What Can You Do to Improve the Peak Shape of ...

Problems with peak shape of basic compounds are fairly common. But there are several simple steps that can dramatically improve the peak shape of troublesome basic compounds. These are reviewed in the discussion below.

1. Select a Base Deactivated Silica Column
Peak tailing of some basic compounds can be eliminated by using base deactivated silica columns. These types of columns are made from silica which is fully hydroxylated and of ultra-high purity, which reduces the acidity of the silica. The result is less interaction of basic compounds with surface silanols and improved peak shape. The analysis of propranolol, shown in Figure 11-1, is an example of the possible improvement in peak shape. Base deactivated silica columns have been available since the late 1980's and are now the preferred choice for separations of basic compounds,

Figure 11-1 Chromatographic Benefits of Base Deactivated Silica Improved Analysis of Propranolol StableBond SB-C18 4.6 x 150 mm, 5 µm ZORBAX ODS 4.6 x 150 mm, 5 µm Mobile Phase: 75% 50 mM KH2PO4, pH 4.4        25% ACN Flow Rate:  1.5 mL/min Sample: Propranolol

2. Use a Low pH Mobile Phase
A low pH mobile phase is preferred for the analysis of basic compounds. Many basic compounds have pKa's of 7.5 or higher. This means that these basic compounds will have a positive charge on them over the normal useable range of silica based HPLC columns. To prevent ion-exchange interactions between the positively charged basic compounds and negatively charged silanols, the mobile phase pH should be low enough to protonate all silanols on the silica surface. The peak tailing which results from ion-exchange interactions is easily avoided, as shown in Figure 11-2. In this case, a phosphate buffer at pH 3 reduces peak tailing over a phosphate buffer at pH 7. At pH 7, ionized surface silanols will exist, but by pH 3, these have been eliminated.

Figure 11-2 Separation of Methamphetamines pH 3.0 USP TF (5%): 1. 1.19 2. 1.12 3. 1.23 4. 1.28 5. 1.21 pH 7.0 USP TF (5%): 1. 1.29 2. 1.91 3. 1.63 4. 2.35 5. 1.57 Column: Eclipse XDB-C8 4.6 x 150 mm Mobile Phase: 85% 25 mM phosphate buffer 15% ACN Flow Rate:  1.0 mL/min Temperature:  35°C Sample: Amphetamines pKa 1. Phenylpropanolamine        9.4 2. Ephedrine 9.6 3.Amphetamine 9.9 4. Methamphetamine 10.1 5. Phenteramine 10.1

3. Increase the Salt Concentration in the Mobile Phase
Increasing the salt concentration can reduce peak tailing for both bases and acids. For ionizable compounds, an increase in ionic strength can suppress solute and silica ionization, as well as secondary interactions between them. Figure 11-3 shows the improvement in peak symmetry achieved by increasing the salt or buffer concentration from 10 mM to 25 mM. Usually it is not necessary, nor is it recommended, to go above 50 mM, due to possible solubility problems of the salt in the organic portion of the mobile phase. An additional benefit of using ~ 25 mM buffer is an increase in long-term method reproducibility.

Figure 11-3 Increasing Buffer Concentration Decreases Tf 10 mM Phosphate, pH 7 USP TF (5%)*: 1. 1.62 2. 1.65 3. 1.63 4. 1.77 5. 1.83 6. 1.12 25 mM Phosphate, pH 7 USP TF (5%)*: 1. 1.41 2. 1.50 3. 1.33 4. 1.39 5. 1.36 6. 1.00 Conditions: Column:  Eclipse XDB-C8, 4.6 x 150 mm, 5µm Mobile Phase: 40% Phosphate Buffer, pH 7  60% ACN Flow Rate: 1.5 mL/min Temperature: 40ºC Sample: Tricyclic Antidepressants 1. Desipramine 2. Nortriptyline 3. Doxepin 4. Imipramine 5. Amitriptyline 6. Trimipramine

4. Select a Double Endcapped Column
If your column selection and mobile phase follow the first three recommendations, but your peak shape is still not what you want, you may need to make another column selection. There are a variety of different bonding processes on top of base-deactivated silica. Among these are double-endcapped columns that provide better peak shape. Figure 11-4 compares the peak tailing of the same compounds on a single endcapped and a double endcapped column. More symmetrical peaks are the result on the double-endcapped column for samples which can strongly interact with residual silanols.

Figure 11-4 Double Endcapping Improves Peak Shape -- pH 7 Conditions: Mobile Phase: 40% 10 mM phosphate buffer, pH 7.0   60% Acetonitrile Flow Rate: 1.5 mL/min Temperature: 40ºC Sample: a. Nortriptyline b. Doxepin c. Amitriptyline d. Trimipramine Each column is made from high purity silica Fewer silanol interactions on the double endcapped column, which also causes less retention

5. Add a Competing Base
Triethylamine (TEA) can be added to the mobile phase as a final step. TEA acts as a competing base and minimizes solute-silanol interactions. Figure 11-5 shows that TEA can improve peak shape. This is usually a final step to try because TEA will reduce retention, modify the column, and complicate the mobile phase.

Figure 11-5 Addition of TEA Modifier -- pH 7 No TEA USP TF (5%)*: 1. 1.29 2. 1.91 3. 1.63 4. 2.35 5. 1.57 10 mM TEA USP TF (5%): 1. 1.19 2. 1.18 3. 1.20 4. 1.26 5. 1.14 Conditions: Column:  Eclipse XDB-C8, 4.6 x 150 mm, 5µm Mobile Phase: 85% 25 mM Na2HPO4 15% ACN Flow Rate: 1.0 mL/min  Temperature: 35ºC Sample: Amphetamines 1. Phenylpropanolamine 2. Ephedrine 3. Amphetamine 4. Methamphetamine 5. Phenteramine

Table of Contents   |    Section 11   |  Section 11: Acids

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