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. 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.

Cuvettes are expensive and fragile (except for “disposable” plastic ones).

Use them properly and carefullya. 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.

3. Do not touch the base of a cuvette or the sides through which light is directed.

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

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

Running a Protein Assay

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 wavelengthB = path length through cuvetteC = 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