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ACCQPrep HP125
Section 5 PeakTrak
5.3.6 Monitoring the Purity
Measure
5.3.7 Viewing runs
5-32
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 dis-
cussion.
If a pure compound is eluting, the absorbance is linearly related
to the concentration of the compound in the solvent. If the com-
pound absorbs differently at different wavelengths, the absor-
bance 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
possible that the detection absorbance trace alone would indicate
a single, valid chromatographic peak. In reality, it is a combi-
nation of two peaks. By monitoring a second wavelength, it may
be possible to reveal the second compound. Because of the slight
shift in time and the different absorbance properties of the two
compounds, the changing ratio during the detected peak would
reveal the impurity. Therefore, one can therefore assume that if
the ratio is not constant for the entire duration of the peak, the
compound eluting may not be pure.
The SHOW SPECTRAL PURITY control measures purity by
using a comparison of UV-spectra measured at differing times.
The algorithm used is the "similarity index". The S
P
D
button allows fractionation based on spectral
URITY
ETECTION
purity. The spectral purity algorithm doesn't will fail on satu-
rated peaks (flat on top due to detector saturation).
To display the ratio, open the M
the Show Ratio option.
After completing a run, the PeakTrak M
display all collected run data. You can also open previous runs to
view the chromatogram and the peak/tube locations. To open a
previous run:
1. From the M
window, select F
AIN
O
button. The FILES window is displayed.
PEN
2. Choose a Run file and click the O
displays the run in the R
Trak R
V
UN
IEWER
• Rack and tube information — The left pane of the
window lists the current rack, a map for that rack, and a
table that lists the peaks and their corresponding tube
numbers. If the window is currently displaying the
collection parameters, click the D
E
window and select
ETHOD
DITOR
window is used to
AIN
> O
ILE
PEN
button. 3.PeakTrak
PEN
V
window. The Peak-
UN
IEWER
window will include:
R
ISPLAY
PECTRAL
, or click the
button to
ACK
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