Spectrophotometry

I. Electromagnetic Radiation = LightA. What is Light?

1. Visible light is a particular kind of electromagnetic radiation

2. X-rays, UV, Infrared, Microwaves, and Radio waves are all light forms

3. Light is a form of Energy

4. All light travels in waves at “the speed of light” = c = 3 x 108 m/s

B. Characteristics of Light

1. Wavelength = distance between two peaks in a wave

a. (lambda) is the symbol

b. Meters = m is the unit

2. Frequency = number of complete waves passing a given point per second

a. (nu) is the symbol

b. Hertz = Hz = s-1 is the unit

3. Amplitude = A = measure of the intensity of the wave, “brightness”

4. The speed of light is constant: c = x a. and are inversely proportional

b. If one increases, the other decreases

c. Their product is always the speed of light = c = x = 3 x 108 m/s

II. Spectroscopy = using light to investigate a compoundA. How the experiment works

B. What color do we see?

a) What we see as the color of a compound is the complementary color to what the compound absorbs

b) Example: Absorbs red, we see green

III. Beer’s LawA. Absorbance tells us how much light is absorbed by the molecule

B. A = lc (y = mx + b) describes the absorption of light in a solution

l = the length of the cell containing the solution, usual 1 cm

c = concentration in mol/L = M

= Molar extinction coefficient = constant for a given molecule at a given wavelength of light = how well the molecule absorbs light

%Tlog2%T

100%logA

100% x I

I%T

I

IlogA

0

0

Percent T

0 10 25 50 75 100

2.0 1.0 0.6 0.3 0.1 0Absorbance

Absorbance

Concentration (mol/L)

1.0

0.5

0.00 0.00001 0.00002 0.00003 0.00004 0.00005

Slope = l [L/mol∙cm][cm]

≈ 18,000 for our red dye

IV. Today’s ExperimentA. Make a Beer’s Law Calibration Curve using a Stock Solution

1. Calculating the concentration of your stock solution: (Ex: 0.0788g/L)

2. Calculations for the calibration curve samples: M1V1 = M2V2

3. An Excel Template is available at faculty.swosu.edu/tim.hubin in the “Shared Folder” link called “Spectrophotometry Lab Excel sheet”

Sample # 1 2 3 4 5 6

mL DyeStock Soln

0.50 1.00 1.50 2.00 2.50 3.00

mL Water 9.50 9.00 8.50 8.00 7.50 7.00

Molarity

Lmolxg

mol

L

g/1059.1

44.496

10788.0 4

LmolxL

LLmolx

L

mol

LL

molL

L

molx

/100.8010.0

00050.01059.1

X

010.0 X

00050.01059.1

6

4

4

B. Find the concentration of an unknown sample of the red dye

1. We will give you an unknown amount of the stock solution

2. Dilute it to 25 ml in a volumetric flask

3. Calculate the absorbance (from %T) on the Spec. 20

4. Use your spreadsheet to calculate the concentration from the absorbance

5. Example Calculation: %T = 15% for your diluted unknown

C. Make 100ml of a solution of a known concentration (2.5 x 10-5 M)

LmolxcmcmmolL

/1057.4)1)(/000,18(

0.824AccA

824.015log2%Tlog2A

5

solutionstock of 7.150157.0

1059.1

100.0 105.2

X

100.0 105.2

X1059.1

VMVM

4

5

54

2211

mlL

Lmolx

LLmolx

L

LL

molxL

L

molx

%100% xA

CAError

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