Retention Time Variability in HPLC
I’m sure we have
all experienced it – that sinking feeling when you realise your analyte
retention times have drifted outside the software ‘window’ and you have a
pile of chromatograms with no quantitative results. Or you are trying
to get that system suitability result to begin your batch of analysis as
you really need to get out of the door fast but your retention times
just won’t settle down. Or you are trying to reproduce Bob from the
R&D centres’ method and his retention times (or chromatogram..!)
look nothing like yours. Or you are trying to validate your method and
the three column lots you are trying give different retention behaviour
to the column that you just developed your method on. Or every time you
do an injection the retention time of the analyte changes just a little –
doesn’t cause anything to fall over, but you just don’t understand why.
Yes – there’s a
whole bunch of retention time issues that cause us problems in HPLC. A
lot of the underlying causes we can do something about – others we just
need to be aware of the cause and put our minds to rest. The remainder
of this technical tip will outline how to overcome, or better control,
all of the situations outlined above.
Overarching rules on retention time variability:
Overarching rules on retention time variability:
- If the void (hold-up) time (t0) and analyte retention time (tR) vary together, suspect a flow rate change. In this scenario the analyte capacity factor (k') will remain constant
- If only the analyte retention time varies, with the void (hold-up) time remaining constant, then k' will change also. In this scenario suspect a change in the selectivity or retentivity of the separation system
Drifting Retention Times
- This is typically due to a change in mobile phase composition, which
can be caused when pre-mixed mobile phases lose organic through
evaporation as the run progresses. Ever noticed this seems to happen
more towards the end of a run? Well of course the organic is being
continually lost to the atmosphere – or as the eluent in the sealed
bottle depletes there is more headspace for the more volatile component
to evaporate into - and of course a small amount of evaporation makes a
bigger overall composition change in the ever diminishing volume of
liquid! This is typically why we see elution times becoming longer
rather than shorter. What to do? Mix eluents (even isocratic ones)
online or at the very least ensure the reservoir you are using is
capped. We may also experience a change in the pH of the aqueous
component of the eluent over time caused by ingress of CO2 –
lowering the eluent pH and changing the retention and perhaps even the
selectivity of the separation….so again, cap your bottle. Do not use
lab film to cover eluent reservoirs – especially when using MS detection
(watch out for ions at 142 Da as you leach the plasticiser from the
film!)
Another related
note on eluents here – if we de-gas pre-mixed mobile phases using
vacuum, the very act of sucking the mobile phase through the filter
under vacuum can cause loss of the more volatile component – which will
obviously lead to irreproducible changes in eluent composition from
batch to batch of eluent. The same is also true when degassing
pre-mixed phases using ultrasonic baths – the warming of the eluent in
the sonic bath can lead to loss of the organic modifier and hence change
retention characteristics!
Of course
temperature is another variable that can alter retention time, changing
not only the viscosity of the eluent but the kinetics of the retention
mechanism, and ionisable compounds tend to be affected by temperature
more than non-ionogenic compounds – so selectivity may also change.
Most systems come with column heaters / chillers these days, but if
yours doesn’t, and you get large temperature variations in the lab, this
can cause retention time variability (especially when the system is
placed directly below your air-con!). Even systems which do have column
heaters work in different ways – some pre-warm the eluent prior to
entry into the column for example and these systems may well give
different retention times to those which heat the column only!
Variable Retention Times
- Equilibrating or priming a column when beginning an analysis can also
show up some strange retention time shifts and variability. Without
going into too much detail, this is due to the stationary phase surface
being modified by your eluent or sample components. Primarily it’s the
‘wetting’ of the surface (especially with more hydrophobic phases such
as C18) as the bonded phase takes on a layer of hydration - a slightly
crass description but one which will do for this short tip. Further –
the polar or ionised silanol (Si-OH) groups on the silica surface can
irreversibly bind with polar analyte components or buffer ions to change
the overall surface polarity. What to do – well you can try to inject a
10x more concentrated sample than you normally would to try and achieve
the equilibration is a shorter time (fewer injections)!
In this category we
must also consider the more esoteric issue of the sample diluent. For
reasons that are too detailed to enter into here, the eluotropic
strength and ionic strength of the sample diluent can sometimes have a
big effect on analyte retention time and peak shape – yes that’s the
sample diluent, the thing you dissolve the sample in – not the HPLC
eluent. You should always strive to match the aqueous / organic ratio
of the eluent (at the start of the gradient if doing gradient elution)
as well as the buffer strength of the eluent . If you need to make the
diluent more highly organic than the eluent (for solubility reasons) –
try to restrict your injection volume to 10 µl or less.
Column to Column Retention Time Irreproducibility
- And what of the situation in which the column you used to develop
your method doesn’t behave like the new shiny ones you just bought to do
your validation? Same as the previous situation really. Everything
you put down the column (eluent and samples) modifies the surface –
sometimes irreversibly, and the same goes for all of the stuff your
colleague also put down the column before you used it to develop your
method. What to do – buy a new column for method development and let it
equilibrate properly before using it. If you have an ion pair reagent
(and remember TFA is an ion pair reagent) and you remove it, use a
different pairing reagent or switch to a different eluent modifier – you
should contact your column supplier before continuing with method
development – it may well be that you need a new column!
Retention Time Issues in Method Transfer
- Matching retention times with Bob from R&D’s method is another
tricky situation. You must make sure that you are preparing the eluent
in the same way – including weighing solid buffers, taking care with
volumetric work, adding organic to aqueous portions and adjusting the pH
using the same acid or base and doing so with a properly calibrated pH
meter. Further, the use of the same buffer is important – and just a
note to all users of phosphate buffers – monosodium dihydrogen
orthophosphate is not the same as disodium monohydrogen orthophosphate
and neither of them has real buffering capacity between pH 3 and 6!
Crucially, if the method involves gradient elution, you REALLY MUST know
the gradient dwell volume for each system and adjust for any
differences prior to repeating the method. If Bob’s dwell volume is
shorter than yours then you need to use an instrument capable of
injection delay (injection occurring after the gradient has started) and
if his dwell volume is larger than yours, add an isocratic hold at the
start of the method equal to the difference in volume x flow rate. If
he was smart when he developed the method, then he would have inserted
an isocratic portion at the start of the gradient which you can adjust
to make sure the gradient dwell volume differences are catered for.
Hardware Related Retention Time Problems
- Pumps pump at a fixed flow rate – until they leak or break! Now most
times poor pump performance will be accompanied by other symptoms such
as low, high or cycling back pressure – but sometimes not. The easiest
way to check your flow rate accuracy is to run the eluent into a 10mL
volumetric cylinder and time it. If you get 10 mL in 10 mins when
operating at 1 mL/min. then all is well – if not – all is bad and you
need to get the system checked out.
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