Spectrophotometer Updated 9/27/2006

I. Outline A. Spectrophotometry Defined B. Electromagnetic Scale C. Waves defined D. Human Eye E. Molecules and Light F. Absorption and Reflection G. 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 wave 5.Trough: Low point in the wave Midpoint of wave where the wave is in equilibrium 7.Amplitude Distance from the midpoint to the crest or trough 8.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 Yellow 575 – 600nm Yellow 600 – 650 nm Orange 600 – 650 nm Orange 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)scotopic 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.photons

f. Materials Every material has a particular arrangement of electrons and of bonds involving electrons.

Colors of Objects Violet Indigo Blue Green Yellow 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 Objects Violet Indigo Blue Green Yellow Orange Red

Colors of Objects Violet Indigo Blue Green Yellow 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 Objects Violet Indigo Blue Green Yellow 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 WavelengthλColorSolution 380 – 430nm VioletYellow Blue 430 – 475nm BlueOrange 505 – 555nm GreenRed YellowViolet 575 – 600nm YellowViolet OrangeBlue 600 – 650nm OrangeBlue 650 – 780nm RedGreen

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

%Transmittance The 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 (I 0 ) passing through a blank is measured. T for transmittance  The 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 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 = II0II0 A = - log 10 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 Absorbance (A) Percent Transmittance (%T)

Recording Absorbance A 260 = 1.6 Absorbance of 1.6 was measured at a wavelength of 260 nm

Absorbance Spectrum of RED Food Coloring

Red Absorbance Curve

Yellow Absorbance Curve

Blue Absorbance Curve

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 G C 16 H 10 N 2O7 S 2 Na 2

What if the solution is clear? Can you measure the absorbance?  Yes  The 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

2 Riboflavin samples

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.

Calibrate by pressing Blue CAL 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.

Calibrate by pressing Blue CAL 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.

Calibrate by pressing Blue CAL 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.

Calibrate by pressing Blue CAL button in the middle of colorimeter

635 nm

Red, Yellow and Blue Absorbance at Various Wavelengths

Red Absorbance Curve

Yellow Absorbance Curve

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. C 1 V 1 =C 2 V 2 V 1 =C 2 V 2 ÷C 1 V 1 = 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. C 1 V 1 =C 2 V 2 V 1 =C 2 V 2 ÷C 1 V 1 = 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. C 1 V 1 =C 2 V 2 V 1 =C 2 V 2 ÷C 1 V 1 = 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. C 1 V 1 =C 2 V 2 V 1 =C 2 V 2 ÷C 1 V 1 = 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. C 1 V 1 =C 2 V 2 V 1 =C 2 V 2 ÷C 1 V 1 = 0.1 ul/ml x 3ml ÷ 1.0 ul/ml

Standard Curve

SmartSpec Plus

A symbolic representation of the SmartSpec Plus optics module.

Keypad interface Display Sample compartment door Printer Printer paper compartment Door SmartSpec Plus

Sample Compartment Cuvette chamber Cuvette storage compartment Cuvette spring Light path

Turn it on Power up the instrument. It will go through system status check and display an error message if there are any problems. Press one of the Assay buttons.

Key Pad Lambda λ

SmartSpec Plus will collect data at up to three different user-specified wavelengths. An optional background wavelength may also be specified and its absorbance will be subtracted from the other wavelengths before they are reported.

Lambda λ This assay allows you to collect absorbance data from up to three different wavelengths simultaneously. When you press λ, SmartSpec Plus will prompt you first for the number of wavelengths to read.

Wavelengths Use the number keys to input the number of wavelengths to be read and press Enter. SmartSpec Plus will then ask for each individual wavelength.

Background Reading After all wavelengths are entered, choose if you want to subtract a background reading from each wavelength.  The same background measurement is subtracted from all readings.

Background Reading If you do not want to subtract a background reading, press Enter and SmartSpec Plus will be ready to collect absorbance data. If you want to subtract a background reading, press Select to toggle from No to Yes and press Enter.

Zero the instrument SmartSpec Plus is now ready to collect absorbance data at the specified wavelengths. For best results, you should first zero the SmartSpec Plus at specified wavelengths by inserting a blank solution and pressing Read Blank.

Zero the instrument. Place a cuvette containing the blank solution into SmartSpec Plus and press Read Blank. Read Blank

SmartSpec Plus will zero the detector in preparation for reading samples. It is important to zero the instrument at each new assay. Insert a cuvette that contains all assay components except the analyte and press Read Blank.

Collect absorbance data. Place cuvette containing sample solution into SmartSpec Plus and press Read Sample. Continue collecting absorbance data until all samples are read. Read Sample

Collect absorbance data. Place cuvette containing sample solution into SmartSpec Plus and press Read Sample. Continue collecting absorbance data until all samples are read. If absorbance or concentration data area is displayed, press Enter to return the Ready screen, or simply put the next sample cuvette into SmartSpec Plus and press Read Sample. After last sample, press left arrow key to exit assay.

A spectral power distribution curve