Choosing Optimal Detection Wavelength by
Diode-Array Data Analysis
Application
Technical
Robert Ricker
Choice of optimal wavelength is a challenge when running HPLC sample-compounds
with widely varying absorbance spectra. Also, baselines can change absorbance over
time, as gradient HPLC is performed. Use of 3-D Diode Array Data can allow the
chromatographer to choose an optimal wavelength, or even switch wavelengths to
provide maximum sensitivity for all sample compounds, with minimal baseline drift.
Highlights
abs
min
nm
pk 4
268
pk 2
220
pk 1
Conditions:
LC: Agilent 1100
Column: ZORBAX Eclipse XDB-C18, 3.5 µm,
4.6 x 30 mm, Agilent Part No. 931975-932
Mobile Phase: A: 10/90, MeOH / H2O + 0.2% H3PO4
B: 90/10, MeOH / H2O + 0.2% H3PO4
Inj. Vol.: 20 mM each compound, 5 µL; 23°C; 3 mL / min
pk 3
• Three-dimensional diode-array data
can be used to reduce background
slope and optimize chromatographic
absorbance for a number of
compounds in a single run.
• By using chromatographic data from
268 nm rather than 220 nm, baseline
drift is eliminated.
Robert Ricker is an application chemist
based at Agilent Technologies, Wilmington,
Delaware.
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www.agilent.com/chem
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Printed in the USA
April 25, 2002
5988-6464EN