Thursday, August 29, 2019
Peak Fronting Tailing
peaks are almost ubiquitous in liquid chromatography (LC) separations. Over the
past 20 years of "LC Troubleshooting," at least a dozen instalments
of this feature have dealt with tailing peaks. In the days of lower-purity
silica, peak tailing was often the result of strong acid-base interactions
between basic sample functional groups, such as amines and acidic silanol
groups on the silica surface. One popular way to combat such tailing was to add
triethylamine to the mobile phase to saturate the stationary phase's acidic
sites. With the widespread use of high-purity silica as the backbone of most of
today's reversed-phase columns, tailing problems have been reduced to the
extent that amine mobile phase additives are seldom used. Today, silanol tailing
remains, but is much less of a problem. Another common cause of peak tailing,
column overload, was discussed in last month's "LC Troubleshooting"
instalment.1 Peak fronting is much more rare than peak tailing.
There is some speculation that complaints are low because peak fronting merely
makes a badly tailing peak look more symmetric, but this is a viewpoint for
cynics. In our laboratory, we rarely see fronting peaks, and this is likely the
problem in most scientists' experience. One classic example of fronting in
ion-pair chromatography showed that peak fronting could be eliminated with a
change in the column temperature.2 A previous "LC
Troubleshooting" column discussed another case in which peak fronting
might have been attributable to a void in the column or the mobile phase
problems, but the cause of the problem was not definitive.3
A satisfactory
chemical model for peak fronting in reversed-phase LC with most samples is
difficult to postulate. However, a problem with the physical structure of the
column is more reasonable. An asymmetric void at the head of the column,
channelling within the column, or a less dense bed structure along the walls
off the column than in the middle, each creates a model that allows one to
visualize the fronting process. If a portion of the sample molecules travel
through this less dense part of the column, they will travel more quickly,
distorting the peak. If the bulk of the peak is retained in the normal fashion,
a fronting peak will occur.
We recently observed
a case of severe peak fronting that appears to fit the hypothesis of a column
void or distortion in the column bed. The LC–tandem mass spectrometry
(LC–MS–MS) method uses a 100 mm × 2.1 mm C18 column packed with 5 μm diameter
particles designed to work well with 100% aqueous phases. Mobile phase A was 10
mM ammonium carbonate (pH 9.0), and mobile phase B was methanol. An isocratic
mobile phase of 5% B was run for 5 min at a flow-rate of 0.5 mL/min, followed
by an 80% methanol flush. Normally, the method produces chromatograms with
symmetric peaks. After approximately 500 injections of extracted plasma
samples, the chromatograms had deteriorated to the degree. Column reversal was ineffective
and normal pressures were observed, suggesting that frit blockage was not the
problem source. The column was replaced and the chromatogram was similar to
initial. We have seen this failure pattern for several columns, indicating that
a column lifetime of approximately 500 injections is typical for this method.
Column
Life Expectancy Replacement of
the column after 500 injections of the sample above might bring some gasps of
horror from some readers. A question we hear frequently relates to how long a
column should last. We have methods in our laboratory for which column lifetime
of 2000 injections are common. Conversely, we've had users tell us of methods
in which column failure is observed after 50 or fewer injections — and they are
not upset.
Before going into
techniques to extend column life, we should consider the cost of the column in
the big scheme of things. We work in a contract research organization (CRO), so
we make our living from running other people's samples. In the CRO world, LC
sample analysis costs generally are $50/sample and up. For our earlier example,
the column costs about $500; at 500 samples/column, this translates to
$1/sample or 2% of the cost. If we could double the life of the column, we
would only reduce the cost to 1%, hardly worth the trouble. However, with a
$500 column, many people seem to treat the column as a capital item instead of
a consumable. Contrast this with a method that uses solid-phase extraction
(SPE) for sample clean-up. SPE cartridges or 96-well plates cost at least
$2/sample. This is twice the cost of the analytical column, yet we think
nothing of throwing the SPE cartridges away after a single use — they are sold
as consumable items. Rather than trying to improve column life, a better investment
for cost reduction is to figure out ways to reduce labour costs or improve
throughput of expensive LC–MS–MS detectors by the use of techniques such as
parallel chromatography.4
Extending
Column Life Although this
discussion should convince us that we do not want to spend too much time trying
to extend column life, there are some simple techniques that will extend the
useful life of a column. The most common mode of failure of columns today is excessive
pressure resulting from build-up of particulate matter at the head of the
column. Make sure that your samples are free of particulates. Filtration of
each sample or centrifugation of samples to eliminate particles will go a long
way towards this goal. Another inexpensive, but very effective tool is a 0.5 μm
porosity in-line filter mounted just downstream from the autosampler. This will
catch the particles that make it past your filtration or centrifugation efforts
so that they do not block the column inlet frit. When the system pressure rises
to an unacceptable level, simply replace the frit in the filter and you should
be back in business. Many workers find that guard columns are beneficial to
improving column life. These small columns upstream from the analytical column
catch both particulate matter and strongly retained materials that might foul
the column packing on the analytical column. If you use guard columns, be sure
to discard them before the contaminants break through onto the main column. Also,
when flushing the system with strong solvent, be sure to flush the guard column
to waste, not onto the analytical column, or you might defeat the purpose of
the guard column.
Sample clean-up is
another technique to extend column usefulness. You need to remember that there
is an economic balance in sample clean-up versus column costs. Does it make
sense to spend $10/sample for clean-up to reduce the per-sample cost of the
column's contribution from $2/sample to $1/sample? In our opinion, one major
goal of sample clean-up should be to improve method ruggedness so that you are
assured of collecting meaningful data from your samples. Some workers perform
the absolute minimum of clean-up, expect their columns to do the clean-up work,
and are satisfied if the column lasts for 100 samples. In other instances,
extensive sample clean-up can lower the background noise and allow a 10-fold
lower limit of quantification; longer column life is only a peripheral benefit.
You have to work out the economics on a case-by-case basis.
Column Conditioning You might have noticed that some
LC methods take a while before they "settle down" and give consistent
results. For example, it may take five or six injections before the retention
time, peak height or peak area stabilizes to a satisfactory level of
variability. What is going on? Many workers think of a reversed-phase
separation as simple partition-like separation between the mobile phase and a
homogeneous stationary phase surface. Unfortunately, this is not the situation.
The closer you look, the less homogeneous the stationary phase appears. In some
instances, there are slow and fast equilibria going on at the same time.
Sometimes the analyte molecules are retained by more than one mechanism.
Loading sample onto the column might allow the slow-equilibrium mechanism to
saturate so consistent results are seen. In other instances, it might be
proteins, polymers or other analytically unimportant matrix materials that must
be loaded onto the column before the method behaves well. Injection of several
mock samples will usually suffice. We have several methods that include five to
ten conditioning injections before injecting the standard curve. This is
alright if the runtime is short, but if the method is a conventional LC–UV
method, the runtime may be 20–30 min, so extensive conditioning might be
unacceptable from a sample-throughput standpoint. Because the conditioning
process is often related to the mass of sample (or matrix) loaded on the
column, one might be able to shorten the conditioning cycle by making the
conditioning injections one after the other without waiting for elution to
occur. For example, if the method has a 20 min runtime, just start the method,
and then make five injections at 30 s intervals rather than waiting for 20 min
for each one. An alternative is to make a single large-mass injection. Try one
or more of these techniques and see if it will help your method settle down
quickly for normal operation.
References 1. John W. Dolan, LCGC
Eur.,18(6), 318–322 (2005).
2. P.A. Asmus, J.B.
Landis and C.L. Vila, J. Chromatogr. 264, 241 (1983).
3. C. Hawkins and
J.W. Dolan, LCGC Eur., 17(1), 32–40 (2004).
4. M.D. Nelson and
J.W. Dolan, LCGC Eur., 17(5), 272–277 (2004).
yours
chormatographically,
kaushik zala
