Retention times and area measurements must be reproducible from run to run. When problems are observed, late, early or variable retention times (and/or peak area values) may be observed. Variation outside of acceptable limits indicates a problem with the sample preparation, method design, function of the HPLC system or a lack of training. Here are several commonly observed reasons why sample (or standard) peak retention times or peak area values may not be reproducible:
(1) TEMPERATURE FLUCTUATIONS:
To
obtain reproducible results, the temperature of the HPLC column must be
kept constant or controlled during each analysis. Laboratory room temperatures can
vary up and down by several degrees during the course of one day and these changes
will often change the retention characteristics of the sample(s). The
'On' and 'Off' cycling of power from an air conditioner or heating unit
will often cause the baseline to drift in a cyclical manner, up and
down, during the day (this can often be seen as a clear sine wave
pattern when you zoom-in to study the baseline trace over time).
Temperature also changes the refractive index of the mobile phase. Optical (Light)
based detectors (i.e. UV/VIS, RI...) will show this change as drift up or
down). In some cases, a temperature change of plus or minus one degree C
from run-to-run can cause changes in retention times which effect
reliability of the method. (1) TEMPERATURE FLUCTUATIONS:
To
reduce temperature fluctuations, you must control the temperature of
the column and mobile phase (if applicable) during the analysis. This is
most commonly done by: (a) using fully equilibrated mobile phase at the start
of the day or analysis, (b) keeping the interconnecting lines as short
as possible (esp. any which exit the column and go to detectors/flow
cells), (c) insulating any stainless steel lines with plastic tubing sleeves to
reduce heat loss and (d) using a thermostatted column compartment to
maintain the column at a single set temperature throughout the day.
Control of the column temperature will remove 'temperature' as a
variable from your analysis. Method analysis temperature should be constant from run to
run, not a variable. Be sure and document the temperature selected as
part of your method.
(2) INADEQUATE MOBILE PHASE MIXING:
Both
high pressure (with separate pumps) and low pressure pumping (one pump
with a proportioning valve module) systems depend on efficient mixing to accurately meter the requested mobile phase composition. For gradient analysis, failure to completely mix the
mobile phase solution before it enters the HPLC column often results in
excessive baseline noise, spikes and poor Retention time reproducibility. If your mobile phase composition changes, then the chromatography will change too (e.g. evaporation of the more volatile organic phase from an open bottle may result in a change in composition). "Mixing" is
often accomplished directly in a mixer installed in the flow path of an
HPLC pump (For more info, please read this article on selecting a mixer). The associated noise and ripple of incomplete mixing can
reduce the limit of detection (LOD) and increase integration error. This
mixer is often a static mixer (a simple 'Tee', a tube filled with
baffles, a frit or beads, valve orifice or microfluidic device) of low
volume design for chromatography use, but allows adequate mixing of the
liquids within a prescribed flow rate range. The best mixers incorporate
longitudinal and radial mixing in-line. A mixer with too low a volume
or of insufficient design can result in poor mixing of the mobile phase
(note: incorrect solvent compressibility settings can also cause mixing, flow instability
and noise problems too). To reduce mixing problems, first insure that
the mobile phases used are fully soluble with each other. Next, make
sure that any mixer used is appropriate for the flow rates and volumes
you will be using. Monitor the baseline for drift, ripple and artifacts
in real time to spot problems and make adjustments to correct them.
(3) INADEQUATE MOBILE PHASE DEGASSING:
For the best
results, continuously degass your mobile phase. Reducing the amount of
gas in solution will also improve signal to noise levels of detection, reduce Retention time drift
and reduce pump cavitation. If you are using an electronic vacuum
degassing module, make sure it is maintained and working 100%. A faulty
degasser may cause more damage to your system and methods. Maintain and
Repair them just as you do for your other instrument modules. Gas
bubbles may cause the inlet or outlet check valves to malfunction (get stuck), baseline
noise spikes to appear randomly, flow rates and/or back pressure values to become
irregular,
detector outputs to show high levels of noise (from air in the flow
cell) and also cause the loss of prime or cavitation in pumps. To
achieve the best balance of low noise levels and high reliability, both
aqueous and organic mobile phases should be fully degassed. This can be
accomplished through stand alone vacuum degassing modules or through
gentle helium gas sparging. In all cases, degassing must be continuous
(not just done one time). Continuous degassing reduces cyclical noise
and signal variations. For this reason, I do not recommend using
ultrasonic baths to degass mobile phase solutions as these are not used
continuously. The mobile phase solution starts to re-absorb gas as soon
as you stop sonicating the solution. This results in continuous baseline
drift.
Removal
of gasses is critical to the function of a modern HPLC pumping system.
The liquids used are compressed to very high levels which forces out
solubilized gas from the solutions. This is best accomplished before the
liquid is transferred into the pump. These gas bubbles must be
minimized to achieve desirable baselines. *Even if you use a high
pressure pumping system, an inline degassing system reduces the amount
of noise and baseline drift. Properly maintain and service your degasser to insure compliant operation. IOW: Always degas your mobile
phase solutions.
(4) SYSTEM LEAKS or FLOW RATE INSTABILITY:
If the peak retention times have increased over time, one possible reason for this change could be a leak. If your flow rate is reduced by a leak, then the retention times will be longer. Always be alert to this pattern of change and check for any signs of leaks on a regular basis. If you find a leak, do not use the HPLC system until it has been repaired. If there is no leak, then the flow rate may not be what you think it is. When the actual flow rate is in question, start by checking it manually (never trust the instrument's display screen or the software value for flow rate. Measure it). An easy way to measure the flow rate involves timing the amount of liquid that exits the HPLC detector line after a defined period of time. For example: If your flow rate is set at 1.000 ml/minute, measure the time it takes to fill a 10ml graduated cylinder. It must take exactly 10.00 minutes.
Inadequate degassing, sticking check valves and/or incorrect solvent compressibility values may also cause flow instability.
(5) COLUMN FOULING:
One of the most common reasons for changes in retention times or area values of well established peak(s) are due to column contamination and fouling of the support material (or of the inlet frit, guard column). The most common reason for this to happen is due to a lack of column flushing or washing after each analysis (esp when running only isocratic methods). Samples that have been poorly prepared, not filtered or were sourced from a complex matrix (i.e. clinical samples) often contain many compounds which are in-addition to the compound of interest. These materials can be retained on the column and not eluted off during each analysis. They build up over time and cause all kinds of strange problems, including changing retention times, new peaks seen and poor overall or wide peak shapes.
Gradient analysis provides an opportunity to make sure you use a strong enough mobile phase to elute everything off the column during the run. Make sure you ramp up to a high enough concentration of solvent and use a "hold time" to insure this.
Isocratic analysis is a worst case situation for this to occur as the mobile phase is not ramped up to a strong solvent at the end of the method to push off any late eluters, Instead, they accumulate on the column.
If you use isocratic methods to analyze samples, then you must follow each analysis run with a second, and separate from your analysis method, "column only wash step". This method does not inject any sample. Instead, it uses a strong wash solution which is compatible with your column AND is well known to dissolve any accumulated material into solution and elute it all off the column. For NP applications an alcohol (e.g. MeOH) may be suitable for this job and for RP applications ACN or even MeCl may be be appropriate. Check with the column manufacturer to find out which wash solutions should be used (do not guess, base it on actual sample solubility).
(6) SAMPLE OVERLOADING (or too large an Injection volume/concentration for the column): If you inject (load) more sample than the column can hold (as determined by a proper loading study), then the peak that results will often be broader in width with more tailing (from diffusion). This will result in a peak which elutes later than expected, fouls the column and results in poor reproducibility. Be sure to inject the sample dissolved in the mobile phase (or a solution that is weaker than the mobile phase).
(7) SAMPLE INJECTION VOLUME VARIATION: The injection volume used must be appropriate for the type of injector used. All injectors have a stated range in which they are most accurate. Make sure you are injecting within this ideal range and not at the extreme ends of the range (larger error). Manual injectors with fixed loops should be overfilled (3x) for best results. Autosampler vials must be correctly chosen to be compatible with the injector used, contain an excess of liquid and have a loose cap to prevent evaporation or a vacuum from forming inside the vial. *Test injector accuracy and reproducibility separately, at the volume used for your analysis, as part of your method development review. *Review my article on HPLC injectors for more information.
(8) Changes in the pH OF the MOBILE PHASE: Samples containing ionizable compounds are strongly effected by the pH of your mobile phase. Solutions should be prepared fresh, each day (*acids in solvent may change over time). Buffer capacity is often overlooked (the ability to resist pH change). It is highest at the pKa of the acid/base. Try to work within ±1 pH unit of the buffers pKa value for the best pH control of the mobile phase. If your mobile phase is buffered too far away from its pKa, then poor peak shape or variable retention times are often the result. Note: Weakly ionizable samples can be very sensitive to changes of as little as 0.1 pH unit.
If you use isocratic methods to analyze samples, then you must follow each analysis run with a second, and separate from your analysis method, "column only wash step". This method does not inject any sample. Instead, it uses a strong wash solution which is compatible with your column AND is well known to dissolve any accumulated material into solution and elute it all off the column. For NP applications an alcohol (e.g. MeOH) may be suitable for this job and for RP applications ACN or even MeCl may be be appropriate. Check with the column manufacturer to find out which wash solutions should be used (do not guess, base it on actual sample solubility).
(6) SAMPLE OVERLOADING (or too large an Injection volume/concentration for the column): If you inject (load) more sample than the column can hold (as determined by a proper loading study), then the peak that results will often be broader in width with more tailing (from diffusion). This will result in a peak which elutes later than expected, fouls the column and results in poor reproducibility. Be sure to inject the sample dissolved in the mobile phase (or a solution that is weaker than the mobile phase).
(7) SAMPLE INJECTION VOLUME VARIATION: The injection volume used must be appropriate for the type of injector used. All injectors have a stated range in which they are most accurate. Make sure you are injecting within this ideal range and not at the extreme ends of the range (larger error). Manual injectors with fixed loops should be overfilled (3x) for best results. Autosampler vials must be correctly chosen to be compatible with the injector used, contain an excess of liquid and have a loose cap to prevent evaporation or a vacuum from forming inside the vial. *Test injector accuracy and reproducibility separately, at the volume used for your analysis, as part of your method development review. *Review my article on HPLC injectors for more information.
(8) Changes in the pH OF the MOBILE PHASE: Samples containing ionizable compounds are strongly effected by the pH of your mobile phase. Solutions should be prepared fresh, each day (*acids in solvent may change over time). Buffer capacity is often overlooked (the ability to resist pH change). It is highest at the pKa of the acid/base. Try to work within ±1 pH unit of the buffers pKa value for the best pH control of the mobile phase. If your mobile phase is buffered too far away from its pKa, then poor peak shape or variable retention times are often the result. Note: Weakly ionizable samples can be very sensitive to changes of as little as 0.1 pH unit.
