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