How to Prepare and Optimise HPLC Mobile Phases: For Accurate and Efficient Separations

How to prepare and optimise HPLC mobile phases is a challenging task for chromatographers!

The HPLC mobile phase is a mixture of aqueous solvent and organic solvent, or a mixture of buffer and organic solvent, or a mixture of organic solvents in different compositions, or an aqueous solvent or organic solvent, depending on the mode of HPLC chromatography.

The HPLC mobile phase plays a crucial role in HPLC (High-Performance Liquid Chromatography) method development, serving as one of the primary factors that influence separation efficiency, retention time, and peak resolution. Even minor inconsistencies in mobile phase preparation can significantly alter elution patterns, potentially compromising the reliability and reproducibility of your analytical results.

In this comprehensive guide, I share my expertise to help you master the fundamentals and best practices of mobile phase preparation. You’ll learn what the mobile phase is, step-by-step preparation techniques, common pitfalls to avoid, tips for optimising cost without sacrificing quality, and real-world case studies to deepen your understanding. Whether you’re a beginner or an experienced analyst, this article provides practical insights and answers to frequently asked questions that will enhance the robustness and efficiency of your HPLC methods.

HPLC Mobile Phase

The mobile phase in High-Performance Liquid Chromatography (HPLC) is the solvent or mixture of solvents, typically comprising an aqueous solution, an organic solvent, or a buffer, that flows through the chromatographic column. Its composition is carefully selected based on the mode of HPLC (e.g., reverse-phase, normal-phase, ion-exchange) to facilitate the separation of components in the sample mixture by influencing their interactions with the stationary phase

Related: How To Control Impurities In Pharmaceuticals: Get Mastery In pharmaceuticals

Type of HPLC Mobile Phase

The following are the different types of mobile phases depending upon the chromatographic mode:

• Reverse Phase HPLC Mobile Phase
• Normal Phase HPLC Mobile phase
• Ion-exchange HPLC Mobile Phase
• Size-exclusion HPLC Mobile Phase

Reverse Phase HPLC Mobile Phase

It consists of a polar mobile phase and consists of a mixture of water or aqueous mixture and organic solvent such as acetonitrile, methanol, etc. The mobile phase composition (water and organic solvent) plays an important role in optimising the separation.

Normal Phase HPLC Mobile phase

It consists of a nonpolar mobile phase and consists of a mixture of nonpolar organic solvents (such as hexane, heptane, etc) and polar organic solvents such as isopropyl alcohol, ethanol, methanol, etc. The mobile phase composition (nonpolar and polar organic solvents) plays an important role in optimising the separation. The following solvents are used in the Normal Phase HPLC Mobile phase:

Ion-exchange HPLC Mobile Phase

The mobile phase is typically an aqueous solution containing buffers to control pH and ionic strength. The mobile phase composition can also include counterions that aid in the separation of charged analytes.

Size-exclusion HPLC Mobile Phase

The mobile phase is usually an aqueous solution or buffer, and its composition is chosen to ensure the sample remains stable and does not aggregate.

The Role of HPLC Mobile Phase in Method Development

The HPLC mobile phase plays a critical role in the separation of analytes based on their interaction with both the stationary phase and the mobile phase. The chemistry of the mobile phase is carefully designed to optimise the separation process, and it can significantly affect retention times, resolution, and selectivity in HPLC analysis.

How to Prepare and Optimise HPLC Mobile Phases?

Mobile Phase Preparation Steps

The following steps play a crucial role in HPLC mobile phase preparation:

1. Selection of Buffer
2. Selection of Ion Pairs
3. Selection of Solvents (aqueous and organic)
4. Selection of Modifiers
5. pH Selection and Adjustment
6. Mobile Phase Composition or Mixing of Solvents
7. Mobile Phase Filtration
8. Mobile Phase Sonication

Selection of Buffer

Buffer concentration and quality play an important role in the separation. Only HPLC-grade chemicals like potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium acetate or any other chemicals should be used for buffer preparation.

Selection of Ion Pairs

Alkyl sulphonates and tetra-butyl ammonium hydroxide are used as the ion pairs in reverse-phase HPLC. Only HPLC-grade ion pairs at a lower concentration of about 0.005M should be used to avoid noise in the chromatogram

Selection of Solvents

• Selection of Aqueous Solvents: Water is often used as a base solvent, particularly in reversed-phase chromatography. Only HPLC-grade water should be used in the mobile phase preparation.
• Selection of Organic Solvents: Organic Solvents, such as methanol, acetonitrile, ethanol, and tetrahydrofuran (THF) used in the reverse phase mobile phase. Only HPLC-grade Organic solvents should be used in the mobile phase preparation.

Selection of Modifiers

Modifiers like triethylamine (TEA), diethylamine (DEA) or tetrahydrofuran (THF) are used to improve peak sharpness and reduce the peak tailing. Only HPLC-grade Modifiers should be used in the mobile phase preparation.

pH Selection and Adjustment

Only HPLC pH adjusters like trifluoroacetic acid, HCOOH, CH₃COOH, NaOH solution, etc, should be used for pH adjustment. Only a calibrated pH meter should be used for pH adjustment.

Mobile Phase Composition or Mixing of Solvents

The mobile phase composition or solvent mixture should be made at low temperatures to avoid evaporation of organic solvents. Any evaporation of the solvent can change the composition of the mobile phase leading to variations in retention times.

Mobile Phase Filtration

The mobile phase should be filtered using suitable filter paper to remove the undissolved particles.

Mobile Phase Sonication

The mobile phase is sonicated to remove the dissolved air. But the mobile phase should be sonicated at a lower temperature to avoid any evaporation of the organic solvent

The HPL Mobile Phase Optimisation

HPLC Mobile Phase Optimisation refers to the process of adjusting the composition and conditions of the mobile phase in High-Performance Liquid Chromatography to achieve the best possible separation of analytes. This involves systematically modifying factors that influence how compounds interact with the stationary phase and elute from the column. The following are the advantages of mobile phase optimisation:

• Improve resolution between peaks
• Reduce analysis time
• Enhance peak shape and symmetry
• Achieve reproducible retention times
• Increase sensitivity and selectivity

The following factors are involved in HPLC mobile phase optimisation:

1. Solvent Composition
2. pH of the Aqueous Phase: Crucial for ionisable compounds; affects ionisation state and interactions
3. Buffer Type and Concentration: Helps control pH and improve reproducibility.
4. Gradient vs. Isocratic Elution: Gradient elution changes solvent strength during the run and can help separate complex mixtures.
5. Flow Rate: Influences peak resolution and analysis time.
6. Temperature: Affects the viscosity of the mobile phase and compound interaction kinetics.

Factors Affecting the HPLC Mobile Phase Chemistry

• Viscosity: The viscosity of the mobile phase affects the pressure needed for the pump, with organic solvents typically having lower viscosities than aqueous solutions.
• Solvent Miscibility: When mixing solvents, ensure that they are fully miscible to avoid phase separation that could cause unstable flow.
• UV Transparency: If using UV detection, ensure that the mobile phase is transparent at the detection wavelength to avoid interference in the signal.

Characteristics of the Mobile Phase

• Cost-effective
• Non-hazardous
• Low viscosity
• Inertness towards analytes and the stationary phase
• Sample components should be fully miscible with the mobile phase
• Low UV cut-off: The detector should not respond to the mobile phase or to changes in the mobile phase composition, as in gradient elution

HPLC Mobile Phase: Common Errors

• Precipitation in the mobile phase: Precipitation generally occurs as the organic solvent increases in the gradient. Care must be taken when optimising gradients.
  • If you are using buffer and acetonitrile in the gradient, do not increase the composition of acetonitrile to more than 80%, or precipitation will occur.
  • If you are using buffer and methanol in gradient elution, do not increase the composition of methanol to more than 85%, otherwise, precipitation will occur.
• Change in solvent composition: Solvent Evaporation during Mobile phase preparation led to the change in the solvent composition
• Air bubble in the Mobile Phase: Remove the air bubbles from the mobile phase by sonication, otherwise, pressure fluctuation can take place or noise may come in the chromatogram

How to Make Cost Cost-Effective Mobile Phase?

• Try to use less costly solvents like methanol during method development in place of acetonitrile
• Prepare the mobile phase as per the analysis need.
• Do not prepare unnecessary amounts of mobile phase that cannot be consumed during the analysis.
• Keep the mobile phase in a closed container at a lower temperature to avoid evaporation

Gradient and Isocratic HPLC Mobile Phase Elution Modes

The following HPLC mobile phase elution modes are used during analysis

1. Gradient elution: The mobile phase composition is gradually changed during the analysis, typically from a low to high concentration of organic solvent. This method is beneficial for complex samples with a wide range of polarities, as it improves the separation of compounds that may otherwise co-elute under isocratic conditions.
2. Isocratic elution: A constant mobile phase composition is maintained throughout the analysis. This approach is simpler and is often used when analytes have similar properties and can be separated under one solvent system. Typical example: Mixture of 0.1% trifluoroacetic acid and methanol in the ratio of 70:30

Impact of Mobile phase on HPLC analysis

A mistake in mobile phase selection or preparation may lead to :

1. Change in retention time
2. Tailing or fronting in the peak
3. Failure in the system suitability
4. Increase in the analysis cost
5. Noise in the chromatogram
6. Ghost peak elution

Case Study: Optimisation of HPLC Mobile Phase for the Analysis of an API-X in Pharmaceutical Tablets

Objective: To develop and optimise an HPLC method for the accurate quantification of API-X in a commercial tablet formulation by optimizing the mobile phase composition for better resolution, retention time, and peak shape.

Background: API-X is a non-steroidal anti-inflammatory drug (NSAID) widely used in over-the-counter medications. Accurate and reproducible HPLC methods are required for quality control and regulatory compliance. Due to the presence of excipients and possible degradation products, mobile phase optimisation is essential to achieve good separation.

Initial Conditions:

• Column: C18, 250 mm × 4.6 mm, 5 µm
• Mobile Phase (Initial Trial): 60:40 Methanol:Water
• Flow Rate: 1.0 mL/min
• Detection Wavelength: 220 nm
• Injection Volume: 10 µL

Observation: Poor peak shape, broad tailing, and insufficient resolution from excipients.

Stepwise Optimisation Process:

• Adjusting Solvent Composition:
  • Tried different ratios: 70:30, 65:35, 50:50 (Methanol:Water)
  • Best peak shape and retention time (~7.5 min) achieved at 70:30

• Switch to Acetonitrile for Comparison:
  • Tested 70:30 Acetonitrile:Water
  • Faster elution (retention time ~4.2 min), but slight peak distortion

• Decision: Continue with Methanol as it offers better selectivity for API-X

• pH Optimization:
  • Adjusted aqueous phase to pH 3.0 using 0.1% orthophosphoric acid
  • Improvement in peak symmetry (pKa of ibuprofen ~4.9; acidic mobile phase suppresses ionisation)

• Buffer Inclusion:
  • Used phosphate buffer (20 mM, pH 3.0) in aqueous phase
  • Enhanced reproducibility and baseline stability
• Final Optimised Mobile Phase:
  • 70:30 Methanol: 20 mM phosphate buffer (pH 3.0)
  • Flow rate: 1.0 mL/min
  • Temperature: 30°C

Results

• Sharp, symmetric peak for ibuprofen
• Retention time ~7.2 min
• Resolution >2.0 from nearest impurity
• RSD of peak area <1% over six injections (excellent reproducibility)

Conclusion:

Mobile phase optimisation significantly improved the quality of chromatographic separation. Proper selection of solvent type, ratio, pH, and buffer led to a robust and reproducible method suitable for routine analysis of API-X in pharmaceuticals.

Conclusion

In HPLC method development, the mobile phase is a critical component that directly impacts separation efficiency, retention time, and peak resolution. Given its significant influence on chromatographic performance, strict consistency in mobile phase composition, preparation, and handling is essential. Careful control of these parameters ensures reliable, reproducible results and maintains the integrity of analytical data..

You may also want to check out other articles on my blog, such as:

• What is the Analytical Method Validation?
• The 7 Key Differences Between Validation and Verification

FAQs

References

• Mobile Phase composition :Science Direct.Com

Abbreviations

• RPC: Reverse phase
• NPC: Normal phase chromatography