Download Spectrophotometry

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.
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.

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
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.
Do not touch the base of a cuvette or the sides through
which light is directed.
3.
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
Running a Protein Assay


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
 = E = absorptivity constant – intrinsic ability of
analyte to absorb light at a particular wavelength
B = path length through cuvette
C = 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