Bio-Rad BioLogic QuadTec™ Detector and Components - Manual (Page 54)

APPENDIX B
WAVELENGTH SELECTION FOR TYPICAL BIOLOGICAL MOLECULES
Wave-
length
Absorbing Species Applications
206 nm Carboxyl groups, Proteins, peptides,
ester links, amide or amino acids,
peptide bonds steroids,
nucleotides,
fatty acids,
carbohydrates
214 nm Peptide bonds Peptides, proteins
224 nm Peptide bonds Peptides, proteins
245 nm Peptide bonds Proteins in the
presence of Triton
X-100
254 nm Nucleotides Nucleotide bases,
260 nm DNA, RNA
280 nm Aromatic amino acids Proteins
313 nm Conjugated ring Certain vitamins,
systems antibiotics
365 nm Conjugated ring Some steroids,
systems NADH,NADPH,
flavoproteins,
bacteriochlorophylls
405 nm Heme group Myoglobin
550 nm Cytochromes
WAVELENGTH SELECTION APPENDIX B
B-1
Comments
Virtually all biological macromolecules will absorb
at this wavelength. Detection at this wavelength
will give high sensitivity and/or permit the
detection of compounds that don’t absorb at other
wavelengths. Some buffers cause a problem with
background absorbance.
This wavelength allows one to use many of the
buffers that might give problems at 206 nm and
yet yields much more sensitivity than 280 nm.
This wavelength detects any compound with
peptide bonds. It is less sensitive than lower
wavelengths but more sensitive than 280 nm; It is
used to overcome the interferences seen with
many buffers at 206 nm.
Triton X-100 absorbs strongly at 280 nm and may
mask true protein absorbance at that wavelength.
These are the best wavelengths for nucleic acids.
This is the traditional wavelength for protein
detection and therefore the most frequently used
by the biochemist. As aromatic amino acids
absorb best at this wavelength (i.e. tryptophan,
tyrosine, and phenylalanine), proteins with few of
these amino acids may not absorb as strongly as
expected.
Flavoproteins exhibit absorption maxima at 280 nm,
350–380nm and 450 nm. Reduction of the flavin
eliminates the absorption at 450 nm.
Reduced cytochrome c absorbs at 550 nm.