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SPECTROPHOTOMETRY IN BIOTECHNOLOGY

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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

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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

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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

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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

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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

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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

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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

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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

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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

lseidman@matcmadison.edu

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

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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

lseidman@matcmadison.edu

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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WHEN OPERATING SPEC

Blank is inserted into the spectrophotometer Instrument is set to 100% transmittance or

zero absorbance

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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.

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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.

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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.

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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.

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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

lseidman@matcmadison.edu

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)

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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

lseidman@matcmadison.edu

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

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LINEAR RANGE

If there is too much or too little analyte, spectrophotometer cannot read the absorbance accurately

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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

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BRADFORD PROTEIN ASSAY

A quantitative colorimetric assay Used to determine the concentration, or

amount, of protein in a sample

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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

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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

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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

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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

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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!

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EQUATION FOR A LINE

A = B Cy = m x + 0

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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

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SLOPE

Slope relates to the absorptivity constant

A = B C

y = m x + 0

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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

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3. Determine the path length for the system (assume 1 cm for a standard sample holder and cuvette)

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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)