SPECTROPHOTOMETRY IN BIOTECHNOLOGY
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 together is called white light
The absence of all light is black Light of slightly shorter wavelengths is
ultraviolet – Eyes do not perceive UV light
WAVELENGTH OF VISIBLE LIGHT AND COLOR
WAVELENGTH
COLOR PERCEIVED
380-430 Violet
430-475 Blue
475-495 Greenish Blue
495-505 Bluish Green
505-555 Green
555-575 Yellowish Green
575-600 Yellow
600-650 Orange
650-780 Red
INTERACTION OF LIGHT WITH MATERIALS IN SOLUTION
When light shines on a solution, it may pass through – be transmitted
OR
Some or all of the light energy may be absorbed
THE ABSORPTION OF LIGHT AND COLOR OF SOLUTIONS
WAVELENGTH OF LIGHT ABSORBED
COLOR OF LIGHT ABSORBED
COLOR OF SOLUTION
380-430 Violet Yellow
430-475 Blue Orange
475-495 Greenish Blue Red-Orange
495-505 Bluish Green Orange-Red
505-555 Green Red
555-575 Yellowish Green Violet-Red
575-600 Yellow Violet
600-650 Orange Blue
650-780 Red Green
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
Are instruments that measure the interaction of light with materials in solution
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
WHEN OPERATING A SPEC
Blank is inserted into the spectrophotometer
Instrument is set to 100% transmittance or zero absorbance
PROPER SELECTION, USE, AND CARE OF CUVETTES
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.
Cuvettes are expensive and fragile (except for “disposable” plastic ones).
Use them properly and carefullya. 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
1. Disposable cuvettes are often recommended for colorimetric protein assays, since dyes used for proteins tend to stain cuvettes and are difficult to remove.
2. 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.
3. Do not touch the base of a cuvette or the sides through which light is directed.
4. Make sure the cuvette is properly aligned in the spectrophotometer.
5. Be certain to only use clean cuvettes.
EXAMPLES
Some examples of qualitative spectrophotometry– The absorbance spectra of various common solvents. Note
that some solvents absorb light at the same wavelengths as DNA, RNA, and proteins
– Hemoglobin bound to oxygen versus carbon monoxide
– Native versus denatured bovine serum albumin (a protein commonly used in the lab)
OVERVIEW OF QUANTITIVE SPECTROPHOTOMETRY
Measure the absorbance of standards containing known concentrations of the analyte
Plot a standard curve with absorbance on the X axis and analyte concentration on the Y axis
Measure the absorbance of the unknown(s)
Determine the concentration of material of interest in the unknowns based on the standard curve
LINEAR RANGE
If there is too much or too little analyte, spectrophotometer cannot read the absorbance accurately
COLORIMETRIC ASSAYS
Quantitative assays of materials that do not intrinsically absorb visible light
Combine the sample with reagents that make the analyte colored
The amount of color is proportional to the amount of analyte present
BRADFORD PROTEIN ASSAY
A quantitative colorimetric assay Used to determine the concentration, or
amount, of protein in a sample
Prepare standards with known protein concentrations
Add Bradford Reagent to the samples and to standards
– Read absorbances – Create a standard curve
Determine the concentration of protein in the samples based on the standard curve
Running a Protein Assay
MORE ABOUT THE CALIBRATION LINE ON A STANDARD CURVE
Three things determine the absorbance of a sample:– The concentration of analyte in
the sample– The path length through the
cuvette– The intrinsic ability of the
analyte to absorb light at the wavelength of interest
BEER-LAMBERT LAW
A = B C
Where:A = absorbance at a particular wavelength = E = absorptivity constant – intrinsic ability of analyte to absorb light at a particular wavelengthB = path length through cuvetteC = concentration of analyte
UV METHODS
These UV methods for estimating concentration and purity of DNA, RNA, and proteins are very commonly used, are very quick, and easy to perform
However, they values obtained are not very accurate – they are rough estimates
CALIBRATION OF A SPECTROPHOTOMETER
Brings the readings of the spectrophotometer into accordance with nationally accepted values
Part of routine quality control/maintenance
CALIBRATION
Two parts:
1. Wavelength accuracy, the agreement between the wavelength selected by the operator and the actual wavelength of light that shines on sample
2. Photometric accuracy, or absorbance scale accuracy, the extent to which a measured absorbance or transmittance value agrees with an accepted reference value
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
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 is difficult to achieve due to different instrument designs and optics
Usually photometric accuracy is not critical if the same instrument is used consistently and if its readings are linear and reproducible
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
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