Corona® CAD® - Charged Aerosol Detector
Comparable Response Between AnalytesDuring Gradient Elution HPLC Using A Make-upInverse Gradient
The response of nebulization-based detectors
A ilent 12
typically varies as a function of mobile phase
volatility. An increase in the organic content of
the eluent leads to more efficient nebulization,an increase in the percentage of analyte
reaching the detector and an increase in
signal. Conversely, highly aqueous mobilephases nebulize less efficiently, with less analyte detected. Such variations in response
during gradient elution can be corrected using
an inverse gradient (Figure 1), so that thedetector is always exposed to an eluent ofconstant composition (Gorecki et al., (2006)
This Technical Note shows a practical example
of how an inverse gradient can be used to
correct for the variation in response for analytes that elute at different times.
Figure 1 Schematic of the Agilent Inverse Gradient System.
HPLC Parameters:Column: 2.1 x 50mm LUNA C18 3µmMobile Phase A: 0.1% Formic Acid in Water
Mobile Phase B: 0.1% Formic Acid in MeOH
Amitriptyline (Ami), Dexamethasone (Dex), N-[(R)-1-(10-
naphthyl)ethyl]phthalamic Acid (Nap Ac) and 2,4,5-Triphenyl-
aniline (TriP) were each made up to a concentration of
Analytical: 5 to 95%B over 8mins, hold for 2mins, back to 5% in 0.1mins. Runtime =13mins
ResultsGradient performance can be evaluated by spiking acetone into
one of the solvents (e.g., B) and monitoring the absorbance at
280nm (Figure 2). The inverse gradient normalizes the gradient
profile, showing that the mobile phase entering the Corona
Four compounds were run at different eluent compositions withor without make-up solvent (Figure 3). The variation ofresponse at different % of MeOH is clearly seen when a make-up solvent is not applied (Figure 3A). This effect is correctedwhen make-up solvent is applied (Figure 3B).
The Corona® CAD® Charged Aerosol Detector Plot of CAD response v Plot of CA D response v %M no rev rse mob Figure 2. 0.1% Acetone was added to mobile phase B (ACN) in
order to trace the gradient profile by monitoring at 280nm. Blue -without make-up solvent. Red - with make-up solvent. Plot of CAD response v Plot of CA D response v %M - with reve th rev rse mobil mobil phase
The use of an inverse gradient can compensate for variation
in response between analytes that elute at different times
during gradient HPLC. In order for this approach to be
successful, it is critical that the volume and back pressure of
analytical and make-up flow paths are equivalent (e.g., by
Figure 3. The four test compounds were run at
different isocratic conditions with the inverse gradient
ESA Biosciences Inc., is grateful to Mr. Ian Mutton and
on (B) or off (A). The graphs show that variation in
Dr. Clare Holyoak (Analytical Chemistry, Molecular Discovery
CAD response due to the % organic in the mobile
Research, GlaxoSmithKline, UK) for permission to use their
phase is corrected when the inverse gradient is used.
The difference in the absolute response between thefour analytes is attributable to variation in volatility.
For more information about this application, the Corona CAD, or chargedaerosol detection visit www.coronacad.com. We are interested in your opinions and are available to answer any questions you may have: pleasecontact a technical representative at 978.250.7000, or if e-mail is more convenient, send your questions to [email protected].
ESA, Inc. is an ISO 9001 and 13485 Certified Company
Corona® and CAD® are registered trademarks of ESA, Inc.
ESA® is a registered trademark of ESA, Inc.
The Corona CAD is covered by multiple patents
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