BASIC LABORATORY METHODS IN A REGULATED ENVIRONMENT

USING SPECTROPHOTOMETRY TO MEASURE LIGHT

LECTURE OVERVIEW

• Light and its Interactions with Matter
• Spectrophotometer Design
• Spectrophotometer Operation
• Calibration of Spectrophotometers

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• Light and its Interactions with Matter
• Spectrophotometer Design
• Spectrophotometer Operation
• Calibration of Spectrophotometers

LIGHT IS A TYPE OF ELECTROMAGNETIC RADIATION

• Imagine electromagnetic radiation like waves on a pond
• But instead of water, electromagnetic radiation is energy moving through space
• Distance from one crest to the next is the wavelength

WAVELENGTH AND COLOR

• Different wavelengths of light correspond to different colors
• All colors blended is called white light
• The absence of all light is black
• Light of slightly shorter wavelengths is ultraviolet
• Human eyes do not perceive UV light

WAVELENGTH OF VISIBLE LIGHT AND COLOR

INTERACTION OF LIGHT WITH MATERIALS IN SOLUTION

• When light shines on a solution, it may pass through – be transmitted – or

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• Some or all the light energy may be absorbed

EXAMPLE, WHITE LIGHT IS SHINED ON A SOLUTION

OF RED FOOD COLORING

WHITE LIGHT IS SHINED ON A SOLUTION OF RED FOOD COLORING AS A SPECTROPHOTOMETER SEES IT

Spectrophotometer shows which wavelengths of light were absorbed

THE ABSORPTION OF LIGHT AND COLOR OF SOLUTIONS

What color is the solution whose absorbance spectrum is shown here?

Example Problem

ANSWER

• This compound has an absorbance peak in the greenish-blue region of the spectrum. Based on the previous table, we would expect it to be orange, and it is, indeed, the dye, Orange G.

BIOLOGICAL SOLUTIONS

• Usually appear clear to our eyes – have no color
• DNA, RNA, most proteins do not absorb any visible light
• But they do absorb UV light, so UV spectrophotometers are useful to biologists
• Example, can use a detector that measures absorbance at 280 nm, or 254 nm to detect proteins

• Spectrophotometers in Biotechnology
• Light and its Interactions with Matter
• Spectrophotometer Design
• Spectrophotometer Operation
• Calibration of Spectrophotometers

SPECTROPHOTOMETERS

• Instruments that measure the interaction of light with materials in solution

Simplified Design of Spectrophotometer

Monochromator Separates Light into Its Component Wavelengths

Modern specs use diffraction gratings,

but a prism is more familiar way to

separate light into its different wavelengths

• Spectrophotometers in Biotechnology
• Light and its Interactions with Matter
• Spectrophotometer Design
• Spectrophotometer Operation
• Calibration of Spectrophotometers

THE BLANK

• Spectrophotometers compare the light transmitted through a sample to the light transmitted through a blank.
• The blank is treated just like the sample
• The blank contains everything except the analyte (the material of interest)
• Contains solvent
• Contains whatever reagents are added to the sample

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COMPARISON LIGHT TRANSMITTED THROUGH SAMPLE SAMPLE AND BLANK

WHEN OPERATING SPEC

• Blank is inserted into the spectrophotometer
• Instrument is set to 100% transmittance or zero absorbance
• Then sample is placed in spectrophotometer and absorbance is measured
• Note that in some specs the sample and blank are measured sequentially; in others at the same time

SPECTROPHOTOMETERS MEASURE TRANSMITTANCE BUT IT IS NOT LINEAR WITH ANALYTE CONCENTRATION

THEREFORE, TRANSMITTANCE IS CONVERTED TO ABSORBANCE

A = - log₁₀ (t)

PROPER SELECTION, USE, AND CARE OF CUVETTES

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• Cuvettes hold samples
• Are made from plastic, glass, or quartz.
• Use quartz cuvettes for UV work.
• Glass, plastic or quartz are acceptable for visible work.
• There are inexpensive plastic cuvettes that may be suitable for some UV work.

PROPER SELECTION, USE, AND CARE OF CUVETTES

• Cuvettes are expensive and fragile (except for “disposable” plastic ones). Use them properly and carefully
• Do not scratch cuvettes; do not store them in wire racks or clean with brushes or abrasives
• Do not allow samples to sit in a cuvette for a long period of time
• Wash cuvettes immediately after use (except if disposable)

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PROPER SELECTION, USE, AND CARE OF CUVETTES

• Disposable cuvettes are often recommended for colorimetric protein assays, since dyes used for proteins tend to stain cuvettes and are difficult to remove

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• Matched cuvettes are manufactured to absorb light identically so that one of the pair can be used for the sample and the other for the blank

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PROPER SELECTION, USE, AND CARE OF CUVETTES

• Do not touch the base of a cuvette or the sides through which light is directed

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• Make sure the cuvette is properly aligned in the spectrophotometer

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• Be certain to only use clean cuvettes

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• Light and its Interactions with Matter
• Spectrophotometer Design
• Spectrophotometer Operation
• Calibration of Spectrophotometers

CALIBRATION OF A SPECTROPHOTOMETER

• Brings the readings of the spectrophotometer into accordance with nationally accepted values
• Part of routine quality control/maintenance,
• Often performed by service technicians

CALIBRATION

Two parts:

• Wavelength accuracy, the agreement between the wavelength selected by the operator and the actual wavelength of light that shines on sample

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• Photometric accuracy, or absorbance scale accuracy, the extent to which a measured absorbance or transmittance value agrees with an accepted reference value

WAVELENGTH ACCURACY

• Wavelength accuracy is determined using certified standard reference materials (SRMs) available from NIST or traceable to NIST
• An absorbance spectrum for the reference material is prepared
• The absorbance peaks for reference standards are known, so the wavelengths of the peaks generated by the instrument can be checked

WAVELENGTH ACCURACY CONT.

• Manufacturers specify the wavelength accuracy of a given instrument
• For example, a high-performance instrument may be specified to have a wavelength accuracy with a tolerance of + 0.5 nm
• A less expensive instrument may be specified to have a wavelength accuracy of + 3 nm

PHOTOMETRIC ACCURACY

• Assures that:
• If the absorbance of a given sample is measured in two spectrophotometers at the same wavelength and under identical conditions
• then the readings will be the same
• and the readings will correspond to nationally accepted values

PHOTOMETRIC ACCURACY

• Photometric accuracy is difficult to achieve due to different instrument designs and optics

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• Usually, photometric accuracy is not critical if the same instrument is used consistently and if its readings are linear and reproducible

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PHOTOMETRIC ACCURACY

• Photometric accuracy is required where values from different labs and instruments are compared
• Required if rely on published absorptivity constants
• Likely required in a GMP-compliant facility

TO DELVE DEEPER INTO THE TOPICS IN THIS LECTURE

• Chapter 21 in Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference, 3^rd Edition has considerably more explanation of spectrophotometry theory. There is also more explanation of how spectrophotometers are maintained.

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