Monitoring The Purity Measure - Teledyne CombiFlash Rf User Manual

Section 6 User Help Reference
isocratic separation. It will require the user to perform
sufficient method development to know when all of the
compounds elute from the column.
As a simple example, assume that a separation is developed
with 2 compounds that elute at 5 and 10 minutes. At the 5
minute point of the separation, the sample loop can be
removed from the flow path and loaded with another sample of
the same materials. The loop can then be placed into the flow
path at the 6 minute point. In this scenario, the first
compound will elute at 5 minutes, the second compound will
elute from the column at 10 minutes. The first compound in
the second injection will elute at 11 minutes (6 minute
injection time plus 5 minutes to elute) and the second
compound from the second injection will elute at 16 minutes.
In this simple example, the 2 injections can be separated in 16
minutes instead of 20 minutes. The process can be repeated as
needed in increase system throughput. While isocratic
separations may typically be slower than a gradient
separation, you are also able to eliminate the time required for
column equilibration after a gradient separation.

6.4.9 Monitoring the Purity Measure

When using two wavelength detection options, a ratio of the
two wavelengths can also be displayed which at times can
provide the best indication of compound purity. Refer to the
following discussion.
If a pure compound is eluting, the absorbance is linearly
related to the concentration of the compound in the solvent. If
the compound absorbs differently at different wavelengths,
the absorbance at each wavelength may be different, but still
linearly related. For example, assume a compound eluting
from the system has an absorbance equal to 2 times the
concentration at 254 nm. This same compound at 220 nm has
an absorbance of 1.5 times the concentration. The ratio of
these signals will be 1.33. Since the relationship of absorbance
to concentration is not variable, the ratio remains steady while
the concentration changes from the beginning to the end of the
peak. During the duration of the peak, the ratio will be 1.33
and this constant value is displayed as a horizontal line.
Now assume a case where there is a second compound eluting,
only slightly shifted in time from the original compound. It is
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