Using the Spectrophotometer
Thermo Scientific
Evolution Pro Spectrophotometer User Guide
9
Note
A question commonly asked of our technical support specialists is “I have a cuvette
with blank solution in the reference beam. Why do I still need to place a cuvette with
blank solution in the sample position and record a blank or baseline? Why can’t I just
measure my sample directly?
The short answer is “That is not how the reference channel works.” A “blank”
measurement is required so that the instrument knows what “100%T” looks like on the
sample detector. The 100%T value is required in order to calculate %T for the sample.
That value is required in order to calculate absorbance.
Most cuvettes have two frosted walls and two polished walls. Try not to touch the polished
walls. Wipe the polished walls with a lint-free cloth to remove dust, droplets of liquid or
fingerprints before making a measurement.
Inserting and Removing Cuvettes
Always insert cuvettes so that the beam passes through the polished walls. Plastic cuvettes
generally have a “V” or other mark at the top of one polished wall. Ideally, you should always
orient this “V” in a consistent direction relative to the beam. Generally, this is towards the
beam.
Using the Reference Beam
For simple measurements it is not generally necessary to put a reference cuvette containing
blank solution in the reference beam. There is no need to “balance the beams” in an
Evolution Pro spectrophotometer. A cuvette in the reference beam is only necessary if:
•
You expect the matrix (for example, a buffer solution) to change absorbance during the
course of the measurement. This typically only applies to kinetics (Rate) experiments that
last several minutes or longer.
•
Where the method calls for placing a reagent blank in the reference beam as part of the
protocol that will effectively subtract out the absorbance of the reagent blank.
•
Where an existing SOP or protocol developed for an older instrument has been validated
and cannot be deviated from. It will do no harm to place a cuvette with blank solution in
the reference beam.
