07031TR

Correcting Peak Tailing Problems in Reversed Phase HPLC

FIGURE 1 FIGURE 2
Effect of Peak Tailing on Accuracy and Precision is
Resolution and Sensitivity Adversely Affected by Peak Tailing
The major causes of peak tailing include:
?injecting sample in a solvent that is significantly stronger than the mobile phase,
?sample mass overload,
?stationary phase silanol interactions with amines,
?adsorption of acidic compounds onto silica, and
?a void in the column packing bed.

Once you have identified the cause of the tailing, you can take action to reduce or eliminate it. (See
Table 1)

TABLE 1
Peak Tailing: Causes and Cures

Causes Cure

Sample solvent stronger than the mobile phase Dissolve sample in mobile phase or at least
reduce the strength of the sample solvent as
much as possible

Sample mass overload Reduce the amount (mass) of sample injected.

Refer to Table 2 for recommended sample
injection size for different column configurations

Silanol interaction with amines ?Reduce mobile phase pH to < 3.0

?Increase mobile phase ionic strength.
25mM to 50mM recommended
?Add a competing amine to the mobile phase.
10 mM TEA is usually sufficient.
?Select a stationary phase with a lower silanol
activity.
See Figure 6 for a ranking of C18 phases by
silanol activity
 Adsorption of acids on silica ?Increase salt concentration in the mobile phase
25 mM to 50 mM is usually sufficient
?Reduce the pH of the mobile phase to < 3.0
?Add a competing organic acid.
1% acetic acid or 0.1% TFA is usually sufficient

Column void Replace the HPLC column.

Attempts to fill-in the void are seldom worth the
effort.

If the sample is dissolved in a solvent that is stronger than the mobile phase, broad and even tailing peaks
can occur. There are several clues to help you identify if this is the cause of peak tailing. The first clue is
that early eluting peaks have more tailing than late eluting peaks. Another clue is that peak tailing
improves if less sample volume is injected or if the sample is diluted with the mobile phase.

If you suspect that mobile phase strength is the cause of your peak tailing, the cure is fairly simple. Either
dissolve your sample in mobile phase, or dilute your sample with mobile phase to the point where the
peak tailing is acceptable.

When the sample mass injected begins to exceed the capacity of the column packing, the peaks will take
on the look of a right triangle. As more sample mass is injected, the front of the peak will become sharper
and the back of the peak will tail more. Another clue that the column is being overloaded is that retention
will decrease as greater sample mass is injected. (See Figure 3)

The cure for peak tailing caused by sample mass overload is to inject less sample. Table 2 provides a list
of column diameters and the recommended maximum sample mass that can be injected before sample
overload appears as a problem. Table 2 provides a range of sample size because the actual amount will
depend on the column packing (packing materials with higher surface area have higher sample loading),
the analyte (larger molecules have lower loading), and other factors such as sample solubility in the
mobile phase.
 FIGURE 3
Sample Mass Overload Causes Peak Tailing

TABLE 2 TABLE 3
Recommended Maximum Sample Commonly Used Buffers for
Mass to Inject onto an HPLC Column Reversed Phase HPLC
A common cause of peak tailing in reversed phase HPLC is the secondary retention that occurs when an
ion-exchange interaction takes place between a positively charged solute (amine) and an acidic silanol on
the surface of silica stationary phase support particles (Figure 4). It is observed most often when using
HPLC columns packed with stationary phases that have significant silanol activity. It usually is worse at
neutral pH (6 to 8) than at acidic pH (<3).

Acidic or neutral compounds are not affected, and some basic compounds are more adversely affected
than others. If you think that silanol interaction is the cause of your peak tailing, there are several steps
you can take to correct the problem. First, make sure that the mobile phase is properly buffered (see
Table 3 above) and operate at a pH below 3, if possible. Using sufficient buffer controls the pH and
reduces ion-exchange interactions. Operating at a pH below 3 protonates silanol groups on the silica
stationary phase support (pKa of silanol is ~ 3.5) and thereby makes the silanols less available for
interacting with solutes.

FIGURE 4
Peak Tailing Interaction
Another solution to peak tailing is to add a competing amine to the mobile phase. Triethylamine (TEA) is
commonly added to mobile phases for this purpose. TEA interacts strongly with silanols and inhibits
them from interacting with amines in your sample. Figure 5 is an example of how TEA improves peak
shape. About 10mM TEA is sufficient for most applications.

Some chromatographers object to adding a competing amine to their mobile phase because it adds
complexity to their method and alters the HPLC column in a way that is not easily reversed. Strong
amines, such as TEA, are difficult to wash off the column. This means that a column thus modified by
TEA is not suitable for applications that do not use TEA in the mobile phase.

FIGURE 5
Adding TEA to the Mobile Phase Often Improves Peak Shape

Column: Eclipse XDB-C8 Temperature: 35

4.6 x 150 mm Sample: Amphetamines
Mobile 85% 25 mM 1. Phenylpropanolamine
Phase: Na2HPO4, 2. Ephedrine
pH 7.0 3. Amphetamine
15% ACN 4. Methamphetamine
Flow Rate:
1.0 mL/min 5. Phenteramine
The use of TEA can often be avoided by selecting stationary phases that have very low silanol activity.
Figure 6 ranks some popular C18 reversed phase columns according to silanol activity. The ranking in
Figure 6 was obtained by measuring the asymmetry of amitriptyline, an amine that is commonly used to
measure silanol activity of stationary phases. The less tailing (lower asymmetry value) exhibited by a
stationary phase when running amitriptyline, the less silanol activity that stationary phase exhibits and
the less peak tailing it will have when separating other basic compounds.

FIGURE 6
Popular C18 HPLC Columns Ranked According to Silanol Activity

Column: Temperature:
Mobile Sample:
Phase:

Flow Rate:
Although much less common than peak tailing of amines, acids can sometimes show peak broadening
and peak
tailing because of adsorption onto the silica stationary phase support. To correct peak tailing in these
cases increase the salt concentration of the mobile phase to suppress secondary interactions, reduce the
mobile phase pH to protonate silanols and solutes and, if necessary, add a competing acid to the mobile
phase (Figure 7).

FIGURE 7
Peak Tailing of Acidic Compounds Can Often Be Corrected
by Adding an Acid to the Mobile Phase

Column:
4.6 x 150 mm, C18
Mobile
40% 5 mM NaH2PO4
Phase:
60% ACN
Ibuprofen
Sample:

Ibuprofen, an acidic compound, tails badly until acetic acid is added to

the mobile phase. The acetic acid preferentially interacts with acidic
silanol groups on the surface of the silica stationary phase support and
inhibits the interaction between ibuprofen and silanols that causes peak
 tailing.

A void at the head of the HPLC column's packing bed will cause peaks to be broad and, sometimes, tail.
Although column voids were common several years ago, manufacturers today have perfected their
packing techniques and the better quality columns seldom experience a void unless there is a contributing
cause, such as operating at extremely high pressures and/or extremely high flow rates. If, however, you
normally get good performance from your HPLC column and then suddenly you start to see tailing on all
of your peaks, you may have a column void. The early eluting peaks will be more affected than late
eluting peaks by a column void. Although some chromatographers will attempt to repair a column by
filling-in the void with a similar stationary phase material, it has been our experience that this is seldom
worth the effort. The best cure for a column that is giving tailing peaks because of a void in the packing
bed, is to replace it. This saves time, money, and frustration.

MAC-MOD Analytical, Inc. -- info@[Link] -- 1-800-441-7508