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Abstract
Understanding the use of the spectrophotometer is useful when
conducting lab experiments where analysis of a solution is required to give
a numerical value for that solution. In situations where the
Order custom research paper on Protein Determination in Powdered Milk (and Understanding of the Use andApplication of the Spectrophotometer)
spectrophotometer is used, the Beer-Lambart equation can be used to find
the concentration of a solution when the absorbance of the solution is
known (which can be extrapolated through the use of the spectrophotometer).
The lab involves the use of a spectrophotometer to analyze the
concentration of protein in a sample of diluted milk reacted with a protein
assay reagent.
Introduction
The general subject we are studying is protein determination in
powdered milk.
We will be using a spectrophotometer, which is an instrument that uses
light energy to determine the concentration of a solute by the solutes
absorption rate. Using standardized protein concentrations and the
spectrophotometer, we will be able to determine the protein concentration
in diluted milk. The basic goal for the experiment is to find out the
concentration of protein in milk.
The subject is important to understand because understanding the
absorption spectrum of a compound or group of compounds can help identify
substance(s) present in solution. Understanding the concentration of
compounds (such as protein) is important to both commercial and domestic
applications. In areas where qualitative information is important,
spectrophotometer analysis helps quantify and qualify information. An
example of a commercial use of compound analysis which applies to domestic
use is the food information labels on the back of food products. The
labels give a numerical summary of the compounds which are contained in the
product. The current trend toward a healthier lifestyle has increased the
awareness of the public to what is being consumed.
Compounds have the ability to absorb and reflect light energy.
Adding the protein assay reagent to the protein standard causes the
solution to turn blue. Setting the spectrophotometer at the maximum
absorption wavelength for the blue form of the protein assay reagent, which
is 55nm, (Lab Manual, pg. 15) will determine the absorbance of the protein
compounds in the solution. Time affects the result of the lab because as
the amount of time passes the more the standard protein solution and the
assay reagent will react together causing the protein to turn a darker blue
thus absorbing more light.
With the varied choices of milk available, determining the
concentration of protein in milk through the spectrophotometer allows us to
compare the amount of protein that is contained in milk. The sample of
skim milk mixed with the protein assay reagent in the spectrophotometer
gave an absorbance number which can be plotted in relation to the
absorbance numbers of the standardized protein solutions. Knowing that
skim plus milk contains 11 g of protein per 8 oz of milk (milk label) and
whole milk contains 8 g per 8 oz, we predict the concentration of skim plus
to be higher than that of whole milk.
Materials and Methods
We first obtained 8 cuvets and numbered them 1 through 8 and placed
them in a test tube holder. Cuvet number 1 contained 0.1 mL of water.
Cuvette contained 0.1 ml of a protein standard with a concentration of
0. mg/ml. Cuvette contained 0.1 ml of a protein standard with a
concentration of 0.4 mg/ml. Cuvette 4 contained 0.1 ml of a protein
standard with a concentration of 0.6 mg/ml. Cuvette 5 contained 0.1 ml of
a protein standard with a concentration of 0.8 mg/ml. Cuvette 6 contained
0.1 ml of a protein standard with a concentration of 1.0 mg/ml. Cuvettes 7
and 8 contained 0.1 mL skim milk plus. Using a 5 mL transfer pipette and a
pipette bulb, place 5 ml of the protein assay reagent into all the cuvets
and cover the cuvets with parafilm. The cuvets are then inverted to allow
the reagent and the protein standard to mix. We then waited 7 minutes to
allow the solution to react. After 7 minutes, we used the
spectrophotometer and recorded the absorbance number for each of the cuvets
after zeroing and standardizing the spectrophotometer.
Results
The graph (Fig 1) shows the linear trend of the absorption rate in respect
to the concentration of the protein standard as the concentration
increases the absorbance of the protein increases. The concentration for
our sample is 56.0 mg/ml since the sample we had was diluted by a factor of
40. The value under concentration for test tubes 7 and 8 (Fig ) is the
concentration of the diluted sample. Compared to Thomas Boes, who got a
protein concentration of .4 mg/ml for whole milk, our concentration of
56.0 mg/ml shows that skim milk plus does in fact have a higher
concentration than whole milk.
Absorption Vs. Concentration
|Test tube number |Absorption |Concentration (mg/ml) |
|Blank |0 |0 |
| |0.40 |0.180 |
| |0.545 |0.61 |
|4 |0.8 |0.570 |
|5 |1.40 |0.866 |
|6 |1.60 |0.85 |
|7 |1.70 |1.04 |
|8 |1.70 |1.04 |
Fig . Table of Concentration and Absorption values for all test tubes
(test tubes -6 are standards and test tube 7 and 8 are skim milk plus
samples)
Discussion
For this experiment, we wanted to find the concentration of protein
in the skim milk compared to the whole milk knowing that an 8 oz glass of
skim milk has more protein than an 8 oz glass of whole milk. Our data did
not support our original hypothesis because we waited longer than the time
listed of five minutes. We waited 7 minutes after placing the assay
reagent into the last cuvette (number 8) then began putting the cuvets into
the spectrophotometer starting with cuvette 1. The assay reagent had more
time to react with the protein standard, changing more protein molecules to
a blue color which absorbed more of the light energy. If this experimented
were repeated, the time that we waited wouldve been shortened to the
recommended amount of five minutes.
Our standard curve will be different from others in our class because
we waited longer than most others before we analyzed the samples. Another
reason for varied results could be the human error involved in the
measurements of both the protein standard and the protein assay reagent.
When the protein standards were dropped into the cuvettes, some syringes
contained air pockets which caused less protein to be dropped into the
cuvets thereby skewing results. Many reasons exist for the varying reasons
why milk samples contained different concentrations of protein. Some milk
manufacturers add protein into their products thereby increasing the
concentration. In other instances, there might have been different volumes
of protein assay used by different groups, dying more protein molecules
which caused an increase in absorption numbers.
Reference
Seagull, Robert W. Krause, Maureen K., 00 General Biology Bio. 001
Laboratory Manual pages 8 to 16
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