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By Rick Lake and Ty Kahler
• Simplifying your mobile and
stationary phase options will
streamline method development.
• USLC® technology effectively
narrows your columns options
from over 600 down to four.
• A scouting gradient makes it
easy to select the best column/
mobile phase combination.
If we’ve learned anything from developing
methods (and probably more from strug-
gling with them), it’s that you will generate
more robust methods in less time if you start
by looking at retention and selectivity. First,
simplify your mobile phase; then, reduce
your column options. Finally, run a scouting
gradient to choose the right column/mobile
phase combination based on your desired
elution profile.
Reduce Your Mobile Phase
Possibilities
When developing a method, the number of
mobile phases you have to choose from is
nearly infinite, so it’s easy to become over-
whelmed. What’s more, using a highly custom-
ized mobile phase may not be necessary—it
could even be detrimental to your data. Long
story short, it’s in your best interest to simplify.
We advise employing a four–mobile phase
system and the recommendations in Table I.
When the time comes for your scouting gradi-
ent, run all four A/B combinations (e.g., A1/B1,
A1/B2, A2/B1, A2/B2) and select your mobile
phase based on the results.
Find the Best LC-MS Column/Mobile Phase Combination
Using a Simple Mobile Phase, USLC® Columns, and a Scouting Gradient
Table I: Run these aqueous solutions and organic solvents using a four–mobile phase system
and our USLC® columns to dramatically simplify mobile phase selection.
Aqueous Solutions Organic Solvents
A1) 0.1% Formic acid in water B1) Acetonitrile (aprotic solvent)
A2) 0.1% Formic acid and 5 mM ammonium formate in water B2) Methanol (protic solvent)
Make the Most of the USLC® Column Set
Unlike with mobile phases, there are“only”around 600 different columns on the market. But, column
phase chemistry can be so similar between product lines and even manufacturers that switching
may do little to alter your results. Instead of wasting time and money running column after column
with nearly identical selectivity—and getting similar results—simply plug the USLC® column set into
your column-switching system. Designed with the method developer in mind, this innovative col-
umn set offers an incredible range of alternate selectivity using just four unique stationary phases.
USLC® phases are so different from each other (i.e., orthogonal) that they offer selectivity and reten-
tion regardless of your target analytes.
Scout for Successful Method Development
Evaluating, or“scouting,”your column/mobile phase combinations will allow you to determine
which works best for your desired elution profile. To perform a scouting gradient, set your instru-
ment to deliver a defined, linear gradient slope over a specified time. Start with the aqueous solution
at 5%, and starting at time 0, begin ramping up to 95% using the flow rate and gradient time listed
in Table II for your column. (If you have sample solubility issues, you can deviate from the starting or
ending ratios, but be sure to keep the gradient defined and linear.) After each gradient, don’t forget
to equilibrate the column using the time in Table II before running the next mobile phase.
Choose Phases Based on Selectivity, Retention, and Elution Profile
When your scouting run is complete, you will have a set of 16 chromatograms (one for each column/
mobile phase combination). To choose the best column/mobile phase combination, you must first
calculate the ideal elution profile for each by looking at the difference in retention time between the
first and last peaks (Δt ) and the gradient time (tG). A Δt/tG less than 0.25 would mean that an isocratic
elution is feasible; a Δt/tG greater than 0.25 would indicate the need for a gradient.
Second, look at your peaks. The column and mobile phase combination that delivers the best reten-
tion, selectivity, and peak shape for your desired elution profile is the one you should choose for
your method (Figure 1). It’s that easy! For an in-depth look at the role of selectivity in reversed phase
separations, check out
At this point, you may find that you are already achieving complete chromatographic resolution
and can continue developing your method without giving another thought to mobile or stationary
phase selection. If, however, your results are less than ideal, visit for
help fine-tuning your mobile phase.