Abstract
The concentrations of a variety of solutions of biological molecules were determined by spectrophotometric methods. The concentration of a solution of NADH was determined by direct application of Beer's Law at 340 nm, with a 2.00 % error. The concentration of solutions of proteins were determined by the Bradford assay, against bovine γ-globulin standards, with a 2.00 % error for a bovine γ-globulin unknown and a 121% error for the protein content of reduced fat milk. The concentration of a solution of Ponceau S in an artificial urine matrix was determined by standard addition, with an error of 9.00%. The identity of a sample of olive oil was determined qualitatively by fluorescence emission.
Introduction
When a beam of monochromatic light passes through a solution containing a substance that absorbs light of that wavelength, some of the light will be absorbed. The amount of light absorbed can be determined by Beer's Law, A = εbc, where A is the absorbance, ε is the absorptivity of the absorbing molecule, b is path length through the solution, and c is the concentration of the absorbing molecule. When the absorptivity is known, concentration may be calculated directly from absorbance measurements using Beer's Law. This is the principle applied to the determination of the NADH concentration, which has a strong absorbance peak at 340 nm in the near UV region.
When the absorptivity is not known, Beer's Law becomes A = kc, where k is the product of constant path length and constant absorptivity and a plot of absorbance versus concentration gives a straight line. If a standard absorbance-concentration plot is determined, the concentration of an unknown solution can be calculated from its absorbance and the trendline equation of the standard plot.
Proteins have no useable wavelengths of absorbance in either the UV or visible regions. The Bradford protein assay is based on the reaction of Coomassie Brilliant Blue G dye with proteins. The complex between the dye and protein absorbs strongly in the visible region and the absorbance is directly proportional to the concentration of protein present.
The use of Beer's Law assumes that the composition of the unknown solution is the same as that of the standards, with no interfering substances. Often, this assumption is not valid. The concentration of the analyte may still be determined by the method of standard addition, in which known quantities of the analyte are added to the aliquots of the unknown solution. From the absorbance of the unknown with and without the addition of the standard, the concentration of the analyte in the unknown may be determined.
Fluorescence occurs when a molecule absorbs a photon of light that excites the electrons in the molecule and causes them to emit light of a lower energy (longer wavelength). A fluorometer is used to measure the emitted light, which is called fluorescence. The wavelength of emitted light is characteristic of the identity of the fluorescing sample and the quantity of emitted light is directly proportional to the amount of fluorescing sample, so Beer's Law can be applied for quantitative analysis of fluorescing molecules.
Materials and Methods
In Part A of this experiment, the absorbance of a solution of NADH of unknown concentration was determined at 340 nm against a water blank in quartz cuvettes using a Spectronic 1201 spectrophotometer.
In Part B, the Bradford protein assay was used to determine the concentration of a protein solution of unknown concentration. A standard curve was prepared by adding 20 uL aliquots of standard bovine γ-globulin solutions to 1.00 mL of Bradford reagent (see Table 1). Similar aliquots of an unknown protein solution and of milk samples previously diluted 1:50 in PBS buffer were treated in the same manner. After a twenty-minute development, the absorbances of the solutions were determined at 595 nm against a Bradford reagent blank in plastic cuvettes using a Spectronic 601 spectrophotometer.
In Part C, the method of standard addition was used to determine the concentration of red Ponceau S dye in a yellow "urine" matrix. A standard curve was prepared by adding aliquots of 1 M Ponceau standard solution to 2.00 mL aliquots of the "urine" sample (see Table 2). The absorbances of the solutions were determined at 540 nm against a water blank.
In Part D, the fluorescence of three olive oil samples of different grades was measured at 405 nm. The fluorescence of two unknown olive oil samples was used to identity the grade of the unknown samples.
Results
Data
Part A - Determination of NADH Concentration
NADH unknown number
N10
Absorbance at 340 nm of unknown NADH solution
0.913
Part B - Bradford Protein Assay
Samples: Bovine γ-globulin protein unknown UP10, reduced-fat chocolate milk, reduced-fat milk
Table 1
Tube Number
Contents
Protein Concentration, mg/mL
Absorbance
blank
PBS buffer
0.000
0.000
1
Bovine γ-globulin
0.125
0.080
2
Bovine γ-globulin
0.250
0.148
3
Bovine γ-globulin
0.500
0.303
4
Bovine γ-globulin
0.750
0.449
5
Bovine γ-globulin
1.000
0.503
6
Bovine γ-globulin
1.500
0.791
7
Bovine γ-globulin
2.000
0.997
8
Bovine γ-globulin unknown UP10
unknown
0.790
9
Reduced-fat milk, 1:50 dilution
unknown
0.943
10
Reduced-fat chocolate milk, 1:50 dilution
unknown
0.957
Part C - Determination of Ponceau S by Standard Addition
Sample: "Urine" unknown #10
Table 3
1 M Ponceau S standard, μL, added
μmoles Ponceau S added
Absorbance
0
0
0.019
2
2
0.040
3
3
0.045
4
4
0.056
5
5
0.064
Part D - Identification of Olive Oil Samples
Samples: Olive Oil Unknown Set 1A and 1B
Sample
Fluorescence at 405 nm
Extra Virgin
0.578
Classic
0.125
Extra Light
0.007
1A
0.569
1B
0.135
Calculations
Part A - NADH Determination
Determination of NADH concentration in unknown N10
A = ε340bC
0.913 = 6200 cm-1M-1 X 1 cm X C
C = 1.47 x 10-4 M = 0.147 mM
Determination of error in NADH determination
% error = [experimental value - actual value]/actual value x 100%
= [0.147 mM - 0.150 mM]/0.150 mM x 100%
= - 2.00%
Part B - Protein Determination by Bradford Assay
Determination of standard curve
Trendline: Absorbance = 0.4968 mg protein/mL + 0.1099
Determination of unknown concentrations using trendline equation.
Determination of protein in bovine γ-globulin UP 10:
Absorbance = 0.4968 mg protein/mL + 0.0285
0.790 = 0.4968 mg protein/mL + 0.0285
mg protein/mL = 1.533 mg protein/mL
Determination of error in UP 10 determination
% error = [experimental value - actual value]/actual value x 100%
= [1.533 mg/mL - 1.500 mg/mL]/1.500 mg/mL x 100%
= + 2.200% error
Determination of protein concentration in milk samples
Reduced-fat milk
Absorbance = 0.4968 mg protein/mL + 0.0285
0.943 = 0.4968 mg protein/mL + 0.0285
mg protein/mL of dil. milk sample = 1.841 mg/mL dil. milk
mg protein/mL of undil. sample = 1.841 mg/mL dil.milk x 50 mL dil.milk/1 mL undil. milk
mg protein/mL of undiluted sample = 92.04 mg/mL = 22.1 g/240 mL
Determination of error in reduced-fat milk determination
% error = [experimental value - actual value]/actual value x 100%
= [22.1 g/240 mL - 10 g/240 mL]/10 g/240 mL x 100%
= + 121% error
Part C - Ponceau S Determination by Standard Addition
Determination of Standard Curve
Trendline: Absorbance = 0.009 umoles of Ponceau S added + 0.0197
Determination of umoles of Ponceau S in urine #10
Absorbance = 0.009 μmoles of Ponceau S added + 0.0197
0 = 0.009 μmoles of Ponceau S added + 0.0197
μmoles of Ponceau S = -2.19 μmoles.
This represents the amount that must be removed from the original sample to have an absorbance = 0.
Therefore, the concentration of Ponceau S in urine #10 is 2.18 μmoles/2.00 mL = 1.09 μmoles/mL
Determination of error in Ponceau S determination:
% error = [experimental value - actual value]/actual value x 100%
= [1.09 μmoles/mL - 1.00 μmoles/mL]/ 1.00 μmoles/mL x 100%
= + 9.00 % error
Results
The concentration of NADH unknown 10 was determined to be 0.147 mM; the percent error from the actual value of 0.150 mM is 2.00%. The concentration of protein unknown #10 was determined to be 1.533 mg/mL; the percent error from the actual value of 1.500 μg/μL is + 2.20%. The concentration of protein in reduced fat milk is determined to be 22 g/240 mL; the percent error from the label value of 10 g/mL is 121%. The concentration of protein in reduced-fat chocolate milk is determined to be 22 g/240 mL; the percent error from the label value of 10 g/240 mL is 121%. The concentration of Ponceau S in urine sample #10 is determined to be 1.09 umoles/mL; the percent error from the actual value is 9.00%. Olive oil sample 1A is extra virgin grade and olive oil sample 1B is classic grade.
Discussion
In Part A, the concentration of NADH is derived directly from the absorbance of the solution and Beer's law. Barring any problems with the spectrophotometer, the accuracy of the concentration of the NADH solution depends only on the transfer of the solution to the quartz cuvette; any spills or smears on the optical surface of the cuvette could increase the absorbance and increase the concentration determined. This is unlikely here, since the concentration determined was lower than the actual value. Water left in the cuvette from the initial rinse would cause a decrease in concentration. This effect can be diminished by rinsing the cuvette with a portion of the sample solution. Another cause of a decreased concentration could be the interaction of NADH with light. If the filled cuvette were allowed to sit exposed to light for any length of time before the absorbance was determined, degradation of the NADH could occur, resulting in an experimental concentration lower than the actual value.
In Part B, the accuracy of the determination of the concentration of protein in the bovine γ-globulin unknown depends upon the linearity of the standard curve. The linearity of the standard curve is very good, as demonstrated by the R2 value of 0.9938; this depends in large part upon the accuracy of pipetting. The agreement of the experimental determination of the bovine γ-globulin unknown with its actual value acts as a control for the experiment. The extremely large deviations of the milk results from the expected values can be attributed to two causes. First, very small pipetting errors during the dilution of the sample could cause the extremely large samples seen. Secondly, the nature of the milk samples themselves is problematic. The protein composition of milk is not solely bovine γ-globulin like the standards used to generate the standard curve; the primary protein in milk is casein. In addition, milk contains many more substances beside proteins which can interfere with the absorbance measurements.
In Part C, the accuracy of the determination of the concentration of Ponceau S "urine" sample depends upon the linearity of the standard curve. The linearity of the standard curve is very good, as demonstrated by the R2 value of 0.9973; again, this depends in large part upon the accuracy of pipetting. The agreement of the experimental determination is relatively good.
In Part D, the identity of the olive oil samples was determined from the corresponding values of emitted fluorescence.
