Spectrophotometer Lab Report

Introduction

The aims of the lab session was to familiarize with the principles, operation and application of a light spectrophotometer. This report discusses an experiment to study the relationship of absorbance and concentration, the interaction of electromagnetic radiant energy(ERE) and matter which is an important aspect of the Beer-Lambert 's Law. This law states that the absorption of a compound is directly proportional to the concentration of that particular compound. This is because (a & b) are constants. In the equation,
A = abc where A is the absorbance, a is the specific absorbance, b is the path length, c is concentration. This report presents the procedures for the experiment, the experiment 's results, and an analysis of those results.

In this experiment, cuvettes containing a reagent blank is inserted into the spectophotometer with the meter adjusted to read zero absorbance. The composition of the reagent blank should be indentical to that of standard or unknown solutions except for the substance to be measured. Standard solutions with known concentrations of substances are then inserted & readings recorded. Finally, a reading is made of the unknown solution, and its concentration is determined by comparison with the readings obtained on the standards.

There were multiple objectives for the experiment. 1.)To determine the concentration of a solution of cytochrome c (cyt c) by comparison of its light absorption with the light absorption of a standard 0.1mg/ml solution. 2.)To plot a spectrum of light absorption against wavelength for cyt c. 3.)Prepare a series of cyt c standard solutions of known concentration and to plot a calibration graph of light absorbance against concentration. 4.)Measure the concentration of a solution of cyt c of unknown concentration.

Materials & Methods

Part A : Preparation of Materials

For the first phase of the experiment, 3ml of dH20 in a cuvette was used as a reference blank. 0.1mg/ml of cyt c solution was then placed in a spectrophotometer to obtain wavelength of peak absorbance.
In order to establish a baseline, 4 samples of stock 0.1mg/ml cyt c solution were diluted to 5ml. The first sample was prepared by pipetting 4ml of cyt c stock solution into 1ml of deionized water up to exactly 5ml.
The next 3 samples were then prepared in the same manner using 3ml of cyt c stock solution, then with 2ml and next with 1ml.

Part B : Collecting the Data

0.1mg/ml of cyt c solution was first placed in a Shimadizu UV-1601 UV-Visible Spectrophotometer at a range of 380nm-435nm to obtain the wavelength of peak absorbance(425nm).
Each prepared sample was then analyzed using the same spectrophotometer at wavelength of 425nm. The absorbance values were recorded and plotted into an excel graph.
After plotting the chart with values of known concentrations, absorbance(y-axis) versus concentration(x-axis). We can construct a standard curve(fig.2). Comparing the standard curve to the data of the unknown allowed the estimation of the concentration of the unknown sample.

Results Figure 1. Standard curve as related to collected data (table 1)

Wavelength(nm)
Absorbance
380
1.087
385
1.207
390
1.314
400
1.509
405
1.593
410
1.661
415
1.721
420
1.744
425
1.768
430
1.762
435
1.741
Table 1. Collected data from spectrophotometer

As shown above (table 1), the wavelength of peak absorbance for cyt c was found to be at 425nm, where absorbance peaks before dipping down at 430nm & 435nm respectively.

Figure 2. Standard curve as related to collected data (table 2)

Final concentration of cyt c (mg/ml)
Absorbance at lambda max
0.08
1.420
0.06
1.076
0.04
0.708
0.02
0.355 Table 2. Tabulated data of the 4 samples after dilutions

As shown in table 2 above, the final concentrations of cyt c were obtained from the dilution formula, C1V1 = C2V2

where C1 is the initial concentration, V1 is the initial volume, C2 is the final concentration, V2 is the final volume. Thus a final concentration of 0.08mg/ml of cyt c for sample 1 can be calculated via,

0.1mg/ml x 4ml = ? x 5ml 0.4/5 = 0.08mg/ml The subsequent figures of the other samples were then plugged into the graph which resulted in a straight line, or standard curve. The graph accurately depicts that as the concentration of cyt c is increased, its absorbance also increases.

Final concentration of cyt c (mg/ml)
Absorbance at lambda max
0.08
1.420
0.06
1.076
0.04
0.708
0.02
0.355
Unknown
0.825 Table 3. Unknown concentration of unknown sample with 0.825 absorbance at lambda max

Figure 3. Cyt c concentration of unknown estimated to be 0.045mg/ml

From figure 3 above, it illustrates that the concentration of the unknown falls between the range of 0.04-0.05mg/ml. Thus it can be concluded that the estimated concentration of unknown sample could be around 0.045mg/ml.

Discussion

In the initial phase of the lab, it demonstrated the use of a spectrophotometer to analyze the optimal wavelength of absorbance for different solutes.
The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. The beam of light consists of a stream of photons. When a photon encounters an analyte molecule, there is a chance the analyte will absorb the photon. This absorption reduces the number of photons in the beam, thereby reducing the intensity of the light beam reaching the detector (N.Blauch.,2014).
Thus cytochrome c, based on this concept of spectophotometry, peak absorbance was found to be at 425nm (table.1). Thus this particular wavelength was chosen to measure the concentration of the unknown cyt c sample.

For the second phase of the lab, it illustrated the direct association between the concentration of particles in a solution and the amount of light that could pass through. This is in accordance to Beer 's Law which states that the absorbance of a species at a particular wavelength of electromagnetic radiation, is proportional to the concentration of the absorbing species(F.Schneider.,2013)
From figure 2 above, the graph shows a straight line, which illustrates the direct proportionality of concentration vs absorbance, whereby increasing concentration of analyte also increases its absorbance. Thus the sample obeys the Beer-Lambert Law.

For the third phase of the lab, applying the principles of Beer 's Law and the concept of spectophotometry. The calibration graph (figure.3) can be used to estimate the concentration of the unknown sample, which is estimated to be at 0.045mg/ml.

Lastly, to calculate the approx % error of the instrument reading.

2 equations will be done. 0.825 +/- 0.02 absorbance units

1.) 0.832 – 0.812/0.812 x 100% = 83.2%
2.)0.792 – 0.812/0.812 x 100% = 79.2%

Conclusion

Other then learning about the principles of spectrophotometry, this lab also showed the importance of accurate measurement. For example, while using the spectrophotometer, equipment has to be throughly checked as any scratched/dirty cuvettes could lead to false readings. Secondly, when using spectrophotometers, its ideal to use the same machine for every subsequent test as different machines could produce even minute differences in results.

References

Blauch, D.N (2014) Spectrophotometry. [online]. Available from: http://www.chm.davidson.edu/vce/spectrophotometry/Spectrophotometry.html [Accessed: 28th /12/2014]
Jeffery, P (2006) Hands on Chemistry. 1St ed. Boston: Mc-Graw Hill
Schneider, R.F (2013) Beer 's Law. [online]. New York: Stony Brook University. Available from: http://www.ic.sunysb.edu/Class/che133/lectures/beerslaw.html [Accessed: 28th/12/2014]
R.Reed, Holmes D, Weyers J, Jones A. (2007) Practical Skills in Biomlecular Sciences. 3Rd ed. United Kingdom: Pearson Education Ltd

References: Blauch, D.N (2014) Spectrophotometry. [online]. Available from: http://www.chm.davidson.edu/vce/spectrophotometry/Spectrophotometry.html [Accessed: 28th /12/2014] Jeffery, P (2006) Hands on Chemistry. 1St ed. Boston: Mc-Graw Hill Schneider, R.F (2013) Beer 's Law. [online]. New York: Stony Brook University. Available from: http://www.ic.sunysb.edu/Class/che133/lectures/beerslaw.html [Accessed: 28th/12/2014] R.Reed, Holmes D, Weyers J, Jones A. (2007) Practical Skills in Biomlecular Sciences. 3Rd ed. United Kingdom: Pearson Education Ltd