Presentation on theme: "Indicator – Quickest method. Indicator solutions change colors when a molecule of interest is present. Allows scientist to detect colorless molecules."— Presentation transcript:
Indicator – Quickest method. Indicator solutions change colors when a molecule of interest is present. Allows scientist to detect colorless molecules in a solution. Examples: Bradford protein reagent Diphenylamine (DPA) or Biuret (NaOH+CuSO 4 ). What kind of data is this?
Spectrophotometer (Spec) An instrument that measures the amount of light that passes through (is transmitted through) a sample.
Uses a type of light to detect molecules in a solution Light is a type of energy, and the energy is reported as wavelengths, in nanometers (nm).
Ultraviolet (UV) Spectrophotometers. Uses ultraviolet light of wave lengths from 200 nm to 350 nm. Visible (VIS) Light Spectrum Spectrophotometers. Uses visible light (white light) of wave lengths from 350 nm to 700 nm.
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Shines a beam of light on a sample. The molecules in the sample interact with the light waves in of 3 ways: Absorb the energy Reflect the energy Transmit the energy between and through the atoms and molecules of the sample.
Blue molecules absorb the other colors of visible light. Blue molecules are blue because they reflect blue light.
Consider blue molecules, all the wavelengths of light are absorbed, except for the blue ones. The blue wavelengths are transmitted or reflected off the molecules. If these blue wavelengths hit a detector (such as in the spectrophotometer or the nerve cells in your eye), they appear blue.
Molecules are whatever color of light that they do not absorb. Green molecules appear green because they absorb most wavelengths of visible light, except the green wavelengths.
The spectrophotometer measures the amount of light transmitted through the sample (Transmittance). By using an equation (Beers law), it converts the transmittance data to an absorbance value. What kind of data is this?
The concentration of an unknown sample can be determined b y comparing the absorbance data to standards of known concentration. The data generated with the set of known standards is called a standard curve.
1. Cuvettes are made from plastic, glass, or quartz. a. Use quartz cuvettes for UV work. b. Glass, plastic or quartz are acceptable visible work. c. There are inexpensive plastic cuvettes that may be suitable for some UV work.
2. Cuvettes are expensive and fragile (except for disposable plastic ones). Use them properly and carefully. a. Do not scratch cuvettes; do not store them in wire racks or clean with brushes or abrasives. b. Do not allow samples to sit in a cuvette for a long period of time. c. Wash cuvettes immediately after use.
Inner parts Lamp Prism or grating that direct light of a specific wavelength.
Visible spectrophotometer white light Contains a tungsten lamp that produces white light. Ultraviolet spectrophotometer Contains a deuterium lamp that produces light in the UV light part of the spectrum.
Outer parts: Sample Holder Display Knobs or buttons used to calibrate the spec to measure the designated molecule. Wavelength Selection
Visible Spectrophotometer White light hits the prism or grating, it is split into the colors of the rainbow (Visible Spectrum). The wavelength knob rotates the prism/grating, directing different color of light toward the sample.
The wavelength of light produced by the tungsten lamp range from about 350 nm (Violet light) to 700 nm (red light). The molecules in the sample either absorb or Transmit the light energy of one wavelength or another.
The detector measures the amount of light being transmitted by the sample and reports that value directly (% transmittance) or converts it to the amount of light absorbed in absorbance units (au) using Beers Law.
A = 2 – log 10 %T
After collecting data for your concentration an absorption spectrum graph is created. These can be used when attempting to identify unknown substances (e.g. CSI)
The absorbance spectrum is a graph of a samples absorbance at different wavelengths.
The spectrophotometer can measure the amount of absorbance or lack of absorbance of different colored light for a given molecule.
The concentration of molecules in a solution affects the solutions absorbance. Remember [ ] is a ratio – when we change one number – it affects the ratio If there are more molecules in one solution than in another, than there are more molecules to absorb the light.
Determines the presence and concentrations of samples. Determines the purity of a sample. Look at the change of samples over time.
A. Measure the absorbance of standards containing known concentrations of the analyte B. Plot a standard curve with absorbance on the X axis and analyte concentration on the Y axis C. Measure the absorbance of the unknown(s) D. Determine the concentration of material of interest in the unknowns based on the standard curve
If there is too much or too little analyte, spectrophotometer cannot read the absorbance accurately