HPLC method development for basic molecules requires special care in selecting the mobile phase and the stationary phase. Use a basic mobile phase with a pH greater than 7 and a column that is stable at high pH.
In this article, I will discuss the three most important HPLC tips for separating basic compounds in reverse-phase chromatographic mode. After reading the article, you will be able to develop an HPLC method for basic pharmaceuticals easily. This article will also enable you to answer several questions.
The principle of separation of basic compounds by HPLC is based on differences in their polarity, hydrophobicity, and ionization state, which affect their interaction with the stationary and mobile phases.
In reversed-phase HPLC, basic compounds are separated mainly by hydrophobic interactions with the non-polar stationary phase. The mobile phase pH is typically kept acidic to suppress ionization of basic groups, enhancing retention and peak shape.
To select the mobile phase and column for HPLC separation of basic compounds:
1. Column selection:
Use a reverse-phase column (e.g., C18) to retain basic compounds through hydrophobic interactions.
2. Choose solvents like acetonitrile or methanol for the organic phase, adjusting polarity for optimal separation.
3. Add buffers (e.g., phosphate or acetate) to maintain pH stability and enhance reproducibility.
4. Adjust gradient: Optimise the gradient to achieve the desired resolution and retention time for basic compounds.
To select modifiers for HPLC separation:
1. Consider solubility: Choose modifiers (e.g., salts, acids, or organic solvents) that enhance the solubility of your analytes in the mobile phase.
2. Match polarity: For reversed-phase HPLC, use organic solvents (e.g., acetonitrile or methanol) to adjust polarity. For normal-phase HPLC, use non-polar solvents.
3. Adjust pH: Select modifiers (like acids or bases) to adjust pH, improving analyte ionisation and separation efficiency.
4. Use buffer salts: Choose appropriate salts (e.g., phosphate, acetate) to maintain a stable pH and reduce sample degradation.
5. Optimise gradient: Modify concentration of organic solvents or additives during the run to improve resolution.
Each modifier should enhance analyte separation without causing baseline drift or interference.
To optimise chromatographic conditions for basic molecules in HPLC:
1. Use an acidic mobile phase (pH 3-4) to prevent ionisation of the basic molecules and improve retention on the stationary phase.
2. Choose a suitable stationary phase (e.g., C18 or mixed-mode) with sufficient interaction for basic molecules.
3. Adjust the organic solvent content: Increase acetonitrile or methanol for better resolution.
4. Optimise flow rate: Faster flow rates reduce retention, while slower rates improve resolution but increase analysis time.
5. Use temperature control to enhance reproducibility and separation efficiency.
Monitor detector wavelength based on the absorption maxima of the basic molecules.
To optimise sample concentration and gradient:
1. Start with a broad range of concentrations to find the optimal one that maximizes signal without saturation or noise.
2. Adjust gradient steepness: A steep gradient is best for resolving closely related samples, while a shallow gradient is good for broader separation.
3. Use trial runs to fine-tune the concentration and gradient to balance sensitivity and resolution.
4. Consider buffer composition and flow rates, as these can influence how samples interact and separate.
5. Monitor detector response to avoid under or overloading the system.
Those organic compounds tending to accept the H+ ion (proton) and remain energetically stable after accepting of proton are called Basic Organic Compounds. For example, compounds like Aniline and Diclofenac sodium.
HPLC columns with non-polar stationary phases with high carbon loading are suitable for the separation of the basic compound in reverse-phase chromatographic mode. Hence, priority should be given to C18 and C8.
Try to maintain the mobile phase pH between 7.5 and 11. In this range, the tendency of basic molecules to accept protons decreases, causing these molecules to become more non-polar and separate in the reverse phase chromatographic mode.
Methanol or Acetonitrile can be used. Considering the cost, methanol should be the preferred choice
Following are the structures of some basic molecules (1-(2-aminophenyl)ethan-1-one and Sodium 2-aminobenzoate):
Note: The above structures have been drawn using ACD/Chemsketch (Freeware) to explain the topic
Both of the compounds can accept the proton and stabilize themselves due to the resonance effect.
Remember to keep the pH of the mobile phase basic, preferably above 7.5. You can use a phosphate or acetate buffer at a concentration between 0.01 M to 0.03 M. For the separation, consider using a column with a C18 stationary phase, high carbon load, and more end-capping.
Methanol or Acetonitrile can be used. Considering the cost methanol should be the preferred choice.
These compounds can be separated either in gradient or isocratic mode gradient or isocratic mode, but gradient mode should be the preferred choice during method development. The trial should be started with a higher aqueous phase of 75% or higher. Based on the elution of each of the molecules, optimisation in the composition of the mobile phase should be done. Example:
| Time | A (0.02% K2HPO4 v/v in water pH 8.0) | B (Methanol) |
| 0 | 75 | 25 |
| 15 | 25 | 75 |
| 20 | 25 | 75 |
| 20.1 | 75 | 25 |
| 25 | 75 | 25 |
Keep the flow rate between 0.5 to 1 ml/minute to get column pressure less than 2000 psi
Keep the sample concentration so that there should not be any column overloading and the peak should be sharp. Sample concentration can be increased or decreased based on requirement e.g sample concentration can be kept like 0.2mg/ml, 0.5mg/ml or 0.7mg/ml
Keep the Injection Volume in such a so that there should not be any column overloading and the peak
should be sharp, e.g. injection volume may be 5μl, 10μl or 20μl.
Prepare the solution of each 1-(2-aminophenyl)ethan-1-one and Sodium 2-aminobenzoate and scan in a UV spectrophotometer or PDA detector. Select a wavelength where each 1-(2-aminophenyl)ethan-1-one and Sodium 2-aminobenzoate have almost equal response.
Inject the standard solution of each 1-(2-aminophenyl)ethan-1-one and Sodium 2-aminobenzoate, and generate the chromatogram. Inject the sample mixture and generate the chromatogram. Based on the elution pattern optimise the mobile phase composition and chromatographic condition to get better separation.
Based on the requirement, use the area % (area normalisation method) or the external standard method to give the result.
Since Sodium 2-aminobenzoate is relatively polar than 1-(2-aminophenyl)ethan-1-one .Therefore, Sodium 2-aminobenzoate will elute first, and after that 1-(2-aminophenyl)ethan-1-one elute.
In addition to 1-(2-aminophenyl)ethan-1-one and Sodium 2-aminobenzoate, the HPLC separation tips mentioned above can also be applied to other basic compounds. Now that you have learned about column selection, mobile phase selection, and chromatographic condition optimisation for the separation of acidic compounds, I hope you can apply this knowledge during method development. That’s all for this post. Please share your thoughts and suggestions related to this post in the comments section.
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