Technical Tip

Effect of pH on Reversed Phase HPLC

Effect of Mobile Phase pH on the stationary phase:

Even after the bonding and endcapping process is completed on high purity silica, there
are still free (unbonded) silanols (Si-O-H) present on the silica surface. These silanols
are weakly acidic, and a pH of less than 3 is typically sufficient to protonate and neutralize most
of them.

Effect of Mobile Phase pH on Analytes:

The ionization state of ionizable functional groups on analytes will depend primarily on the pH of
the mobile phase. Recommended is to control the mobile phase pH 2 units above or below their
pKas.

Some Chromatographic symptoms relative to pH:

1. Peak Tailing: This is most common with basic analytes with amine functionalities that
can be protonated and positively charged. The observed tailing is due to secondary ion-
exchange interactions with the weakly acidic silica surface. Acidifying the mobile phase
pH below 3 with an appropriate buffer is the most common remedy. On some newer
technology materials such as Gemini and Gemini-NX that have an extended pH stability
to basic pHs up to pH 12, it is also possible to control the pH of the mobile phase at a
high pH, above that of the analyte’s pKa. This allows for neutralization of the basic
functionality on the analyte itself. This will also improve its reversed phase retention, as
neutral compounds are more hydrophobic than their ionized counterparts.
2. Retention Time Shifting: This can also be related to pH, particularly if the mobile phase
pH is not controlled with a proper buffer that has capacity to resist pH changes in the
desired region. In such cases, the ionization states of the analytes who’s retention times
are shifting may be fluctuating with a changing mobile phase pH.
3. Broad peaks and/or peak splitting: Running at a pH close to the pKa of an analyte’s
functional group can cause its peak shape to be broad, or in some cases split into to two
peaks. Here the analyte is in two different ionization states, to some extent two different
compounds (not completely accurate since the ionization states will be in dynamic
equilibrium … a broad peak is a more common than splitting in these cases).

Example:

A solution containing seven compounds was injected, but only 6 peaks observed (mobile phase
buffer used was 10mM ammonium bicarbonate ph 10.3):
DAD1 A, Sig=254,8 Ref=360,100 (SK0518\NIC00004.D)
11.187

mAU

250

200

150
8.923

100
11.001
4.195

12.685
3.949

50

2 4 6 8 10 12 14 16 min

Key words: HPLC, peak tailing, split peaks, peak shape, retention time shifting, missing peak, pH, mobile phase, buffer,
capacity
Author: Scott Krepich
© 2009 Phenomenex, Inc. All rights reserved.
Web: www.Phenomenex.com Email: Info@Phenomenex.com Page 1 of 2
 Technical Tip

Increased the mobile phase pH to 11.2 and all 7 expected peaks are now observed. Top
chromatogram is an individual injection of nornicotine and bottom chromatograms collection of all
seven analytes:
DAD1 A, Sig=254,8 Ref=360,100 (SK0524\NIC00010.D)
mAU

100

80

10.603
60

40

13.222
11.463
11.128
20

2 4 6 8 10 12 14 min
DAD1 A, Sig=254,8 Ref=360,100 (SK0524\NIC00003.D)

9.107

11.448
mAU

100

80

11.279
3.770

10.588

13.012
60
3.557

40

13.265
20

2 4 6 8 10 12 14 min

Diagnosis:

In this case, the missing peak was Nornicotine, and the original mobile phase pH of 10.3 is fairly
close to the pKa of Nornicotine. Subsequently, the peak was broad and difficult to decipher from
the baseline. Increasing the mobile phase pH to 11.2 was sufficient to neutralize Nornicotine and
visualize a sharp, corresponding peak.

Key words: HPLC, peak tailing, split peaks, peak shape, retention time shifting, missing peak, pH, mobile phase, buffer,
capacity
Author: Scott Krepich
© 2009 Phenomenex, Inc. All rights reserved.
Web: www.Phenomenex.com Email: Info@Phenomenex.com Page 2 of 2