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Inverse gradient.qxp

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 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
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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