 Acids Poor
peak shape for acidic compounds is less common than with basic compounds but can still be
problematic. Suggestions on how to improve the peak shape of acidic compounds, like
Ibuprofen, are highlighted in the discussion below.
1. Increase the Salt Concentration in the Mobile Phase
The USP method for Ibuprofen
recommends using a relatively high salt mobile phase of sixty percent acetonitrile and
forty percent 50 mM NaH2PO4. A high salt
concentration can improve the peak shape of acidic compounds by suppressing solute and
silica ionization as well as secondary interactions between them. Figure 11-6 shows that
Ibuprofen tails considerably when using a low salt concentration, 5mM NaH2PO4,
mobile phase; the tailing factor is 3.9.
Figure 11-6
pH 4.4 -- 5mM NaH2PO4
 |
|
Solute-Mobile Phase Evaluation
Before we proceed with other experiments, let's look more critically at this method and
solute.
- Ibuprofen has a prominent carboxylic acid functional group; its pKa is 4.4.
- 50 mM NaH2PO4 has a pH of 4.4, which is
outside the phosphate buffering range (1.1 - 3.1).
At a pH of 4.4, the Ibuprofen carboxylic acid group is found equally in its ionized and
non-ionized form. This acid group, especially the ionized form, can exchange or compete
with hydrogens on the silica surface, potentially increasing tailing and retention. To reduce these interactions, let's move the mobile phase pH away from the
pKa. |
|
2. Reduce the Mobile Phase pH
Byreducing the mobile phase pH to
3*, the peak shape is improved (Figure 11-7). Ibuprofen is in a single protonated form and
is less likely to interact with the protonated silanols on the silica surface. At both low
pH and low salt conditions, the tailing factor for Ibuprofen reduces to 1.8, suggesting
that secondary interactions have been minimized but not eliminated. To
further improve peak shape, let's try an additive.
3. Add a Competing Organic Acid
Next, 1% acetic acid was added to
the mobile phase to minimize solute-silanol interactions by acting, in this case, as a
competing acid. The results were remarkable, as Ibuprofen eluted as a perfectly gaussian
band (Figure 11-8), having a tailing factor of 1.00. To
simplify the mobile phase, let's eliminate the phosphate salt altogether.
4. Substitute 0.1% Trifluoracetic Acid
The relatively high concentration
of acetic acid resulted in the noisy base line of Figure 3. Using 0.1% TFA as the aqueous
modifier results in a more transparent mobile phase and still elutes Ibuprofen as a very
symmetrical band (Figure 11-9).
Figure 11-7
pH 3 --
5mM NaH2PO4
|
|
Figure 11-8
pH 3 -- 5mM NaH2PO4
1% acetic acid
|
|
Figure 11-9
pH 2.5 --
0.1% TFA+
|
|
Column: StableBond® SB-C18, 4.6 x 150 mm;
Mobile Phase: 40% A, 60% Acetonitrile;
Flow Rate: 1.0 mL/min;
Room Temperature |
*At pH
3, the mobile phase is now buffered, but the buffer capacity is low. Increasing salt
concentration to 10 - 20 mM will improve chromatographic reproducibility over time and, as
a bonus, improve peak shape even more.
+Equivalent to 13 mM TFA. |
5. Select the Best Column for Your Sample
These hints on improving peak shape for ionizable compounds should all be implemented
after checking the pKa of the sample and reviewing the capabilities of the columns
selected for the analysis. Several columns are available which are expected to give good
peak shape for acids, bases and neutrals. One example is shown in Figure 11-10. These
columns should be the starting place for most of today's samples.
Figure 11-10
Separation of an Acid, Base, and Neutral Mix on Eclipse XDB-C18
Column:
Eclipse XDB-C18
5 µm, 4.6 x 150 mm
Mobile Phase:
40% 20 mM KH2PO4, pH 3.1
60% Acetonitrile
Flow Rate: 1.0 mL/min
Temperature: R.T. |
Sample:
1. Uracil
2. Pyridine
3. Phenol
4. N,N-dimethylaniline
5. 4-butylbenzoic acid
6. Toluene |
|
For Technical
Assistance Call |
|
| 1-800-441-7508 |
Table of Contents
| Section 11 | Section 11: Bases
MAC-MOD Analytical, Inc. -- info@mac-mod.com -- 1-800-441-7508
|
