reagent blank
• Filtration or centrifugation before reading
absorbance is required if the reaction
mixture is not clear
• A standard curve of concentration versus
absorbance is constructed using bovine
serum albumin (BSA)
BSA : 0, 20, 40, 60, 80, 100, 120 g/mL
4. ADVANTAGES
• Lebih murah daripada metoda Kjeldahl,
cepat (dapat selesai dalam < 30 menit),
paling sederhana
• Deviasi warna jarang terjadi dibanding
dengan metoda Lowry, absorpsi UV, atau
turbidimetri
• Senyawa yang dapat mengganggu analisis
hanya sedikit sekali
• Tidak mendeteksi nitrogen dari
sumbernonprotein
5. DISADVANTAGES
1. Kurang sensitif bila dibandingkan dengan
metoda Lowry; perlu minimal 2 – 4 mg
protein untuk analisa
2. Absorbansi dapat berasal dari pigmen
empedu, bila pigemen tersebut ada di
dalam sampel
3. Garam amonium dengan konsentrasi
tinggi dapat mengganggu reaksi
4. Warna yang dihasilkan bervariasi dengan
perbedaan jenis protein. Misal : gelatin
menghasilkan warna pinkish-purple
5. Kondisi “tak tembus cahaya” dapat terjadi
pada larutan final apabila terdapat lipida
atau karbohidrat dalam jumlah banyak
6. Bukan merupakan metoda yang absolut :
warna harus distandardisasi dengan
protein yang diketahui (misal BSA) atau
dicocokkan dengan metoda Kjeldahl
Hubungan antara absorbansi pada 540
nm pada Biuret method dengan kadar
protein menurut metoda Kjeldahl
% Protein (Kjeldalh)
C. LOWRY METHOD
1. PRINCIPLE
• The Lowry method combines the biuret
reaction (see point B) with the reduction
of the Folin-Ciocalteau phenol reagent
(phosphomolybdic-phosphotungstic acid)
by tyrosin & tryptophan residues in the
protein
• The bluish color developed is read at 750
nm (high sensitivity for low protein
concentration) or 500 nm (low sensitivity
for high protein concentration)
Protein reaction with cupric ion under
alkaline condition
2a. PROSEDUR
• Terdapat 2 macam reagen Lowry, yaitu
- Lowry A (mengandung fosfotungstat-fosfo
molibdat 1 : 1)
- Lowry B (mengandung Na-karbonat 2%
dalam NaOH 0,1N serta Cu-sulfat dan NaK-tartrat 2%)
• Cara :
- 1 mL larutan protein sampel + 5mL
reagen Lowry B, gojog dan biarkan 10
menit
- Kemudian tambahkan 0,5 mL reagen
Lowry A dan biarkan 20 menit
- Baca nilai absorbansi pada 600 nm
2b. PROCEDURE
1. Proteins to be analyzed are diluted to an
appropriate range (20 – 100 g)
2. K Na Tartrate-Na2CO3 solution is added
after cooling and incubated at room
temperature for 10 min
3. CuSO4-K Na Tartrate-NaOH solution is
added after cooling and incubated at
room temperature for 10 min
4. Freshly prepared Folin reagent is added,
then the reaction mixture is mixedand
incubated at 50oC for 10 min
5. Absorbance is read at 650 nm
6. A standard curve of bovine serum
albumin (BSA) is carefully constructed for
estimating protein concentration of the
unknown
• Cu++ in alkaline solution to form complexity with
protein.
A at
750 nm
• Cu++ catalyses oxidation of phenol group of
tyrosine with phosphomolybdic-phosphotungstic
acid.
 g of protein (Kjeldahl)
3. ADVANTAGES
1. Very sensitive :
a. 50 – 100 times more sensitive than biuret
method
b. 10 – 20 times more sensitive than 280nm
UV absorption method
c. Similar sensitivity as Nesslerization;
however, more convenient than
Nesslerization
2. Less affected by turbidity of the sample
3. More specific than most other methods
4. Relatively simple, can be done in 1 – 1.5 hr
4. DISADVANTAGES
1. Color varies with different proteins to a
greater extent than biuret method
2. Color is not strictly proportional to protein
concentration
3. The reaction is interfered with to varying
degrees by sucrose, lipids, phosphate
buffers, monosaccharides, and hexoamines
4. High concentration of reducing sugars,
ammonium sulfate, and sulfhydryl
compounds interfere with the reaction
D. TITRASI FORMOL
1. PRINSIP
• Larutan protein dinetralkan dengan basa
(NaOH), kemudian ditambah formalin
sehingga terbentuk dimetilol
• Dengan terbentuknya dimetilol maka
gugus amino protein sudah terikat dan
tidak mempengaruhi reaksi antara asam
(gugus karboksil) dengan basa NaOH
sehingga akhir titrasi dapat diakhiri
dengan tepat
• Indikator yang dipakai adalah PP
• Akhir titrasi ditandai saat terjadinya
perubahan warna menjadi merah muda
yang tidak hilang dalam 30 detik
• Titrasi formol baik untuk digunakan untuk
evaluasi proses terjadinya pemecahan
protein (misal : pada fermentasi protein
pada tempe, kecap, tauco, dzsb.)
• Proses hidrolisis protein ditandai dengan
meningkatnya titrasi formol
2. REAKSI PADA TITRASI FORMOL
O
H O
R–CH–C–OH + NaOH  R–C–C–O–
NH2
NH3+
pada pH netral
H O
H
R–C–C–O– + CH2O

R–C–COOH
NH3+
(formalin)
HOH2C–N–CH2OH
(dimetilol)
H
R–C–COOH
H O
HOH2C–N–CH2OH + NaOH 
R–C–C–ONa
(dimetilol)
HOH2C–N–CH2OH
3. PROSEDUR
• 10 ml larutan protein sampel + 20 ml aquades + 0,4 ml larutan K-oksalat jenuh (Koksalat : air = 1 : 3) dan 1 ml indikator PP
1%. Diamkan selama 2 menit
• Titrasi larutan tersebut dengan 0,1N NaOH
sampai terbentuk warna pink atau warna
standar (10 ml susu + 10 ml aquades + 0,4
ml K-oksalat jenuh + 1 tetes indikator
rosanilin-khlorida 0,01%)
• Setelah warna tercapai, tambahkan 2 ml
formaldehid 40% dan titrasilah kembali
dengan larutan NaOH sampai warna
seperti warna standar tercapai lagi.
Catatlah nilai titrasi kedua ini.
• Dibuat titrasi blanko yang terdiri dari : 20
ml aquades + 0,4 ml larutan K-oksalat
jenuh + 1 ml indikator PP + 2 ml formaldehid, dan titrasilah dengan larutan NaOH
• Titrasi formol = titrasi terkoreksi
= titrasi kedua – titrasi blanko
• Bila nilai titrasi formol akan digunakan
untuk menentukan kadar protein, maka
harus dibuat percobaan serupa dengan
menggunakan larutan yang telah diketahui
kadar proteinnya (misalnya dengan
metoda Kjeldahl)
• Selanjutnya ditentukan hubungan antara
titrasi formol dengan % protein.
• Misal :
- % protein susu = 1,83 x ml titrasi formol
- % kasein = 1,63 x ml titrasi formol
E. DUMAS (NITROGEN
COMBUSTION) METHOD
1. PRINCIPLE
• Sample are combusted at high temperature (700 – 1.000oC)
• The nitrogen released is quantitated by
gas chromatography using thermal
conductivity detector (TCD)
• The nitrogen determined is converted to
protein content in the sample
2. PROCEDURE & APLICATION
PROCEDURE
• Sample (100 – 500 mg) are weighed into a
tin capsule and introduced to a combuston reactor in automated equipment
• The nitrogen released is measured by a
built-in gas chromatograph
APLICATION
• It is an alternative to the Kjeldahl method
• It is suitable for all type of foods
3. ADVANTAGES & DISADVANTAGES
ADVANTAGES
• Requires no hazardous chemicals
• Can be accomplished in 3 minutes
• Recent automated instrument can analyze
up to 150 samples without attention
DISADVANTAGES
• Expensive equipment is required
• Measures total organic nitrogen, not just
protein nitrogen
F. BICINCHONINIC ACID
(BCA) METHOD
1. PRINSIP
• Protein mampu mereduksi ion kupri (Cu2+)
menjadi ion kupro dalam suasana alkalis
• Dengan reagen BCA (berwarna applegreenish), ion kupro tersebut membentuk
kompleks berwarna purplish, yang
intensitasnya dapat ditera pada 562 nm
• Intensitas warna purplish tersebut proporsional dengan kadar protein
2. PROSEDUR
• Mix (one step) the protein solution with
the BCA reagent, which contain BCA
sodium salt, Na-carbonate, NaOH, and Cusulfate pH 11.25
• Incubate at room temperature for 2 hr, or
60oC for 30 min. A higher temperature
gives a greater color respon
• Read the solution at 562 nm against a
reagent blank
• Construct a standard curve using BSA
3. APPLICATION
• BCA method had been used in protein
isolation and purification
• The suitability of BCA method for
measuring protein in complex food has not
been reported
4. ADVANTAGES
1. Sensitivity of the micro BCA method (0.5
– 10 g) is better than Lowry method
2. One-step mixing is easier than in the
Lowry method
3. The reagent is more stable than for the
Lowry method
4. Nonionic detergent and buffer salts do
not interfere with the reaction
5. DISADVANTAGES
1. Color is not stable with time. The analyst
needs to carefully control the time for
reading absorbance
2. Any compound capable of reducing Cu2+
to Cu+ will lead to color formation
3. Reducing sugar interfere to a greater
extent than in Lowry method
4. Color variation among proteins are similar
to those in the Lowry method
The Basic Principles of Some Methods to
Measure Protein Content
II. Based on the UV absorptivity
of protein
ULTRAVIOLET (UV) 280 nm
METHOD
1. PRINCIPLE
• Protein show strong absorption at 280 nm,
primarily due to tyrosine (Tyr) and
tryptophan (Trp) residues in the protein
• Because the content of Tyr & Trp in
protein from each food source is fairly
constant, the A280 could be used to
estimate the concentration of protein
2. PROCEDURE
• Protein are solubilized in buffer or alkali
• Absorbance of protein solution is read at
280 nm against a reagent blank
• Protein concentration is calculated
according Beer’s law : A = abc
• A = absorbance, a = konstanta (molar absorp-
tivity for individual protein), b = cuvette
path length, c = concentration
• Bisa juga memakai standar BSA
3. APPLICATION
• It has been used to determine the protein
content of milk and meat products
• It has not been used widely in food system
• It is better applied in a purified protein
system
• It is also better applied in proteins that
have been extracted in alkali or denaturing
agents such as 8M urea
4. ADVANTAGES
1. Rapid and relatively sensitive (at 280 nm,
+ 100g protein is required, several times
more sensitive than the Biuret method)
2. Nondestructive : sample can be used for
other analysis after protein determination
3. No interference from ammonium sulfate
and other buffer salts
4. DISADVANTAGES
1. Nucleic acid also absorb at 280 nm.
2. The solution must be clear and colorless.
Turbidity due to particulates in the
solution will increase absorbance falsely
3. A relatively pure system is required to use
this method
The Basic Principles of Some Methods to
Measure Protein Content
III. Based on the dye-binding
capacity of protein
A. Dye : acid orange-12
B. Dye : coamassive brillian blue G-250
A. DYE BINDING METHOD
with acid orange-12 as a dye
Principle:
at low pH, basic groups of protein are (+) charged.
These will quantitatively bind a (-) charged dye.
What are these basic groups?
NH3
+
CH 2
CH 2
CH 2
CH 2
Lysine
CH 2
H
N
CH
N
H
C
C
O
CH 2
NH 2
NH2
O
CH 2
Arginine
C
CH
CH
N
H
C
CH 2
N
H
C NH+
HC
H
N
Histidine
+
Acid Orange 12:
HO
N=N
Procedure:
1.
Mix protein, dye, buffer pH = 2.  protein
+ dye : membentuk komplex tidak larut
2.
Filter or centrifuge.
3.
Measure optical density (OD) or
absorbancy of filtrate (filtrat mengandung
dye sisa yang tak bereaksi dengan protein)
SO3
-
Absorbance of dye bound by protein
= Absorbance of initial dye – Absorbance
of filtrate
Kadar protein  maka intensitas warna filtrat 
Skim milk
6
8
10
12
% Protein (Kjeldahl)
14
16
Factors Influencing Dye Binding determination:
1.
Temperature
2.
Non-proteins.
3.
Buffers systems
4.
Protein quality
B. DYE BINDING METHOD
with coamassie brillian blue G-250
as a dye
• PRINSIP : dye + protein  kompleks larut
• Yang ditera : absorbansi senyawa kompleks
yang larut tersebut
• Oleh karena itu : bila kadar protein  maka
absorbansi juga 
• Dengan tabel konversi yang menunjukkan
hubungan antara cat yang terikat protein
dengan kadar protein  kadar protein sampel
dapat diketahui
• Dapat pula dibuat garis regresi yang yang
menunjukkan hubungan antara cat yang terikat
protein dengan kadar protein
COMPARISON OF METHODS
1. SAMPLE PREPARATION
• Kjeldahl & Dumas methods require little
preparation  sample particle size of 20
mesh is satisfactory
• Other methods require fine particles for
extraction of protein from the complex food
systems
2. PRINCIPLE
• Kjeldahl & Dumas methods measure
directly the total amount of organic N in
the foods
• Other method measure the various
properties of protein. For examples :
- The Biuret method measures peptide
bonds
- The Lowry methos measures a
combination of peptide bonds and the
amino acids Tyr & Trp
3. SENSITIVITY
• Kjeldahl, Dumas, and Biuret method are
less sensitive than Lowry, BCA, or UV
method
4. SPEED
• Speed of determination in spectrophotometric method and the Dumas method are
faster than with the Kjeldahl method
AKHIR DARI
KULIAH DENGAN
POKOK BAHASAN
PROTEIN
TERIMA KASIH