Welcome to BIO 260Molecular 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

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Terms to know for the spectrophotometer:TransmittanceAbsorptionQuality Assurance

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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.

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A spectrophotometer consists of two instruments: A spectrometer for producing light of any

selected color (wavelength)A photometer for measuring the intensity of

light.

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Beer’s 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.

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Beer’s 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.

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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 1000% T is all light absorbed100%T is no light absorbed.

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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.

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Absorbtion:Light of a specific wavelength is used to pass

through a solution or suspensionThe light may be absorbed or scatteredThe 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

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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

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Light source visible: incandescent bulbLight source UV: deuterium-discharge lampsMonochromator : wave selecting deviceCuvette: round or square, come in sets, quartz must be

used for UVPhotoresistor: converts radiant energy to electrical energyAmplifier:

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http://viewpure.com/xHQM4BbR040 ncbionetwork a bit boring, but

decent, quick presentation of a Vis Spec

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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.

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ChromatographyThe 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.

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Chromatography is commonly used in

biotechnology for purifying biological molecules,

like proteins, for medicine or other uses.

Chromatography separates individual components

from complex mixtures.

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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.

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Molecules travel through the stationary

phase at different rates because of their

chemistry.

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Some Common Types of Chromatography

Gel filtration chromatography

Affinity chromatography

Ion exchange chromatography

High Performance Liquid Chromatography

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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 column—also referred to as the exclusion limit of a column.

Sample

Column bed

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Buffer

http://viewpure.com/Z54ec_G12QE column chromatography by Gen Ed

http://viewpure.com/Hb791WsC78s affinity urd2urd

http://viewpure.com/kz_egMtdnL4 HPLC Royal Society

of Chemistry

http://www.ncbionetwork.org/chromatography/ interactive tutorial by BioNetwork – could be used as an assignment includes affinity, ion, size chromatography

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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.  

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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.

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http://viewpure.com/WBTO9skgVP4

pretty simple, but could be used elsewhere???

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