5/3/24, 17:42 How Much Retention Time Variation Is Normal?

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How Much Retention Time Variation Is

Normal?
August 1, 2014
John W. Dolan

Article LCGC North America

LCGC North America

LCGC North America-08-01-2014
Volume 32 Issue 8
Pages: 546–551

Small changes in retention time with an LC method are normal. At what point is
a problem suggested?

Small changes in retention time of a liquid

chromatography (LC) method are normal. At what point is
a problem suggested? Retention time shifts can be
frustrating when you can't figure out if a shift is something
you caused or if there is another reason for it.

It is interesting how problems tend to cluster — in the last few

weeks I've received several questions related to retention time
shifts with liquid chromatography (LC) methods. Some of these
correspond to the preparation of new batches of mobile phase,
some are from one day to another, some are from within a
batch of samples, and some result from a change in
instruments. In this month's "LC Troubleshooting," I will discuss
some of the factors that influence small changes in retention.

Most LC methods run on modern instrumentation with a good

quality column will have quite stable retention times. I
generally expect to see run-to-run variation in the second
decimal place of the retention time, such as ±0.02–0.05 min.
However, the historical behavior of the method should be used
to determine what is normal for that method. For example,
with large biological molecules, such as proteins, that use
shallow gradients, the variability can be an order of magnitude
or more larger.

Mobile-Phase Organic Concentration

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One of the most common causes of shifts in retention time in

reversed-phase LC separations is a minor change in the
concentration of the organic solvent, usually methanol or
acetonitrile. This can happen from a minor error in formulation
or a change in the mobile-phase composition if one solvent
evapo rates over time.

Figure 1: Plots of log k versus %B for compounds of 500 Da

(red), 5000 Da (blue), and 50 kDa (green). See text for details.

For small molecules (arbitrarily <1000 Da), we can use the

"Rule of Three" to estimate the effect of a change in organic
solvent, or %B. This rule states that the retention factor, k,
changes approximately threefold for a 10% change in %B. This
rule derives from plots, such as that in Figure 1, where k is
plotted versus %B on a semi-log plot. The red line in Figure 1
represents the retention behavior for a 500 Da analyte.
Retention changes from ~29 min at 40% B to ~4 min at 60%.
For practical purposes, this relationship can be considered
linear and represented in the standard y = mx + b format as

where kw is the (theoretical) retention in 100% water, S is the

slope of the plot, and Φ is the %B as a decimal (0.5 = 50%).
Values of S can be determined from two experimental runs
using equation 1. Empirical observations indicate that S can be
estimated as

where MW is the molecular weight (Da). Thus for a 500 Da

analyte, S ≈ 5.6. If we consider S ≈ 5 as an average for sub-
1000 Da molecules, we can then estimate how k changes with
%B with

where Δk is the change in k value for a Φ change in organic. If

S = 5, a 10% change in organic gives Δk ≈ 105×0.1 = 3.16 ≈ 3.
This is the basis of the Rule of Three.

As an example, our 500 Da analyte in Figure 1 has k = 5 at 50%

B. We can convert this to retention time, tR, by rearranging the
equation for k,

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where t0 is the column dead time (also abbreviated tM). If we

assume a 150 mm × 4.6 mm column operated at 1 mL/min, t0
≈ 1.5 min, so tR = 1.5(1 + 5) = 9.0 min. The Rule of Three would
suggest that k ≈ 3 × 5 = 15 for a change to 40% B, which
would correspond to tR ≈ 24 min. In fact, S = 5.16 for our
analyte, so k = 18.1 and tR = 28.7 min — close enough for a
rule of thumb.

Mobile-phase formulation errors should not be in the 10%

region if you are at all careful, so what happens for smaller
changes? We can use equations 3 and 5 to determine the
effect of a 0.1%, 0.5%, and 1% error in formulating our 50% B
mobile phase for our "average" S = 5 small molecule. I'll leave
the calculations to you, but the results summarized in Table I
show that these correspond to retention shifts of
approximately 0.1, 0.4, and 0.9 min, respectively (I have
rounded values for display, so if you try to repeat my
calculations, your results may vary somewhat). So, you can see
that it takes only a minor error in mobile-phase formulation to
shift retention times enough to notice the change.

Table I: Effect of small errors in mobile-phase composition on

the retention for analytes of different S values

With larger molecules, the problem is magnified, because S

increases markedly with an increase in molecular weight. This
makes the log k versus %B plots steeper, as is seen for 5000 Da
(blue) and 50,000 Da (green) compounds, such as peptides or
proteins, respectively. Steeper plots mean that these
compounds are much more sensitive to minor changes in the
mobile-phase composition. Our Rule of Three for <1000 Da
samples becomes the Rule of 60 at 5000 Da, and the Rule of
400 at 50,000 Da. This extreme sensitivity of the retention of
large molecules to small changes in %B means that isocratic
separation is not practical for most separations of these
analytes.

Column Temperature

Linear plots, as in Figure 1, are also observed if you plot log k

versus reciprocal temperature (in kelvins [K], where K = °C +
273). This means that small changes in column temperature
can also affect retention times. A rule of thumb for small
molecules is that retention changes by ~2% for each 1 °C
change in temperature. In Figure 2, you can see that a 10 °C

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change in temperature changes the retention of the last peak

from approximately 12 min to 10 min, as expected. Most
workers know that retention can shift with a change in
temperature, but many overlook the fact that relative retention
also can change. In the (specially selected) sample of weak
acids and bases of Figure 2, there are three reversals in
retention for a 10 °C change in temperature. Smaller changes
in temperature will have smaller, but noticeable effects on the
appearance of the chromatogram. Such problems can be
exacerbated by two common factors. Operation with the
column at ambient temperature can result in several degrees
change in column temperature during a single day, with
subsequent retention shifts. In my observation, the
temperature of the mobile phase within the column can vary
several degrees from the column oven setting and if the oven
is not properly calibrated, or of different design, switching from
one column oven to another can give similar temperature
differences. Usually, temperature differences cause shifts in
retention times of all peaks to earlier or later times, and small
adjustments in the temperature setting can correct for
instrument-to-instrument differences. Because of the wide
variation in ambient temperatures that are possible, I strongly
recommend that you always use a column oven.

Figure 2: Effect of a change in column temperature of 10 °C

or 0.2 pH units for a sample of weak acids and bases. Data
from reference 1; see text for details.

Mobile-Phase pH

A change in the mobile-phase pH can have a dramatic effect

on the appearance of the chromatogram when ionizable
compounds are present, but it makes no difference for neutral
samples. The behavior of retention with a change in pH will
vary depending on how close the mobile-phase pH is to the
pKa of the analyte, and in any case, is not a linear relationship,
so a general rule of thumb for the effect of pH on retention is
not possible. The lower chromatogram of Figure 2 illustrates
how sensitive retention can be to changes in pH. A change in
pH of 0.2 units gives similar retention changes as a 10 °C
change in temperature for this sample of weak acids and bases.
Typical laboratory practice of adjusting the mobile-phase pH
with the help of a pH meter can result in errors of ±0.1 pH
units, so special care needs to be taken if the separation is
particularly pH sensitive, as is that of Figure 2. To minimize pH-
related problems, use buffers within ±1 unit of their pKa, make

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buffers by blending equimolar portions of acid and base,

ensure that the buffer is sufficiently concentrated (usually 20–
25 mM is adequate), and make sure to use a column oven.

Flow Rate

Any change in flow rate of the pump will directly affect

retention for isocratic separations, whereas the effect on
gradients can be a bit more complicated. I did an informal on-
line survey of the specifications of seven instrument models
from three major LC manufacturers. This shows that the typical
specifications for flow accuracy are ±1% and for precision are
±0.07% relative standard deviation (RSD) or ±0.02 min,
whichever is greater. This means that it would not be unusual
for the retention to vary by 0.1 min (~1% of our k = 5, tR = 9
min sample used in the examples above) between instruments.
Between-run variation of 0.02 min should be considered
reasonable. With quaternary, low-pressure mixing systems,
flow will not affect the mobile-phase composition;
specifications for such systems are for maximum retention
variations of ±0.02–0.04 min because of proportioning errors.
However, with binary high-pressure mixing systems, flow rate
will affect the solvent proportions, and thus retention.

Any pump malfunction, such as problems with check valves,

pump seals, leaks, or bubbles, will also be reflected in analyte
retention times. Because such changes will only reduce the
flow rate, increases in retention would be observed when these
problems exist.

When Good Isn't Good Enough

Sometimes the instrument can be working within its

specifications and you have taken care to control the column
temperature and carefully formulate the mobile phase, but
retention time variations still are not acceptable. An example
was given in an earlier "LC Troubleshooting" column (2) where
a freshly serviced instrument (new check valves and pump
seals) still resulted in retention times that differed by up to 1
min between runs, as can be seen at the top of Figure 3. The
sample was a peptide sample run with a very shallow gradient
(0.17%/min). The instrument was specified to generate mobile
phase mixtures within ±0.1%, but it can be seen that this
variation was more than half of the gradient change per
minute. Although the system was working as specified, the
demands of the method were too great. In this case, instead of
using 100% buffer as solvent A and 100% acetonitrile as the
solvent B, the solvents were blended so A comprised 10:90
buffer–acetonitrile and B comprised 30:70 buffer–acetonitrile.
The gradient programmed into the instrument was modified to

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give the same actual gradient as the original, and consecutive

injections varied by <0.1 min in retention. By premixing the
mobile phase, the effective precision of the instrument was
increased from ±0.1% to ±0.02%, which allowed the method to
be run acceptably. This problem was partly related to
demanding more of the LC system than it was capable of and
the extreme sensitivity of the peptide sample to small changes
in the mobile phase, as discussed at the beginning of this
article.

Figure 3: Run-to-run retention variation for three consecutive

injections of a peptide sample separated with a 0.17%/min
gradient. Mobile phases: A = buffer and B = acetonitrile (top);
A = 10:90 buffer–acetonitrile and B = 30:70
buffer–acetonitrile (bottom). Data from reference 2, see text
for details.

Quality by Design to the Rescue

All of the above discussion centers around normal variations

that can be expected with a well-maintained instrument and
reasonably careful laboratory practice. Small errors in method
conditions are inevitable, so LC methods should be designed to
tolerate them, and areas of specific sensitivity should be noted
in the method document. These areas are addressed by the
International Conference on Harmonization in two places, a
document about method validation (3) and one on quality by
design (QbD) (4). When a method is validated, its robustness
should be demonstrated. Robustness is the tolerance of the
method to small, but normally expected, changes in
operational variables. This might encompass ±1% change in
%B, ±2 °C, ±0.2 pH units, and so forth. QbD principles state
that quality should be designed into a method, in particular its
sensitivity to changes in operational variables. This means that
during method development, experiments should be run to
determine how each variable (for example, %B) influences the
separation, as well as how interactions that may occur when
changes in more than one variable take place. Based on such
studies, a design space can be established. The design space is
a multidimensional space bounded by the limits of each of the
operational variables. As long as the method is operated within
the design space, it should work properly. Thus, a method can
include instructions about how large a change in each variable
can be made, while allowing the method to produce
acceptable results. Part of this information could include
tolerances in retention time or relative retention. Armed with
this kind of information, it would be much easier to know when

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an observed change in retention was a problem and when it

was normal. In addition, if an unacceptable change occurred,
the QbD information in the method would describe how to
adjust the method to bring it back into compliance.

Summary

We've seen that even small changes in the operational

variables of a method can result in noticeable changes in
retention time. Such changes in retention need to be evaluated
to determine if they have any negative impact on the quality of
the analytical results, and if so, corrective action can be taken.
The observation that most methods operate without problems
of excessive retention variation is an indicator of generally
good laboratory practice and reliable instrumentation. So keep
up the good work, but be watchful for potential problems.

References

(1) J.W. Dolan, LCGC North Am. 25(5), 448–452 (2007).

(2) D.H. Marchand, P-L Zhu, and J.W. Dolan, LCGC North Am.
14(12), 1028–1033 (1996).

(3) International Conference on Harmonization, Validation of

Analytical Procedures: Text and Methodology, Q2(R1) (ICH,
Geneva, Switzerland, 2005).

(4) International Conference on Harmonization, Q8:

Pharmaceutical Development (ICH, Geneva, Switzerland, 2006).

John W. Dolan "LC Troubleshooting" Editor John Dolan has

been writing "LC Troubleshooting" for LCGC for more than 30
years. One of the industry's most respected professionals, John is
currently the Vice President of and a principal instructor for LC
Resources in Walnut Creek, California. He is also a member of
LCGC's editorial advisory board. Direct correspondence about
this column via e-mail to [email protected]

John W. Dolan

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