Welcome to BIO 260 Molecular Techniques Unit 8 – Spectrophotometry and Chromatography

Objectives for Spectrophotometry & Chromatography Unit: Explain how a spectrophotometer works. Contrast the difference between absorption and transmittance. Explain the difference between using a spectrophotometer and a nanodrop. Explain how chromatography works Explain what is chromatography used for. List three common methods of chromatography MATC - BIO U1

Terms to know for the spectrophotometer: Transmittance Absorption Quality Assurance MATC - BIO U1

Principle of the spectrophotometer The spectrophotometer is a very simple but beautiful instrument. It is employed to measure the amount of light that a sample absorbs. Simply put, the instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. MATC - BIO U1

A spectrophotometer consists of two instruments: A spectrometer for producing light of any selected color (wavelength) A photometer for measuring the intensity of light. MATC - BIO U1

Beers Law When monochromatic light (light of a specific wavelength) passes through a solution there is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light, that is, the more concentrated the specimen is, the less light is transmitted through it. MATC - BIO U1

Beers Law: The concentration of a substance in directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light. MATC - BIO U1

Transmittance: % Transmittance is the ratio of the radiant energy transmitted (T) divided by the radiant energy incident on the sample (I): %T=T/I x 100 0% T is all light absorbed 100%T is no light absorbed. MATC - BIO U1

Before beginning, the electrical readout (of transmittance) is set arbitrarily at 100%T with a blank in place. (The blank is the solvent without the constituent). Next the sample constituent (with the absorbing molecules) is placed in the light path. The difference in amount transmitted by the blank and the sample is due only to the presence of the absorbing molecules of the constituent. %T = measurement of transmitted light/ blank measurement. MATC - BIO U1

Absorbtion: Light of a specific wavelength is used to pass through a solution or suspension The light may be absorbed or scattered The amount of light that gets through is a measure of the concentration of the solution or suspension Absorption is the property of a solution to absorb the light that comes to it MATC - BIO U1

Absorbance is measured by a spectrophotometer. The range of absorbance is 0 (no light absorbed) to around 2 (all light absorbed). A spectrophotometer can be set for a specific wavelength and provides an absorbance value for a solution at that wavelength MATC - BIO U1

Light source visible: incandescent bulb Light source UV: deuterium-discharge lamps Monochromator : wave selecting device Cuvette: round or square, come in sets, quartz must be used for UV Photoresistor: converts radiant energy to electrical energy Amplifier: MATC - BIO U1

ncbionetwork a bit boring, but decent, quick presentation of a Vis Spec MATC - BIO U1

Quality Assurance 3 important checks: Wavelength accuracy - the wavelength on the dial is the wave length of the light passing through the monochromator. Standard absorbing solutions or filters are used to check this. Stray light – Wavelengths that pass outside band transmitted by the monochromator (scratches on surface of monochromator or dust. The effect is absorbance errors. Correct with cutoff filters which eliminate all radiation other than the one chosen by the monochromator. Linearity - is evidenced by the straight-line calibration curve from a change in concentration of solute. Linearity is effected by stray light. Colored solutions can be used as standards. MATC - BIO U1

Chromatography The Science of Separation The separation of molecules based upon their chemical and physical properties. Used for purification, isolation, and characterization of inorganic, organic, and biological compounds. There are many types of chromatography, based upon molecular size, charge, and shape. MATC - BIO U1

Chromatography is commonly used in biotechnology for purifying biological molecules, like proteins, for medicine or other uses. Chromatography separates individual components from complex mixtures. MATC - BIO U1

Chromatography consists of two phases: mobile phase (solvent and the molecules to be separated) stationary phase either, in paper (in paper chromatography) or glass beads, called resin, (in column chromatography), through which the mobile phase travels. MATC - BIO U1

Molecules travel through the stationary phase at different rates because of their chemistry. MATC - BIO U1

Some Common Types of Chromatography Gel filtration chromatography Affinity chromatography Ion exchange chromatography High Performance Liquid Chromatography MATC - BIO U1

Principles of Size Exclusion Chromatography (SEC) The mass of beads within the column is often referred to as the column bed. The beads act as traps or sieves and function to filter small molecules which become temporarily trapped within the pores. Larger molecules pass around, or are excluded from, the beads. This is called fractionating. As the liquid flows through the column, molecules below the chosen Dalton size enter the beads and pass through the column more slowly. The smaller the molecules, the slower they move through the column. Larger molecules pass around the beads and are excluded from the columnalso referred to as the exclusion limit of a column. Sample Column bed MATC - BIO U1 Buffer

column chromatography by Gen Ed affinity urd2urd HPLC Royal Society of Chemistry interactive tutorial by BioNetwork – could be used as an assignment includes affinity, ion, size chromatography MATC - BIO U1

The liquid used to dissolve the biomolecules to make the mobile phase is usually called a buffer. The mixture of biomolecules dissolved in the buffer is called the sample. The sample is placed on the column bed and the biomolecules within the buffer enter the top of the column bed, filter through and around the porous beads, and ultimately pass through a small opening at the bottom of the column. MATC - BIO U1

For this process to be completed additional buffer is placed on the column bed after the sample has entered the bed. The mobile phase liquid is collected, as drops, into collection tubes which are sequentially ordered. A set number of drops is usually collected into each tube. The larger molecules which pass quickly through the column will end up in the early tubes or fractions. The smaller molecules which penetrate the pores of the stationary phase end up in the later fractions. MATC - BIO U1

pretty simple, but could be used elsewhere??? MATC - BIO U1