Biochem

EXPERIMENT REPORT
Determining The Percentage of Protein in

Milkfish By Biuret Method
Submitted by
DESIANA ANGGRAENI
(12030194234)
INTERNATIONAL CHEMISTRY EDUCATION 2012

THE STATE UNIVERSITY OF SURABAYA
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
DEPARTMENT OF CHEMISTRY
2014
I.
TITLE OF EXPERIMENT:
Determining The Percentage of Protein in Milkfish By Biuret Method
II.
DAY/DATE OF EXPERIMENT:
Friday, 31th October 2014
III.
PURPOSE:
To determine the percentage of protein in milkfish by biuret method
IV.
BASIC THEORY:
PROTEIN
Proteins are polymers of amino acids. Twenty different types of
amino acids occur naturally in proteins. Proteins differ from each other
according to the type, number and sequence of amino acids that make up
the polypeptide backbone. As a result they have different molecular
structures, nutritional attributes and physiochemical properties. Proteins
are important constituents of foods for a number of different reasons. They
are a major source of energy, as well as containing essential amino-acids,
such as lysine, tryptophan, methionine, leucine, isoleucine and valine,
which are essential to human health, but which the body cannot
synthesize. Proteins are also the major structural components of many
natural foods, often determining their overall texture, e.g., tenderness of
meat or fish products. Isolated proteins are often used in foods as
ingredients because of their unique functional properties, i.e., their ability
to provide desirable appearance, texture or stability. Typically, proteins are
used as gelling agents, emulsifiers, foaming agents and thickeners. Many
food proteins are enzymes which are capable of enhancing the rate of
certain biochemical reactions. These reactions can have either a favorable
or detrimental effect on the overall properties of foods. Food analysts are
interested in knowing the total concentration, type, molecular structure and
functional properties of the proteins in foods.
Protein Solubility
The percentage of protein in milkfish by biuret method
Page 1
Proteins are macromolecules which are soluble in water. Proteins

are least soluble in water at their isoelectric points and are more soluble at
higher or lower pH's. The solubilitiy at pH's different than the isoelectric
point appears to be due to the presence of an excess of cationic groups or
anionic groups on the surface of the protein. If the protein is, for instance,
negatively charged at a pH larger than the isoelectric point, when two
proteins bump into each other, the net negative charge repels them and
they do not aggregate as one would expect from large molecules.
However, at the protein's isoelectric point there is no net charge. When a
negative end of one protein bumps into a positive end of another protein,
electrostatic attraction causes the two proteins to stick together. Other
proteins run into the "dimer" and join the group. Eventually, enough
proteins aggregate together that the protein precipitates.
MILKFISH
Milkfish are euryhaline, stenothermic fish. They occur and can be
cultured in freshwater, brackishwater, and marine waters but only in the
tropical and subtropical Indian and Pacific oceans (rare in eastern Pacific
from
southern
California
Peru)
to
where
temperature is
>20C.
Adult
milkfish (315
years, 50150 cm TL, 414 kg) are found in the open sea and spawn near
coral reefs and small islands, but metamorphose from ribbonlike larvae in
brackishwater. First sexual maturity occurs at 34 years. Newly hatched
larvae are about 3.5 mm TL and have a large yolk sac, unpigmented eyes
and no mouth. The yolk is completely absorbed on day 5. Two to three
week old fry (1013 mm TL) reach inshore waters via active migration or
through passive advection. Milkfish larger than 20 mm have the
characteristic shape and morphology of the adult and are considered
Page 2
juveniles. The life history of milkfish has been described in detail by

Bagarinao (1994). In aquaculture terminology, juveniles <10 cm long are
called fingerlings. Twenty one day-old fry caught from the wild or
obtained from a hatchery are ready to be stocked into nursery ponds. After
3045 days in the nursery, fingerlings are grown to marketable size (250
500 g) in ponds, pens, and cages. They are important food fish in
Southeast Asia, specifically in the Philippines, Indonesia, and Taiwan.
Protein and Amino Acids in Milkfish
(Nutrition facts and analysis,2014)
Determination of Protein Concentration

Determining the exact quantity of proteins in a solution is very
often necessary in the biochemical practice. There are many ways to
Page 3
measure protein concentration. In chromogenic methods, the absorbance

of a coloured product formed by the protein and an organic molecule is
measured. Protein concentration can also be determined from the proteins
own (intrinsic) UV absorbance. Note, however, that these methods may
give different results for different proteins of the same concentration. Also,
different methods can yield somewhat different results for the same
protein. There is no absolute photometric protein concentration assay. All
methods have advantages and disadvantages and we must choose among
them by taking the following aspects into consideration: specificity,
sensitivity, the measurable range of concentration, the accuracy, the nature
of the protein to be examined, the presence of materials interfering with
the measurement, and the time required for the measurement.
1) Biuret test
Molecules with two or more peptide bonds react with
Cu2+ ions in alkaline solution and form a purple complex. Nitrogen
atoms of the peptide bonds form a coordination bond with the
metal ion. The quantity of the complexes formed is proportional to
the number of peptide bonds.
The Biuret Test is a general test for proteins. When a
protein reacts with copper(II) sulfate (blue), the positive test is the
formation of a violet colored complex.
Page 4
The Biuret Test works for any compound containing two or

more of the following groups.
In practice, the determination of protein concentration is

done using a calibration curve created using samples of known
concentration. The protein treated with biuret reagent is measured
at 540 nm after the purple product is formed.
The advantages of the method include that only few
materials (e.g. Tris and amino acid buffers) interfere with it, it can
be done in a short time and does not depend on the amino acid
composition of the protein. Its disadvantages are its low sensitivity
and that it requires at least 1 mg of protein.
2) Lowry (Folin) protein assay
In this sensitive technique, a coloured product is formed
similarly to the biuret reaction, but a second reagent (FolinCiocalteu reagent) is used in addition to strengthen the colour. The
strong blue colour is created by two reactions: (1) formation of the
coordination bond between peptide bond nitrogens and a copper
ion and (2) reduction of the Folin-Ciocalteu reagent by tyrosine
(phosphomolybdic and phosphotungstic acid of the reagent react
with phenol). The measurement is carried out at 750 nm.
As in the biuret reaction, a calibration curve is created (for
example using BSA, bovine serum albumin), and the concentration
of the unknown protein is determined from the curve.
The advantages of the method include that it is quite
sensitive and is able to detect even 1 g of protein. Its
Page 5
disadvantages are that it takes rather long to carry out, is disturbed

by various materials (including ammonium sulphate, glycine and
mercaptans) and that the incubation time is critical. As different
proteins contain different amounts of tyrosine, the amount of the
coloured product will also be different. As a consequence, this
method is more suited to compare the concentration of solutions of
the same protein than to absolute measurement.
3) Bradford protein assay
Despite being relatively new, probably this is the most
widely used protein assay. The method is based on the ability of
the Coomassie Brilliant Blue dye to bind to proteins in acidic
solution (via electrostatic and van der Waals bonds), resulting in a
shift of the absorption maximum of the dye from 465 to 595 nm.
The advantages of the method include that it is highly
sensitive, is able to measure 1-20 g of protein and is very fast.
Only relatively few materials interfere with it (it works even in
presence of urea or guanidine hydrochloride) but, importantly,
detergents do. Even traces of detergent (e.g. cleaning products) can
invalidate the results. Its disadvantages are that it depends strongly
on amino acid composition and that it stains the cuvettes used.
4) Spectrophotometry based on UV absorption
This method is based on the fact that two of the aromatic
amino acids, tryptophan and tyrosine, show a peak in absorbance
around 280 nm. It has the advantage of being quick and easy. Since
it needs no chemical reaction to be performed, it is widely used for
detection of proteins or peptides during their separation by
chromatography. As proteins contain different ratios of aromatic
amino acids, per se it is more suited to the comparison of solutions
of the same protein and less to absolute measurement. The latter
Page 6
requires the knowledge of the molar extinction coefficients of

proteins. For many proteins, these were determined and can be
found in the literature. Moreover, if we know the number of
tyrosine and tryptophan amino acids in the protein of interest, since
their absorption values are additive, it is possible to calculate the
molar extinction coefficient.
Using a Spectronic 20 spectrophotometer
The Spectronic 20 spectrometer is widely used in teaching
laboratories. The specific instructions will differ with other models, but the
principles remain.
1.
The instrument must have been warm for at least 15 min. prior to
use. The power switch doubles as the zeroing control.
2.
Use the wavelength knob to set the desired wavelength. Extreme

wavelengths, in the ultraviolet or infrared ranges, require special
filters, light sources, and/or sample holders (cuvettes).
3.
With the sample cover closed, use the zero control to adjust the
meter needle to "0" on the % transmittance scale (with no sample
in the instrument the light path is blocked, so the photometer reads
no light at all).
4.
Wipe the tube containing the reference solution with a lab wipe and
place it into the sample holder. Close the cover and use the light
control knob to set the meter needle to "0" on the absorbance scale.
5.
Remove the reference tube, wipe off the first sample or standard
tube, insert it and close the cover. Read and record the absorbance,
not the transmittance.
6.
Remove the sample tube, readjust the zero, and recalibrate if

necessary before checking the next sample.
Spectrophotometric Analysis
Page 7
Analysis relies on the interaction of electromagnetic radiation

(light)with the matter of interest. Strictly speaking, every compound has a
distinct absorption spectrum which allows its identification, in many cases,
in the presence of other compounds. In addition to the identification of a
compound, it is also possible to determine quantitatively the concentration
of that compound. The relationship between absorbance and concentration
is given by the Lambert-Beer Law and is written mathematically as:
A=ec
where A is the absorbance, a unit-less quantity;e is the molar absorptivity
constant ( a constant of the compound having units ofL*mol-1*cm-1); and
is the path length over which the light interacts with the sample in cm. In
analytical spectrophotometry, the molar absorptivity of the unknown
compound may not be known. It is therefore a common practice to
generate a calibration or standard curve. A standard curve is generated by
measuring the absorbance of a series of samples for which the
concentration is known. Then, since the Lambert -Beer Law shows a linear
relationship between absorbance and concentration, a linear least squares
fit of the standard curve will yield a mathematical relationship between the
absorbance and concentration. This relationship in turn can them be used
to calculate the concentration of the unknown sample.
Standard curve
Standard curves are used to determine the concentration of
substances. They are obtained by relating a measured quantity to the
concentration of the substance of interest in "known" samples, i.e.
Standards of known concentration. These standards provide a reference to
determine unknown concentrations. Thus amounts chosen of standards
need to span the range of concentrations expected to be found in the
"unknown" sample concentration.
Page 8
The
quantity
(assay
measurements
as
i.e.
luminescence,
radioactivity, fluorescence, and optical density of various known

concentrations of a substance) graphed on y-axis and standard
concentrations on x-axis. Data analyzed by fitting a line on curve (see
Figure below).
To determine the unknown concentration of a substance in a

sample (with same assay as for standards used), intersect across the assay
measurement on y with standard concentration, and down to x. The
concentration of substance in unknown sample is the value on x.
Quantitative estimation of the amount of proteins present in

an unknown solution
To determine protein content of an unknown requires:
1. an assay in which measurable quantity is related to concentration
2. standards for comparison
Visible Spectrum
Page 9
Page 10
V.
TOOLS AND MATERIALS:
Tools
1. Test tube
Materials
9 units
1. Standard protein
solution
2. Spectronic-20
1 unit
2. Biuret reagent
3. Pipette
9 units
3. Milkfish
4. Mortal and alu
1 unit
4. Aquadest
5. Spatula
2 units
6. Digital neraca
1 unit
7. Knife
1 unit
8. Paper filtrate
1 unit
9. Beaker glass
2 units
10. Volumetric flask
1 unit
11. Pipette volume
1 unit
12. Funnel
1 unit
13. Measure glass
1 unit
Page 11
VI.
PROCEDURE
1. Sample Preparation
0.8 gram of Milkfish
-blended
-added with aquadest
-poured into volumetric
flask
-filtrated
Residue
Filtrate
2. Standard Preparation
Standard protein solution
Continues diluted
1 mg/ml
3 mg/ml
2 mg/ml
4 mg/ml
5 mg/ml
-entered each conc into different test

tube
-added 4ml of biuret reagent
-shaked
-incubated at 37oC for 10 minutes
-measured the absorbance by spec20 520nm
Absorbance value
3. Determining Absorbance of Blanco Solution

1ml of aquadest
-shaken
-incubated at 37oC for 10
minutes
-measured the absorbance by
spec-20 520nm
Absorbance of sample
Page 12
4. Determining Absorbance of Sample Solution

1ml of sample filtrate
-shaken
-measured the absorbance by spec-20
520nm
Absorbance of sample filtrate
Page 13
VII.
No.
1.
RESULT OF EXPERIMENT
Procedures
Sample Preparation
0.8 gram of Milkfish
-blended
-added with aquadest
-poured into volumetric flask and
added aquadest until limit line
-filtrated
Residue
Observation
Hypothesis/Reaction
Conclusion
Before
Filtrate of milkfish is
Milkfish = pale pink
colorless
Aquadest = colorless
After
Filtrate of milkfish =
colorless
Filtrate
Page 14
2.
Standard Solution Preparation
Before
Standard protein
solution
-performed serial dilutions
1
mg/
ml
2
mg/
ml
3
mg/
ml
4
mg/
ml
5
mg/
ml
-entered each conc into

different test tube
-added 4ml of biuret
reagent
-shaked
-incubated at 37oC for
10 minutes
-measured the
Absorbanc
absorbance by spec-20
e value
520nm
Protein in the alkaline environment reduce Cu2+ to

+
Std 1(1mg/ml)= WL
Biuret reagent =
Cu , which forms a coordination complex with
0.049
color solution
proteins, leading to a blue to light violet color change
Std 2(2mg/ml)= WL
Filtrate of milkfish =
0.108
colorless
Std 3(3mg/ml)= WL
All of Standard
0.154
solution = colorless
Std 4 (4mg/ml)= WL
After
0.196
Standard solution
Std 5 (5mg/ml)= WL
+biuret reagent
0.215
Std 1(1mg/ml)= blue

Std 2(2mg/ml)=
purplish blue
Std
3(3mg/ml)=violet
(+)
Std 4 (4mg/ml)=
violet (++)
Std 5 (5mg/ml)=
Page 15
violet (+++)
3.
Determining Absorbance of Blanco
Before
Solution
Aquadest = colorless
1ml of aquadest
Blanco not contain protein
Blanco not contain

protein
Biuret reagent =
blue color solution

-shaken
-incubated at 37oC for 10
minutes
-measured the absorbance by
spec-20 520nm
After
Aquadest+biuret
reagent= light blue
Absorbance of sample
Page 16
4.
Determining Absorbance of Sample

Solution
1ml of sample filtrate
-shaken
-measured the absorbance by spec-20
520nm
Before
Protein in the alkaline environment reduce Cu2+ to

+
Wavelength
Filtrate of milkfish=
Cu , which forms a coordination complex with
Milkfish 1= 0.034
colorless
proteins, leading to a blue to light violet color change
Milkfish 2=0.032
After
Milkfish 3=0.034
Filtrate+biuret
Absorbance
reagent= light blue
Bandeng 1=1.2381
Bandeng 2= 1.19048
Bandeng 3= 1.2381
Absorbance of sample filtrate
Page 17
VIII.
DATA ANALYSIS
1. Sample Preparation
In the sample preparation, 0.8 gram of milkfish thats have pale
pink color is blended smoothly. The blended result is white turbid solution
. To make the result of blended before homogenous, added aquadest and
poured into volumetric flask and diluted with aquadest until limit line and
shaked to make it more homogenous, so we will get more filtrate. In our
experiment, we just need the filtrate to test the protein, so we filtrated with
paper filtrated. The filtrate of milkfish is colorless, its means there arent
residue again.
2. Standard Solution Preparation

Using the protein standard solution we prepared a five point standard curve by
performing serial dilutions of protein between a range of 1mg/ml, 2mg/ml,
3mg/ml, 4mg/ml, 5mg/ml. Biuret reagent is added to solutions which containing
"standards" samples of the substance to be assayed, at 1-5 mg/ml concentrations.
The biuret reagent is a solution of copper sulphate (CuSO4) and sodium hydroxide
(NaOH), the NaOH is there to raise the pH of the solution to alkaline levels, the
crucial component is the copper II ion Cu2+ from CuSO4. When peptide bonds are
present in this alkaline solution, the Cu2+ ions will from a coordination complex
with four N atoms from peptide bonds. The complex of Cu2+ ions and nitrogen
atoms make the color of CuSO4 solution changes from blue to violet. The color
change is dependent on the number of peptide bonds in the solution, so the more
protein, the more intense the change. Std 1(1mg/ml)= blue, Std 2(2mg/ml)=
purplish blue, Std 3(3mg/ml)=violet (+), Std 4 (4mg/ml)= violet (++) and Std 5
(5mg/ml)= violet (+++). The estimate reaction :
Page 18
After added biuret reagent, the standard solution are incubated for
10 minutes at 37oC, the aim for incubation is support the reagent react with
the standard solution rapidly and mixed completly. The absorbances
measured with specnometer-20 with 520nm, from the solutions of the
standard are plotted against the concentration of the standard to construct a
"standard curve".
Standard Solution Curve

Absorbance
0.25
y = 0.0446x - 0.0359
R = 0.9807
0.2
0.15
0.1
0.05
0
0
Concentration
From the standard curve we got y = 0.044x - 0.035, this equation used as
calculation the percentage protein in milkfish by imagine y as the
absorbance of milkfish.
3. Determining Absorbance of Blanco Solution

The preparation of blanco solution is same with standard solution
treatment, but blanco solution made from aquadest and the next treatments
are same. The treatment are added with biuret reagent and next incubated
for 10 minutes at 37oC. After that this blanco solution measured the
absorbance. The measuring of blanco solution to give zero/standard
absorbance.
4. Determining Absorbance of Sample Solution

From the first experiment, the filtrate divided into 3 tube and given same
treatment. The filtrate added biuret reagent and incubated for 10 minutes.
Three of the filtrate are light blue. The biuret reagent is a solution of
copper sulphate (CuSO4) and sodium hydroxide (NaOH), the NaOH is
Page 19
there to raise the pH of the solution to alkaline levels, the crucial

component is the copper II ion Cu2+ from CuSO4. When peptide bonds are
present in this alkaline solution, the Cu2+ ions will from a coordination
complex with four N atoms from peptide bonds. The complex of Cu2+ ions
and nitrogen atoms make the color of CuSO4 solution changes from blue
to violet. The color change is dependent on the number of peptide bonds in
the solution, so the more protein, the more intense the change.
The aim for incubation is support the reagent react with the standard
solution rapidly and mixed completely. Finally measure the absornbance
with specnometer-20 with 520nm, this wave length suitable with the color
blue-violet like a this theory
The result of the wave length and calculation of absorbance from the third
experiment (y = 0.044x - 0.035) in sample filtrate milkfish are :
Wavelength
Absorbance
Milkfish 1= 0.034
Milkfish 1=1.2381
Milkfish 2=0.032
Milkfish 2= 1.19048
Milkfish 3=0.034
Milkfish 3= 1.2381
From the absorbance we could calculated the percentage protein in

milkfish, the percentage are Milkfish 1 is 19.6 %, Milkfish 2 is 19.03 %,
milkfish 3 is 19.6 %. We used data milkfish 1 and milkfish 3 as a
Page 20
comparison from the theory, our percentage that we got is 19.6% and
theory is 29%(FAO data).
Page 21
IX.
DISCUSION
From our experiment we found some differences about our
experiment and theory. The percentage protein in milkfish from theory
(FAO data) is 29% and our experiment is 19.6%. from that we learned
about experience, the regression should be 1(0.99) from theory, our
regression is 0.98. it is slightly differ but differ is differ. This was caused
when doing series dilution of standard solution not accurate, so its make
the other value follow it and when we prepared the filtrate is not quite
smooth. So, the filtrate that we got decrease.
Page 22
X.
CONLUSION
From the experiment that we done is the percentage protein of milkfish
filtrate is 19.6%
Page 23
XI.
QUESTIONS AND ANSWERS
1. Make curve of standard concentration vs absorbance. By the curve,

determine the percentage of protein!

Absorbance
0.25
y = 0.0446x - 0.0359
R = 0.9807
0.2
0.15
0.1
0.05
0
0
Concentration
Calculation The Percentage Protein in Milkfish Sample (y = 0.044x - 0.035)

Milkfish 1
y1 = 0.044x - 0.035
0.034 = 0.044x - 0.035
0.034 + 0.035 = 0.044x
x=
x1 = 1.56818
Milkfish 2
y2 = 0.044x - 0.035
0.032 = 0.044x - 0.035
0.032 + 0.035 = 0.044x
x=
x2 = 1.52273
Milkfish 3
y3 = 0.044x - 0.035
0.034 = 0.044x - 0.035
0.034 + 0.035 = 0.044x
x=
x3 = 1.56818
The percentage protein in

milkfish 1 =

milkfish 2 =

milkfish 3 =
=19.6 %
=19.03 %
=19.6 %
2. Will peptide give a positive reaction with biuret reagent? If it true, how to
determine the percentage of protein that are mixed with peptide?
It will its proven from violet color result, to determine the percentage of protein
that are mixed with peptide by using equation curve ; y = 0.044x - 0.035. this
equation taken from the result of the absorbance of standard solution.
Page 24
REFERENCES
FAO.2014. Aquaculture Feed and Fertilizer Resources Information
System.(online) http://www.fao.org/fishery/affris/speciesprofiles/milkfish/faqs/en/ accessed on 1/11/2014
Scopes, Robert K.1994. Protein Purification: Principles and Practice.United
States of America:Springer Science+Business Media,LCC
Tim Dosen.2014.Petunjuk Praktikum Biokimia.Surabaya:Unesa
USDA.2014.Nutrition
Facts:Fish,
Milkfish,Raw.
(online)
http://nutritiondata.self.com/facts/finfish-and-shellfish-products/4079/2#
accessed 1/11/2014
Page 25
ATTACHMENT
No.
1.
Procedures
Pictures
Sample Preparation
Milkfish
Fresh Milkfish
Milkfish blended
Crushing the milkfish (0.8 gram)
Page 26
Diluted in volumetric flask
Turbid in white solution

Filtrated
Filtrate of milkfish
Colorless filtrate
2.
Standard Solution Preparation
Page 27
Standard solution+biuret reagent
violet
After incubated
Violet
3.
Determining Absorbance of Blanco Solution

Aquadest + biuret reagent
Light blue
Page 28
After incubated
4.
Determining Absorbance of Sample Solution

Filtrate of sample
Colorless solution
Filtrate + biuret reagent
light blue
Page 29
After incubated
blue
Page 30
ATTACHMENT
Standard Solution
Concentration
Wavelength
0
1 mg/ml
2 mg/ml
3 mg/ml
4 mg/ml
5 mg/ml
0
0.049
0.108
0.154
0.196
0.215
Absorbance

0.25
0.2
0.15
0.1
0.05
0
y = 0.0446x - 0.0359
R = 0.9807
Concentration
Sample of Milkfish Filtrate

Sample ID
Concentration
Wavelength
Milkfish 1
0.729
0.034
Milkfish 2
0.677
0.032
Milkfish 3
0.716
0.034
Calculation The Percentage Protein in Milkfish Sample (y = 0.044x - 0.035)
Milkfish 1
y1 = 0.044x - 0.035
0.034 = 0.044x - 0.035
0.034 + 0.035 = 0.044x
x=
x1 = 1.56818
Milkfish 2
y2 = 0.044x - 0.035
0.032 = 0.044x - 0.035
0.032 + 0.035 = 0.044x
x=
x2 = 1.52273
Milkfish 3
y3 = 0.044x - 0.035
0.034 = 0.044x - 0.035
0.034 + 0.035 = 0.044x
x=
x3 = 1.56818

milkfish 1 =

milkfish 2 =

milkfish 3 =
=19.6 %
=19.03 %
=19.6 %
Page 31

Biochem

Uploaded by

Copyright:

Available Formats

You might also like

Biochem

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Biochem

Uploaded by

Copyright:

Available Formats

EXPERIMENT REPORT

Determining The Percentage of Protein in

INTERNATIONAL CHEMISTRY EDUCATION 2012

The percentage of protein in milkfish by biuret method

Proteins are macromolecules which are soluble in water. Proteins

The percentage of protein in milkfish by biuret method

juveniles. The life history of milkfish has been described in detail by

Protein and Amino Acids in Milkfish

(Nutrition facts and analysis,2014)

Determination of Protein Concentration

The percentage of protein in milkfish by biuret method

measure protein concentration. In chromogenic methods, the absorbance

The percentage of protein in milkfish by biuret method

The Biuret Test works for any compound containing two or

In practice, the determination of protein concentration is

disadvantages are that it takes rather long to carry out, is disturbed

The percentage of protein in milkfish by biuret method

requires the knowledge of the molar extinction coefficients of

Use the wavelength knob to set the desired wavelength. Extreme

Remove the sample tube, readjust the zero, and recalibrate if

The percentage of protein in milkfish by biuret method

Analysis relies on the interaction of electromagnetic radiation

The percentage of protein in milkfish by biuret method

radioactivity, fluorescence, and optical density of various known

To determine the unknown concentration of a substance in a

Quantitative estimation of the amount of proteins present in

The percentage of protein in milkfish by biuret method

The percentage of protein in milkfish by biuret method

TOOLS AND MATERIALS:

4. Mortal and alu

10. Volumetric flask

11. Pipette volume

13. Measure glass

The percentage of protein in milkfish by biuret method

-entered each conc into different test

3. Determining Absorbance of Blanco Solution

The percentage of protein in milkfish by biuret method

4. Determining Absorbance of Sample Solution

Absorbance of sample filtrate

The percentage of protein in milkfish by biuret method

Milkfish = pale pink

The percentage of protein in milkfish by biuret method

Standard Solution Preparation

-entered each conc into

Protein in the alkaline environment reduce Cu2+ to

Cu , which forms a coordination complex with

proteins, leading to a blue to light violet color change

Std 1(1mg/ml)= blue

The percentage of protein in milkfish by biuret method

Determining Absorbance of Blanco

Blanco not contain protein

Blanco not contain

-added 4ml of biuret reagent

The percentage of protein in milkfish by biuret method

Determining Absorbance of Sample

Protein in the alkaline environment reduce Cu2+ to

Cu , which forms a coordination complex with

proteins, leading to a blue to light violet color change