Uv/Vis Spectroscopy

Theory
Molecules absorb visible light at a particular wavelength which is dependent upon their chemical nature, transition metal ions and highly conjugated chemical structure. Ultraviolet-Visible spectroscopy uses light in the visible and adjacent near ultraviolet regions to analyze molecules undergoing electronic transition. Absorption measures the electronic transitions of photons from the ground state to the excited state.

UV/Vis spectrophotometer
It contains many elements: Diffraction Grating or monochromator that separates the different wavelength of light A beam of light from a visible and/or UV light source (colored red) is separated into its component wavelengths by a prism or diffraction grating. Half mirrored device that splits the beam into two equal intensity beams (color red to color magenta and color blue) A cuvette containing a solution of the compound being studied. Electronic detectors that measured and compared the intensities of these light beams.

Uv/Vis Spectrophotometer
The spectrophotometer measures the intensity of light passing through a sample (I), and compares it to the intensity of light before it passes through the sample (Io). The ratio I / Io are called the transmittance, and are usually expressed as a percentage (%T). The absorbance A, is based on the transmittance: A = log (%T / 100%). Ultraviolet region scanned is normally from 200 to 400 nm, and the visible portion is from 400 to 800 nm

Beer Lambert Law

Beers law describes how much light a solution containing a molecule will absorb. A= bc is the molar extinction coefficient, b is the distance the light travel to the solution c is the concentration. If all light passes through a solution without any absorption, then absorbance is zero and percent transmittance is 100%. If all light absorbed, then percent transmittance is zero and absorption is infinite.

Purpose
In this lab, we used a Cary 50 UV/Vis to look absorbance of Tyrosine and Phenlyalanine. Using these concepts are very important for biochemistry because many biomolecules absorb light. Some of the four biopolymers such as proteins, RNA, and DNA absorb light but complex carbohydrates do not absorb light.

Procedure
Sample Prep. The first step of this experiment is to make up the two samples of Phe and Trp amino acids that will be used for this experiment. This is done exactly as it was for the HPLC lab. Pour 2 separate 10mL of TRIS buffer solution in graduate cylinder Weight out 0.01g of L-Tryptophan and 5,5diphenylhyantoin Add the solids to 2 separate beakers and add the 2 solutions TRIS to each Stir the two solutions so the solid has dissolved

 Now you are able to load your samples into the Cary-50 UV/Vis spectrophotometer. There will be 3 different samples that will be looked at Phe, Trp will both be ran with the addition of water only. Trp will be ran an additional time this time with NaOH as well as with water. The total volume for each sample will be 1mL. Add 900uL of water will be added to the quartz cuvette, this must be run in the machine prior to adding any of the amino acids in order to blank or zero. Once your sample has been zero add 100uL of your Phe solution to the cuvette for a final volume of 1mL. This will be run 3 separate times.

Procedure

 Repeat the same procedure for the Trp. For the Trp with NaOH we are looking at how the pH difference affects the concentration of it in solution. To do this, add 700uL and then 200uL of NaOH to get 900uL zero your graph and then add your 100uL Trp. This again will be done 3 separate times. Collect the graphs for each sample that has been ran, a total of 9 graphs should be seen. The graphs provide you with the absorbance of each amino acid within the wavelength range of 250-300nm, you are now able to solve the Beers Law (A=abc) in order to find the corresponding concentrations of each and compare your results.

Procedure

Data Collected
Tyrosine + Water

275nm 275nm 275nm

Abs
0.600 0.567 0.589

Phenylalanine + Water 259.9nm 259.9nm 269.9nm Abs 0.964 0.897 0.878

Calculations
From the data collected you can use A= bc to calculate the solutions concentration. Ex: 1 Tyr + W @ 274nm A= bc
= 1,405 M-1 cm-1 0.600 = (1,405 M-1 cm-1 ) X (1cm) X (c) b= 1cm 0.000427 M = c A= 0.600 This is the conc of Tyr in your sample! c= Concentration ? Do these calculations for as many samples you tested, in our case 3 samples.

Data Calculated
Ttests Concentrations (M) Tyr + W Phe + W 0.000427 0.004778 0.000415 0.004679 0.000399 0.004235 Mean : StDev: T-Test: 0.000413 0.004618 3.86E-05 0.000167 1.80E-06 Once all the concentrations are calculated, you can set up a table in excel. This will allow you to compare the concentrations of your two different samples with a Ttest. The P-value should be 0.01 for a One Tailed Ttest. A value in this range tells you that the concentrations being compared are Different Should the P-value be above 0.01, this would not indicate anything specific about the data analyzed more evidence is needed to confirm they are different

Data Calculated
Ttests should be calculated to compare the following: Concentrations of Tyr to Phe Absorbencies collected of Tyr to Phe Absorbencies collected of Tyr +W to Tyr+ NaOH Confidence Limits Take the mean of your measured absorbencies for Tyr + W and Tyr + NaOH Compare them using Excels Confidence Interval

Confidence Limits
First you should calculate the Molar Ext. Coeff of Tyr + NaOH as follows: A= bc Use Ave Conc from your calculations of Tyr + W c = 0.000413M b = 1 cm 1.315 = ()( 1) (0.000413 ) A = Tyr + NaOH data 1.315 = 3184 M-1 cm-1 =?

Confidence Limits
Do the same calculations for the three samples.
Calculate the Mean (4378) & SD Use excel to calculate Confidence Limit for 5 different Zvalues or (excel) Excel requests these three numbers to calculate CL = 0.45, 0.70, 0.85, 0.925, 0.99 ( one at a time) Standard Deviation Sample Size = 3 The result is an expression as follows 4378 13.04 at 99 % Confidence Limit Which means we are 99% sure the Molar ext Coeff is between 4364.96 and 4391.04 for your Tyr + NaOH sample

Conclusions
For our analysis
Ttests helps us identify whether the concentrations and the absorbencies were the same for our 3 samples The Confidence Limits gave us ranges as to how confident we were that the Molar Extinction Coeff. changed as we added NaOH the pH increased.