SPECTROPHOTOMETRY IN BIOTECHNOLOGY

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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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• 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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WAVELENGTH OF VISIBLE LIGHT AND COLOR
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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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THE ABSORPTION OF LIGHT AND COLOR OF SOLUTIONS
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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

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SPECTROPHOTOMETERS

• Are instruments that measure the interaction of
  light with materials in solution

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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

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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

• Cuvettes are made from plastic, glass, or quartz.
  
• 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.

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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

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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

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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
• ? E absorptivity constant intrinsic
  ability of analyte to absorb light at a
  particular wavelength
• B path length through cuvette
• C 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 samples 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 C
• y 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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• 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
• 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)

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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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UV SPECTROPHOTOMETRY

• DNA, RNA and proteins are commonly analyzed with
  UV spectrophotometry because these molecules
  absorb UV light

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UV METHODS

• Used to evaluate the quality of DNA or RNA in a
  sample
• Used to estimate the quantity of DNA or RNA in a
  sample
• Procedure Take single wavelength readings of
  samples at 260 and 280 nm

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CONCENTRATION

• The absorbance at 260 nm is related to the
  concentration of DNA or RNA in the sample
• Pure, double-stranded DNA has an absorbance of
  about 1 at 260 nm when it is at a concentration
  of about 50 micrograms/mL
• Pure, single-stranded DNA has an absorbance of
  about 1 at 260 nm when it is at a concentration
  of about 33 micrograms/mL
• Values for proteins vary considerably from
  protein to protein

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PURITY

• The ratio of the absorbance at 260 and 280 nm is
  related to the purity of the sample
• An A260/A280 ratio of 2.0 is characteristic of
  pure RNA
• An A260/A280 ratio of 1.8 is characteristic of
  pure DNA
• An A260/A280 ratio of 0.6 is characteristic of
  pure protein

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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

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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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CALIBRATION OF A SPECTROPHOTOMETER

• Brings the readings of the spectrophotometer into
  accordance with nationally accepted values
• Part of routine quality control/maintenance

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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

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• 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

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• 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

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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

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• 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

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• 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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PROBLEM

• Assume that a spectrophotometer is able to read
  accurately in the range from 0.1 to 1.8 AU. The
  molar absorptivity constant for NADH is 15,000
  L/mole-cm at 260 nm. Using Beer's Law, calculate
  the concentration range of NADH that can be
  accurately quantitated at 260 nm based on the
  limits of the spectrophotometer.

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ANSWER

• This involves calculation of the molar
  concentrations which will produce absorbances of
  0.1 and 1.8. From Beer's Law
• C A
• e b
• Substituting 0.1 and 1.8 into the equation

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• C 0.1
  6.7 X 10-6 mole/L
• (15,000 L)(1 cm)
• Mole-cm
• C 1.8
  120.0 X 10-6 mole/L
• (15,000 L)(1 cm)
• mole-cm

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• Thus, the range of NADH concentrations that can
  be detected at this wavelength with this
  spectrophotometer is from 6.7 X 10-6 mole/L to
  120.0 X 10-6 mole/L. These are very dilute
  solutions of NADH.