Spectrophotometer
Updated 9/27/2006
I. Outline
A. Spectrophotometry DefinedB. Electromagnetic ScaleC. Waves definedD. Human EyeE. Molecules and Light F. Absorption and ReflectionG. Spectrophotometer
A. Spectrophotometry Defined
1. Quantifies a given sample in a solution
2. Concentration in a volume of solution
3. Spectro=sight
4. Photo=light waves
5. Metry=measurement
Types of Electromagnetic
Radiation
B. Electromagnetic Scale
1. Electromagnetic Spectrum (visible light)
C. Waves
λ
Crest
trough
midpoint
4. Crest: High point in the wave5. Trough: Low point in the wave
6. --------Midpoint of wave where the wave is in equilibrium
7. Amplitude Distance from the midpoint to the crest or trough8. The higher the amplitude the stronger the wave
1. Light waves
λ
λ
λ
a) The wave length of electromagnetic radiation varies greatly depending on its type.
b) X-ray are measured in nanometers, whereas, radio waves 10,000 meters.
c) They also vary in the amount of energy they carry.
d) The shorter the wavelength, the more energy is carried by it.
e) X-ray have very short wavelengths and carry a great deal of energy. Radio waves have long wavelengths and photons with much less energy
D. Human Eye & Vision
380 – 430nm Violet 430 – 475nm Blue 505 – 555nm Green 575 – 600nm YellowYellow 600 – 650 nm600 – 650 nm OrangeOrange 650 – 780nm Red
1. Sensing Light
a) Humans have two light detectors.
b) Do you know what they are called?
Rods and Cones!!!
Color Vision
c) Cones Current understanding
is that the 6 to 7 million cones can be divided
into "red" cones (64%), "green" cones (32%), and "blue" cones (2%)
d) Rods not sensitive to color. They are responsible
for our dark-adapted, or scotopic, vision (night vision)
e) Sensitive to light. The rods are
incredibly efficient photoreceptors.
More than one thousand times as sensitive as the cones,
they can reportedly be triggered by individual photons under optimal conditions.
f. Materials
Every material has a particular arrangement of electrons and of bonds involving electrons.
Colors of ObjectsViolet
IndigoBlue
GreenYellow
Orange
Red
The color of an object is determined by which wavelengths of light it reflects.
If the object absorbs light of a particular color, then that color does not reach our eyes when we look at that object.
Colors of ObjectsViolet
IndigoBlue
GreenYellow
Orange
Red
Colors of ObjectsViolet
IndigoBlue
GreenYellow
Orange
Red
The color of an object is determined by which wavelengths of light it absorbs. If the object absorbs light of a particular color, then that color does not reach our eyes when we look at that object.
An object appears orange if it absorbs all the colors except orange.
Colors of ObjectsViolet
IndigoBlue
GreenYellow
Orange
Red
Color Wheel
Absorption of Light by a Solution of RED Food Coloring
Incoming light=green
solution appears red
or red-orange.
The Absorption of Light of Particular Wavelengths and Color of Solutions
Wavelength λColor Solution
380 – 430nm Violet Yellow
430 – 475nm BlueBlue Orange
505 – 555nm Green Red
575 – 600nm YellowYellow VioletViolet
600 – 650nm OrangeOrange BlueBlue
650 – 780nm Red Green
Spectrophotometer
Used to measure the effect of a sample on a beam of light.
Updated 9/30/2005
Basics of Spectrophotometry
Blank
The blank contains the solvent and any reagents that are added to the sample.
Sample
Well-mixed No air bubbles No particulate Avoid fingerprints on
cuvette
%TransmittanceThe ratio of the amount of light
transmitted through a sample to that of the blank
t = Light transmitted through sample
Light transmitted through blank
First, the intensity of light (I0) passing through a blank is measured. T for transmittanceThe intensity is the number of photons per second.
The blank is a solution that is identical to the sample solution except that the blank does not contain the solute that absorbs light. This measurement is necessary, because the cell itself
scatters some of the light.
Second, the intensity of light (I) passing through the sample solution is measured. (In practice, instruments measure the power rather
than the intensity of the light. The power is the energy per second, which is the
product of the intensity (photons per second) and the energy per photon.)
E=hf or hc/λ; c is the speed of light and h is 6.63 x 10-
34 E is the energy of one photon
Third, the experimental data is used to calculate two quantities: the transmittance (T) and the absorbance (A).
T = I
I0
A = - log10 T
Transmittance If t ≤ 1 (less than or equal to) then the
amount of light transmitted through the sample is less than the blank.
In another situation, where both the sample and the blank transmit the same amount of light t = 1
In a sample that transmits no light at all then t = 0
Transmittance range 0 to 1
Percent Transmittance%T = t x 100%
When both the sample and the blank transmit the same amount of light . %T = 100%
When a sample transmits no light at all the %T = 0%
Transmission vs. Absorption
transmission- pass without interaction through the material.
Absorption- gives up some or all of its energy to the material. Light energy is converted to heat energy.
Absorbance or Optical Density (OD)
Amount of light absorbed by the sample.
A = -log 10 (t)
1.6 or 1.6A or 1.6 AU or OD 1.6
Relationship between %Transmittance and Absorbance of Light and Concentration of Analyte.
A ↑ = t ↓
Concentration Concentration
Ab
sorb
ance
(A
)
Per
cent
Tra
nsm
itta
nce
(%T
)
Recording Absorbance
A260 = 1.6
Absorbance of 1.6 was measured at a wavelength of 260 nm
Absorbance Spectrum of RED Food Coloring
Red Absorbance CurveRed Coloring
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
400 450 500 550 600 650 700
Wavelength in Nanometers
Ab
so
rpti
on
Red
Yellow Absorbance CurveYellow Coloring
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
400 450 500 550 600 650 700
Wavelength in Nanometers
Ab
sorp
tio
n
Yellow
Blue Absorbance CurveBlue Coloring
0
0.5
1
1.5
2
2.5
3
3.5
4
400 450 500 550 600 650 700
Wavelength in Nanometers
Ab
sorp
tio
n
Blue
What color is this solution?
This compound has an absorbance peak in the greenish-blue region of the spectrum. So this solution would be orange.
It is the dye Orange G.
Orange GC16H10N2O7S2Na2
What if the solution is clear?
Can you measure the absorbance?YesThe material may not absorb light in the
visible range of the EM spectrum.Proteins and nucleic acid absorb in the UV
range of EM Spectrum
Riboflavin
Every material has a particular arrangement of electrons and of bonds involving electrons.
Riboflavin
Riboflavin
DNAProtein
Absorbance spectra for DNA
Absorbance spectra for Protein BSA
Absorbance Spectra for DNA and Protein
Distinct peaks for DNA and Protein Can not Be resolved.
Set wavelength
to 430 nm
Blank
The blank contains the solvent and any reagents that are added to the sample.
Calibrateby pressing BlueCAL button in the middle of colorimeter
Sample
Well-mixed No air bubbles No particulate Avoid fingerprints on
cuvette
Set wavelength to 470 nm
Blank
The blank contains the solvent and any reagents that are added to the sample.
Calibrateby pressing BlueCAL button in the middle of colorimeter
470nm
Set wavelength
to 565 nm
Blank
The blank contains the solvent and any reagents that are added to the sample.
Calibrateby pressing BlueCAL button in the middle of colorimeter
565 nm
Set wavelength
to 635 nm
Blank
The blank contains the solvent and any reagents that are added to the sample.
Calibrateby pressing BlueCAL button in the middle of colorimeter
635 nm
Red Yellow Blue430 2.077 1.275 1.092470 2.378 1.952 1.131565 0.734 0.063 1.393635 0.043 0 1.901
Red, Yellow and Blue Absorbance at Various Wavelengths
Red Absorbance CurveRed Coloring
0
0.5
1
1.5
2
2.5
430 470 565 635
Wavelength in Nanometers
Ab
so
rpti
on
Red
Yellow Coloring
0
0.5
1
1.5
2
2.5
430 470 565 635
Wavelength in Nanometers
Ab
sorp
tio
n
Yellow
Yellow Absorbance Curve
Blue Coloring
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
430 470 565 635
Wavelength in Nanometers
Ab
sorp
tio
n
Blue
Blue Absorbance Curve
Standard Curves
Answer Essential Questions
What is the identity or nature of the components of a sample?
Qualitative How much of an analyte is present in a
sample?
Quantitative
Uses
How much DNA is present in a cellular extract?
How pure is the protein in an enzyme preparation?
What is the effectiveness of an enzyme? What is the active ingredient in a drug
formulation?
Standard Curve The concentration of the
stock solution is 1 ul/ml. You want to create a series
of dilutions with the following concentrations: 1 ul/ml, 0.8 ul/ml, 0.6 ul/ml, 0.4 ul/ml, 0.2 ul/ml and 0.1 ul/ml.
You want the final volume of each dilution to be 3 ml.
0.8 uL/mL The concentration of the stock
solution is 1 ul/ml. You want the final volume of
each dilution to be 3 ml. C1V1=C2V2
V1=C2V2÷C1
V1= 0.8 ul/ml x 3ml ÷ 1.0 ul/ml 2.4 ml stock solution 0.6 ml water
0.6 uL/mL The concentration of the stock
solution is 1 ul/ml. You want the final volume of
each dilution to be 3 ml. C1V1=C2V2
V1=C2V2÷C1
V1= 0.6 ul/ml x 3ml ÷ 1.0 ul/ml 1.8 ml stock solution 1.2 ml water
0.4 uL/mL The concentration of the stock
solution is 1 ul/ml. You want the final volume of
each dilution to be 3 ml. C1V1=C2V2
V1=C2V2÷C1
V1= 0.4 ul/ml x 3ml ÷ 1.0 ul/ml 1.2 ml stock solution 1.8 ml water
0.2 uL/mL The concentration of the stock
solution is 1 ul/ml. You want the final volume of
each dilution to be 3 ml. C1V1=C2V2
V1=C2V2÷C1
V1= 0.2 ul/ml x 3ml ÷ 1.0 ul/ml 0.6 ml stock solution 2.4 ml water
0.1 uL/mL The concentration of the stock
solution is 1 ul/ml. You want the final volume of
each dilution to be 3 ml. C1V1=C2V2
V1=C2V2÷C1
V1= 0.1 ul/ml x 3ml ÷ 1.0 ul/ml
Standard Curve
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Concentration
Ab
so
rba
nc
e