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Transcript of SPECTROPHOTOMETRY IN BIOTECHNOLOGY. [email protected] TOPICS Spectrophotometers in...
SPECTROPHOTOMETRY IN BIOTECHNOLOGY
TOPICS Spectrophotometers in Biotechnology Light and its Interactions with Matter Spectrophotometer Design Spectrophotometer Operation Qualitative Spectrophotometry Quantitative Spectrophotometry UV Spectrophotometry of DNA, RNA and Proteins Calibration of Spectrophotometers
BIOTECHNOLOGY PROCESS
Find gene that codes for useful protein
Isolate gene
Insert gene into vector
Insert vector into cells (transform/transfect cells)
Grow cells, cells manufacture protein product
Purify product
Sell product
BIOTECHNOLOGY PROCESS
Find gene that codes for useful protein
Isolate gene
Insert gene into vector
Insert vector into cells (transform/transfect cells)
Grow cells, cells manufacture protein product
Purify product
Sell product
Estimate DNA [ ]
Check cell density
Check proteinactivity
Check proteinconcentrationCheck protein purity
Spectrophotometers in Biotechnology
Light and its Interactions with Matter
Spectrophotometer Design Spectrophotometer Operation Qualitative Spectrophotometry Quantitative Spectrophotometry UV Spectrophotometry of DNA, RNA and Proteins 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 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
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
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 Qualitative Spectrophotometry Quantitative Spectrophotometry UV Spectrophotometry of DNA, RNA and Proteins Calibration of Spectrophotometers
SPECTROPHOTOMETERS
Are instruments that measure the interaction of light with materials in solution
Monochromator Separates Light into Its Component Wavelengths. Modern Specs Use Diffraction Gratings
Spectrophotometers in Biotechnology Light and its Interactions with Matter Spectrophotometer Design
Spectrophotometer Operation Qualitative Spectrophotometry Quantitative Spectrophotometry UV Spectrophotometry of DNA, RNA and Proteins 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
WHEN OPERATING 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.
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.
3. Disposable cuvettes are often recommended for colorimetric protein assays, since dyes used for proteins tend to stain cuvettes and are difficult to remove.
4. 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.
5. Do not touch the base of a cuvette or the sides through which light is directed.
6. Make sure the cuvette is properly aligned in the spectrophotometer.
7. Be certain to only use clean cuvettes.
Spectrophotometers in Biotechnology Light and its Interactions with Matter Spectrophotometer Design Spectrophotometer Operation
Qualitative Spectrophotometry Quantitative Spectrophotometry UV Spectrophotometry of DNA, RNA and Proteins Calibration of Spectrophotometers
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)
Spectrophotometers in Biotechnology Light and its Interactions with Matter Spectrophotometer Design Spectrophotometer Operation Qualitative Spectrophotometry
Quantitative Spectrophotometry UV Spectrophotometry of DNA, RNA and Proteins Calibration of Spectrophotometers
OVERVIEW OF QUANTITIVE SPECTROPHOTOMETRY
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
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
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 = absorptivity constant – intrinsic ability of analyte to absorb light at a particular wavelengthB = path length through cuvetteC = concentration of analyte
APPLYING THE EQUATION
Suppose you have a sample: And you know the path length And you know the absorptivity constant for the
analyte of interest at a particular wavelength Then, measure the sample’s absorbance at
the specified wavelength
Can calculate the concentration of the analyte from the Beer-Lambert equation
A = B C But this is a shortcut that may give inaccurate
results!
Y intercept should be zero because of the blank Blank has no analyte (zero concentration) and is
used to set transmittance to 100% = absorbance to zero
DETERMINATION OF THE ABSORPTIVITY CONSTANT
1. Prepare a calibration line based on a series of standards
Plot concentration on the X axis and absorbance on the Y axis
2. Calculate the slope of the calibration line:Y2 – Y1
X2 - X1
3. Determine the path length for the system (assume 1 cm for a standard sample holder and cuvette)
A = B C
y = m x + 0
3. Slope = absorptivity constant X path length Absorptivity constant = slope
path length
(Observe that the constant has units that depend on how concentration was expressed in the standards)
