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Separation and Identification of Amino Acids present in

Casein from Milk by Paper Chromatography and


Micro-Bradford Assay

Sy, Bernadette ; Takatsu, Risa ; Tiu, Claribel ;


Torres, Alyssa ; Torres, Jade

Group 7, 2G Pharmacy, Faculty of Pharmacy, University of Santo Tomas

____________________________________________________________________________________

ABSTRACT

In this experiment, qualitative analysis through paper chromatography was done and Bradford
total protein assay for quantitative analysis. Paper Chromatography is a type of partition chromatography
that uses a specialized paper made up of polymeric carbohydrate cellulose (filter paper) as a support
medium for the stationary phase. It is generally used for the separation of water-soluble organic and
inorganic compounds or highly polar compounds such as amino acids and sugars. This method is useful
for separating complex mixtures of compounds having similar polarity, like amino acids. In this experiment
10 amino acid standards were used to compare and determine as to which amino acid is present in the
protein casein obtained from milk. The Rf values were computed and it was seen that the enzymatic
hydrolysate obtained from casein has the same Rf value with proline which means that casein contains is
proline. The Micro-Bradford Protein Assay, on the other hand, is a rapid and accurate method for the
estimation of protein concentration. A total of 6 test tubes were prepared with different volumes of the
standard and water, and the other test tube contained the unknown sample, respectively. In a microplate
(96-well), each cuvettes contained the BSA standard and were filled with a right amount of each test tube
prepared and was immediately subjected to the UV/Vis Spectrophotometer. The absorbance of each
sample was determined and plotted into a linear graph. The concentration of the sample was also
computed using the linear regression method and resulted to 0.7843 mg/mL.

INTRODUCTION

Chromatography is an analytical in a solvent. It is a powerful method in industry,


technique commonly used for separating a where it is used on a large scale to separate and
mixture of chemical substances into its individual purify the intermediates and products in various
components, so that the individual components syntheses.[3] Paper Chromatography is a type of
can be thoroughly analyzed.[1] There are many partition chromatography that uses a specialized
types of chromatography but all types employ paper made up of polymeric carbohydrate
[2]
the same basic principles. The basic principle cellulose (filter paper) as a support medium for
is that components in a mixture have different the stationary phase.[4] it is generally used for
tendencies to adsorb onto a surface or dissolve the separation of water-soluble organic and
inorganic compounds or highly polar standards at the same concentration as that
[5]
compounds such as amino acids and sugars. found in the sample. The presence of base in
This method is useful for separating complex the assay increases absorbance by shifting the
mixtures of compounds having similar polarity, equilibrium of the free dye toward the anionic
like amino acids. The setup has three form. This may present problems when
components: the mobile phase which is a measuring protein content in concentrated basic
solution that travels up the stationary phase, due buffers. Guanidine hydrochloride and sodium
to capillary action. The mobile phase is generally ascorbate compete with dye for protein, leading
an alcohol solvent mixture, while the stationary to underestimation of the protein content. [7] This
phase is a strip of chromatography paper, also method is based on the observation that the
called a chromatogram. The amino acids are absorbance maximum for an acidic solution of
carried along with the solvent up the paper at Coomassie Brilliant Blue G-250 shifts from 465
varying speeds, depending on its differential nm to 595 nm when binding to protein occurs.
affinity between the stationary and mobile Both hydrophobic and ionic interactions stabilize
phases. As a result, the amino acids are the anionic form of the dye, causing a visible
separated and spread out on the chromatogram color change of blue. The assay is useful since
[6]
as spots or bands. the extinction coefficient of a dye-albumin
The Micro-Bradford Protein Assay, on complex solution is constant over a 10-fold
the other hand, is a rapid and accurate method concentration range.[8]
for the estimation of protein concentration . This The objectives of this experiment are:
technique is simpler, faster, and more sensitive (1) determine the amino acid components of the
than the Lowry method. Moreover, when protein casein using paper chromatography and
compared with the Lowry method, it is subject to (2) quantitatively determine the protein
less interference by common reagents and non- concentration of a given sample using the Micro-
protein components of biological samples simple Bradford Protein Assay Method.
because The Bradford assay is relatively free
MATERIALS AND METHODS
from interference by most commonly used
biochemical reagents. However, a few chemicals Materials
may significantly alter the absorbance of the To facilitate the paper chromatography,
reagent blank or modify the response of proteins the materials needed are filter paper, 1000mL
to the dye. The materials that are most likely to beaker, capillary tubes, enzymatic hydrolysate
cause problems in biological extracts are obtained from milk, 1% Ninhydrin solution spray,
detergents and ampholytes. These can be 1-bButnaol:acetic acid:water in the ratio of 4:1:5
removed from the sample solution by gel respectively and lastly the amino acid
filtration, dialysis, or precipitation of protein with standards : 2% w/v tryptophan, arginine, proline,
calcium phosphate. Alternatively, they can be cysteine, serine, aspartic acid, tyrosine,
included in the reagent blank and calibration histidine, glycine and alanine.
To facilitate the Micro-Bradford Total
Protein Assay the materials needed are Bradford
reagent (Coomassie Brilliant Blue G-250),
Blank 0 1000
Bovine Serum Albumin (BSA), test tubes, 1 50 950
cuvettes, microplate (96-well), microplate reader, 2 100 900
3 250 750
micropipetors, and the UV/Vis
4 350 650
Spectrophotometer. 5 500 500

Methodology Using a micropipetor, a 75L of Bradford

For Paper Chromatography, a 12x15cm reagent were added to each cuvettes in the

of filter paper was prepared and from the bottom microwell plate (Figure 1), enough volume of the

of the longer edge, a 1.5 cm margin was made samples from the test tubes made were then

to serve as the origin of the amino acid transferred to the cuvettes with Bradford reagent

standards and the enzyme hydrolysate to be and was mixed well. The first cuvette served as

dropped. On the line made, 11 equidistant points the blank while the rest of the cuvettes

were marked for the spotting of the respective contained the samples. The albumin sample

samples to be used. Using a capillary tube, the which was made by mixing a 75L of Bradford

amino acids standards were dropped 5 times reagent and 100L of the BSA standard, was

and the enzymatic hydrolysate 10 times. After all also pipetted into the cuvettes three time to

the samples were dropped and let dried, the obtain an accurate result. The microplate was

chromatogram was stapled properly together then placed inside the spectrophotometer and

forming a cylinder and was placed inside a was read at the absorbance 595nm. The

1000mL beaker with the solvent butanol:acetic albumin standard curve was then constructed by

acid:water in the ratio of 4:1:5 just below the plotting the absorbance at 595nm against

origin and was covered with a watch glass until concentration and the concentration of the

the solvent front was approximately 0.5 cm from protein sample was determined.

the top edge of the plate. The solvent front was


immediately marked with a pencil line and was
allowed to dry. Then it was lightly sprayed with
1% Ninhydrin solution and was dried. All the
observed spots that has a color of blue, purple
or yellow were encircled and the Rf value of
each were computed accordingly.
For the Micro-Bradford Total Protein
Assay, a series of test tubes were prepared as
follows:

Test Tube BSA Std (L) H2O (L)


Figure 1. Microplate (96-well) containing the the Rf value, the shorter the distance. The
samples prepared solvent front always moves further than any of
the compound in the mixture. However, the
RESULTS AND DISCUSSIONS
compounds migrate differently depending on the
In paper chromatography, the following
type of mobile phase used because the
results were obtained as shown in Figure 2.
migration speed depends on some
characteristics of the solvent, such as polarity.
The Rf value is characteristic for any chemical
compound.[11] The Rf values of the amino acid
standards and enzyme hydrolysate were
computed using the formula:

distance traveled by the solute


Rf =
Figure 2. Chromatogram
distance traveled by the solvent

The spots appeared as colors blue,


where the distance traveled by the solvent was
violet, or yellow after being sprayed with 1%
measured 7cm.
ninhydrin solution which implies that all the
samples contains an alpha-amino group Table 1. Computed Rf Values of the samples
because ninihydrin is a test for detecting the
presence of an alpha amino group that gives a
blue to blue violet color for a positive result. [9]
However it can be observed that the amino acid Sample Distance Traveled Rf Value

proline gave a yellow color instead of a blue or (cm)

violet color this is because the bluish-purple


result is usually associated with primary amino
Tryptophan 4.6 0.66
acids. In the other amino acids, the N is free to
react with ninhydrin. However, in proline, the N
Arginine 5.1 0.73
is not available for reaction as it is locked in a
five membered ring. Therefore no ammonia is Proline 5.2 0.74
[10]
produced, so no blue color is presented.
Cysteine 5.3 0.76
The Rf value or retention factor
quantifies the distance that each compound of Serine 5.1 0.73
Aspartic
the analyzed mixture has traveled. The higher
Acid 5.0 0.71
the Rf value, the longer the distance that a
specific compound has traveled and the lower
Tyrosine 4.1 0.56 In the Micro-Bradford Total Protein
Assay the following data as shown in Table 2
Histidine 4.9 0.70 were obtained after undergoing the UV/Vis
Spectrophotometer. It can be seen that two trials
Glycine 5.0 0.71
were done for more accurate results. The

Alanine 5.3 0.76 Absorbance of the samples were then computed


Enzymatic by using the formula:
Hydrolysat 5.2 0.74
e Acorrected = Astd Ablank

Table 1 shows the computed Rf values Table 2 Micro-Bradford Assay Results


of the samples. It can be seen from the table
that the amino acids Proline has the same Rf
Sample Con A595 (Tria1) A595 (Trial2)
values with the hydrolysate sample which
c
means that the protein casein contains the
amino acid proline. Blank 0 0.369 0.365
1 0.10 0.608 0.582
In terms of the polarity of the sample, 2 0.20 0.648 0.658
3 0.50 0.791 0.752
the results obtained are not reliable because 4 0.70 0.838 0.873
due to lack of time, the solvent front did not 5 1.00 0.944 0.933
travel that much as seen in Figure 2. However, it
Table 3. Acorrected of the samples
should be expected for the amino acid
tryptophan to be farthest from the origin as it is Sample Conc. Acorrected Acorrected Acorrected
the most non-polar sample and histidine to be (Trial 1) (Trial 2) Average
nearest the origin as it is the least non-polar or
the most polar.
Blank 0 0 0 0

The Bradford assay is commonly used 1 0.10 0.2390 0.2170 0.2280


to determine the total protein concentration of a
2 0.20 0.2790 0.2930 0.2860
sample. The method is based on the binding
of Coomassie Brilliant Blue G-250 dye to 3 0.50 0.4220 0.4170 0.4195
proteins. The acidic environment of the reagent
4 0.70 0.4690 0.5080 0.4885
results in a spectral shift from the reddish brown
of the dye, which has a maximum absorbance of 5 1.00 0.5750 0.5680 0.5715
465 nm, to the blue form of the dye, which has a
Table 4. Albumin Sample
maximum absorbance of 610 nm. The difference
between the two forms of the dye is greatest at Abs 595nm Acorrected Conc. (mg/mL)
595 nm.
where is the extinction coefficient, b is
Trial 1 0.8220 0.4550 0.6474 the path length, c is the concentration and A is

Trial 2 0.8140 0.4470 0.6265 the absorbance. The Beer-Lambert law relates
the attenuation of light to the properties of the
Trial 3 0.8300 0.4630 0.6683
material through which the light is traveling. This
Average 0.465 0.6474 page takes a brief look at the Beer-Lambert Law
and explains the use of the terms absorbance
In determining the total protein
and molar absorptivity relating to UV-visible
concentration of the sample, the linear
absorption spectrometry.[12]
regression (y=mx+b) was used to compute for
the slope and the y-intercept then the
concentration which is the variable x. The slope
having a value of 0.3823, 0.2075 for the y-
intercept and 0.7843 for the concentration of the CONCLUSION
protein sample. However, the total protein
From the experiment performed, based
concentration can also just be manually
on paper chromatography it can be concluded
inspected and in this experiment it gave a
that the protein casein isolated from a non-fat
concentration of 0.6474mg/mL.
skimmed milk contains the amino acid proline
because proline has the same Rf values with the
enzymatic hydrolysate obtained from casein.
While in the Micro-Bradford Total Protein Assay,
it can be concluded that the protein
concentration of the unknown sample is
0,7843mg/mL when computed and 0.6474 when
manually inspected.

REFERENCES

[1]
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The absorbance of each sample were
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[5] [10]
What is Paper Chromatography: Types, Yellow Results for Ninhydrin Test?. (2013).
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