Spectrophotometry in Biotechnology

Spectrophotometry in Biotechnology

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 [email protected]

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 [email protected]

BIOTECHNOLOGY PROCESS Find gene that codes for useful protein Isolate gene Estimate DNA [ ] Insert gene into vector Check cell density Insert vector into cells (transform/transfect cells)

Grow cells, cells manufacture protein product Purify product Sell product [email protected] Check protein activity Check protein concentration Check 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 [email protected] 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 [email protected] 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 [email protected] 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 [email protected] 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 [email protected]

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 [email protected] SPECTROPHOTOMETERS

Are instruments that measure the interaction of light with materials in solution [email protected] 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 [email protected] 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 [email protected] WHEN OPERATING SPEC Blank is inserted into the spectrophotometer Instrument is set to 100% transmittance or zero absorbance

[email protected] PROPER SELECTION, USE, AND CARE OF CUVETTES Cuvettes are made from plastic, glass, or quartz. 1. a. b.

c. Use quartz cuvettes for UV work. Glass, plastic or quartz are acceptable visible work. There are inexpensive plastic cuvettes that may be suitable for some UV work. [email protected] 2. 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. b. Do not allow samples to sit in a cuvette for a long period of time. c. Wash cuvettes immediately after use. a. [email protected] 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. [email protected] 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. [email protected]

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

[email protected] 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) [email protected]

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 [email protected] 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 [email protected] LINEAR RANGE If there is too much or too little analyte, spectrophotometer cannot read the

absorbance accurately [email protected] 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 [email protected] BRADFORD PROTEIN ASSAY

A quantitative colorimetric assay Used to determine the concentration, or amount, of protein in a sample [email protected] 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

[email protected] 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 [email protected] 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 wavelength B = path length through cuvette C = concentration of analyte [email protected] 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 samples absorbance at the specified wavelength

[email protected] 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!

[email protected] EQUATION FOR A LINE A = B C y = m

x +0 [email protected] 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

[email protected] SLOPE Slope relates to the absorptivity constant A = y = B m

C x +0 [email protected] 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 [email protected] 3. Determine the path length for the system

(assume 1 cm for a standard sample holder and cuvette) [email protected] A = y = 3. B m

C x +0 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) [email protected]

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