Who doesn't want more speed? Whether you are looking at a new motorcycle, examining your times for a 10K run, or developing
a liquid chromatography (LC) method, faster usually is better. Face it, most of us who work as chromatographers get paid,
either directly or indirectly, by the number of samples we run. A faster method allows us to run more samples or get the sample
set done more quickly so we can move on to something else. In the previous installments of this series on efficient development
of LC methods (1–5), we have concentrated on improving resolution by modifying the mobile phase, choosing a different stationary
phase, or changing some other condition, such as column temperature. In this month's "LC Troubleshooting" installment, we're
going to look at trading some of that resolution for a faster separation.
One More Time
Throughout this series on efficient LC method development we have been using equation 1 as a guide. Usually our goal (1) is
to develop a method that gives baseline resolution, Rs, for all components of interest. If it is to be a method used under the oversight of one of the regulatory agencies, Rs > 2.0 is recommended. As a starting point (2), we chose a reversed-phase C8 or C18 column, because this chromatographic mode
has a high probability of success with most samples. A 150 mm × 4.6 mm column packed with 5-μm diameter particles or a 100
mm × 4.6 mm, 3-μm dp column was used, because these columns generate approximately 10,000 theoretical plates, N, which is sufficient to separate most sample mixtures. As a bonus, these column sizes can be run at 1.5–2.0 mL/min for a
reasonable run time without much concern about excessive pressure.
As soon as we had our starting conditions, we worked our way through equation 1 in an effort to develop a separation with
the necessary resolution. First we tried adjusting the retention factor, k, which is most easily controlled by changing the mobile phase strength (3). We started with a strong mobile phase, such as
90:10 acetonitrile–water (or buffer) or methanol–water, then worked in a step-wise fashion to weaker mobile phases (more aqueous
phase) until k was in the 1 < k < 20, or better 2 < k < 10, region. Because a change in k also results in a change in selectivity, α, for many sample mixtures, adjustment of the mobile phase strength may be enough
to obtain the required resolution. If mobile phase strength changes are not sufficient, we can add more power to the process
by concentrating on α through adjustments in the chemistry of the mobile phase (4) by changing solvents from acetonitrile
to methanol (or vice versa), or changing the pH, temperature, or mobile phase additives. Selectivity also can be changed with
a change in the column packing type (5), although this option often is reserved for later in the development process, because
of the expense of purchasing additional columns.
At this point in the process, we hopefully have the resolution we need through the adjustment of k and α using a column that generated a sufficient number of theoretical plates. If the resolution is satisfactory and the
run time is acceptable, we should be ready to validate the method. If resolution is larger than is needed, we can trade some
of that resolution for shorter run times. If resolution is smaller than is needed, we may be able to adjust N to gain a little resolution.
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