In Chromatography What Is The Mobile Phase

In chromatography, the mobile phase acts as the carrier, transporting the mixture of substances you're trying to separate through the stationary phase. It's a crucial element in achieving effective separation and analysis. The choice of mobile phase significantly influences the interaction between the different components of the mixture and the stationary phase, thus dictating the separation process.

Understanding the Mobile Phase in Chromatography

The mobile phase can be a liquid (in Liquid Chromatography or LC), a gas (in Gas Chromatography or GC), or even a supercritical fluid (in Supercritical Fluid Chromatography or SFC). Its primary role is to dissolve the sample and carry it through the column, interacting with the stationary phase along the way. The effectiveness of a chromatographic separation hinges on the correct selection and optimization of the mobile phase.

Key Functions of the Mobile Phase

• Solubilization: The mobile phase must be able to dissolve the components of the sample you intend to analyze. If a compound doesn't dissolve in the mobile phase, it won't be carried through the column and therefore won't be separated.
• Elution: The mobile phase is responsible for eluting, or washing, the separated components off the stationary phase. Different components interact differently with both the mobile and stationary phases. Components that interact more strongly with the mobile phase will elute faster, while those that interact more strongly with the stationary phase will elute slower.
• Differential Migration: This is the core function of the mobile phase. By interacting differently with the various components of the mixture, it facilitates their differential migration through the stationary phase. This differential migration is what leads to separation.
• Detection Compatibility: The mobile phase should be compatible with the detection method being used. For example, in UV-Vis detection, the mobile phase should not absorb strongly in the UV-Vis range to avoid interference with the analyte signal.

Types of Mobile Phases

The nature of the mobile phase varies depending on the type of chromatography being employed:

• Liquid Chromatography (LC): The mobile phase is a liquid, typically a solvent or a mixture of solvents. Common examples include water, acetonitrile, methanol, and tetrahydrofuran (THF). The choice of solvent depends on the polarity of the analytes and the stationary phase.
• Gas Chromatography (GC): The mobile phase is a gas, often referred to as a carrier gas. Common carrier gases include helium, nitrogen, argon, and hydrogen. The carrier gas must be inert and compatible with the detector.
• Supercritical Fluid Chromatography (SFC): The mobile phase is a supercritical fluid, most commonly carbon dioxide (CO2). Supercritical fluids possess properties intermediate between liquids and gases, offering advantages in terms of both dissolving power and diffusivity.

Factors Influencing Mobile Phase Selection

Choosing the right mobile phase is critical for achieving optimal separation. Several factors need to be considered:

• Polarity: The polarity of the mobile phase must be compatible with the polarity of both the sample components and the stationary phase. In reversed-phase chromatography, a non-polar stationary phase is used with a polar mobile phase (e.g., water and acetonitrile). In normal-phase chromatography, a polar stationary phase is used with a non-polar mobile phase (e.g., hexane and ethyl acetate).
• Solvent Strength: The solvent strength of the mobile phase refers to its ability to elute the analytes from the stationary phase. A stronger solvent will elute analytes more quickly. In LC, solvent strength is related to polarity; in GC, it is related to the boiling point of the carrier gas.
• Viscosity: The viscosity of the mobile phase affects the pressure drop across the column and the efficiency of the separation. Lower viscosity mobile phases are generally preferred as they allow for faster flow rates and reduced backpressure.
• UV Cutoff: If using UV-Vis detection, the mobile phase must have a low UV cutoff, meaning it should not absorb UV light at the wavelengths used for detection. This prevents interference with the analyte signal.
• Compatibility with the Detector: The mobile phase must be compatible with the detector being used. For example, certain solvents can quench fluorescence, making them unsuitable for fluorescence detection.
• Sample Solubility: As mentioned earlier, the mobile phase must be able to dissolve the sample components.
• Chemical Reactivity: The mobile phase should be chemically inert and not react with the sample, the stationary phase, or the components of the chromatographic system.
• Cost and Availability: The cost and availability of the mobile phase are also practical considerations. Some solvents are more expensive or difficult to obtain than others.

Mobile Phase Optimization in Liquid Chromatography (LC)

In LC, optimizing the mobile phase composition is a common strategy to improve separation. This often involves adjusting the ratio of two or more solvents to fine-tune the polarity and solvent strength of the mobile phase.

Isocratic vs. Gradient Elution

• Isocratic Elution: In isocratic elution, the mobile phase composition remains constant throughout the separation. This is suitable for separating mixtures of compounds with similar polarities.
• Gradient Elution: In gradient elution, the mobile phase composition is changed over time. This is useful for separating complex mixtures of compounds with a wide range of polarities. A common approach is to start with a weak solvent (e.g., water in reversed-phase chromatography) and gradually increase the proportion of a strong solvent (e.g., acetonitrile) to elute the more retained compounds.

Mobile Phase Additives

Adding small amounts of additives to the mobile phase can significantly improve peak shape and separation. Common additives include:

• Acids: Acids, such as formic acid, acetic acid, and trifluoroacetic acid (TFA), are often added to the mobile phase to protonate basic analytes and improve their peak shape. They can also help to suppress ionization of silanol groups on the silica stationary phase, which can lead to peak tailing.
• Bases: Bases, such as ammonia and triethylamine (TEA), are added to the mobile phase to deprotonate acidic analytes and improve their peak shape.
• Buffers: Buffers are used to maintain a constant pH in the mobile phase. This is important for analytes that are pH-sensitive, as their retention and ionization can change with pH.
• Ion-Pairing Reagents: Ion-pairing reagents are used to improve the retention and separation of ionic compounds. They form ion pairs with the analytes, which can then be separated by reversed-phase chromatography.

Examples of Mobile Phase Optimization in LC

• Separating a mixture of polar and non-polar compounds: A gradient elution method might be used, starting with a high percentage of water and gradually increasing the percentage of acetonitrile to elute the less polar compounds.
• Improving the peak shape of a basic analyte: Adding a small amount of formic acid to the mobile phase can protonate the analyte and improve its peak shape by reducing tailing.
• Separating a mixture of peptides: Using a gradient of water and acetonitrile with the addition of TFA can improve the resolution and peak shape of the peptides.

Mobile Phase Considerations in Gas Chromatography (GC)

In GC, the mobile phase, or carrier gas, plays a less direct role in the separation process compared to LC. The separation is primarily determined by the boiling points of the analytes and their interaction with the stationary phase. However, the choice of carrier gas can still affect the efficiency and speed of the separation.

Common Carrier Gases

• Helium: Helium is the most commonly used carrier gas in GC due to its inertness, high efficiency, and compatibility with most detectors.
• Nitrogen: Nitrogen is less expensive than helium but has a lower efficiency. It is often used in packed columns and with certain detectors that are less sensitive to changes in flow rate.
• Hydrogen: Hydrogen offers the highest efficiency and allows for faster analysis times. However, it is flammable and requires careful handling. It is also not compatible with all detectors.
• Argon: Argon is used with specific detectors, such as the pulsed discharge helium ionization detector (PDHID).

Factors Influencing Carrier Gas Selection in GC

• Efficiency: The efficiency of the carrier gas is related to its diffusivity. Gases with higher diffusivities, such as hydrogen and helium, provide better resolution and allow for faster analysis times.
• Detector Compatibility: The carrier gas must be compatible with the detector being used. For example, helium is often preferred for use with mass spectrometers (MS) because it produces less background noise.
• Safety: The carrier gas must be safe to use. Hydrogen is flammable and requires special precautions.
• Cost: The cost of the carrier gas is also a consideration. Nitrogen is the least expensive, while helium is more expensive.

Optimizing Carrier Gas Flow Rate

The flow rate of the carrier gas affects the separation efficiency and analysis time.

• Van Deemter Curve: The relationship between plate height (a measure of column efficiency) and carrier gas flow rate is described by the Van Deemter curve. This curve shows that there is an optimal flow rate that minimizes plate height and maximizes efficiency.
• Practical Considerations: In practice, the optimal flow rate is often determined empirically by running a series of separations at different flow rates and evaluating the resolution and peak shape.

Mobile Phase in Supercritical Fluid Chromatography (SFC)

SFC combines the advantages of both LC and GC. The mobile phase, typically supercritical carbon dioxide (CO2), possesses properties intermediate between liquids and gases, offering both good dissolving power and high diffusivity.

Advantages of Supercritical CO2

• Tunable Solvent Strength: The solvent strength of supercritical CO2 can be easily adjusted by changing the pressure and temperature.
• High Diffusivity: The high diffusivity of supercritical CO2 allows for faster analysis times compared to LC.
• Environmentally Friendly: CO2 is a non-toxic and environmentally friendly solvent.
• Compatibility with Various Detectors: SFC is compatible with a wide range of detectors, including UV-Vis, fluorescence, and mass spectrometry.

Modifiers in SFC

While supercritical CO2 is a versatile mobile phase, it is often necessary to add small amounts of modifiers to improve its dissolving power and selectivity. Common modifiers include methanol, ethanol, and acetonitrile.

Applications of SFC

SFC is used in a variety of applications, including:

• Pharmaceutical Analysis: SFC is used for the separation and purification of chiral drugs and other pharmaceutical compounds.
• Polymer Analysis: SFC is used for the characterization of polymers and other high-molecular-weight compounds.
• Food Analysis: SFC is used for the analysis of fats, oils, and other food components.
• Environmental Analysis: SFC is used for the analysis of pollutants and other environmental contaminants.

Troubleshooting Mobile Phase-Related Problems

Problems with the mobile phase can lead to various issues in chromatography, such as poor resolution, peak tailing, baseline drift, and ghost peaks.

Common Problems and Solutions

• Poor Resolution:
  • Problem: Mobile phase is too strong or too weak.
  • Solution: Adjust the mobile phase composition or use a gradient elution method.
• Peak Tailing:
  • Problem: Silanol interactions in LC, insufficient derivatization in GC.
  • Solution: Add an acid or base modifier to the mobile phase in LC, perform derivatization in GC.
• Baseline Drift:
  • Problem: Contaminated mobile phase, temperature fluctuations.
  • Solution: Use high-purity solvents, control the temperature of the system.
• Ghost Peaks:
  • Problem: Contamination of the mobile phase or system.
  • Solution: Use high-purity solvents, clean the system thoroughly.
• High Backpressure:
  • Problem: Viscous mobile phase, clogged column.
  • Solution: Use a less viscous mobile phase, flush the column.

Importance of Mobile Phase Purity

The purity of the mobile phase is crucial for obtaining accurate and reliable results. Impurities in the mobile phase can lead to baseline noise, ghost peaks, and reduced sensitivity. It is essential to use high-purity solvents and to filter the mobile phase before use.

Conclusion

The mobile phase is an integral component of any chromatographic separation. Its careful selection and optimization are paramount for achieving effective separation, accurate quantification, and reliable results. Understanding the different types of mobile phases, the factors influencing their selection, and common troubleshooting strategies will enable you to optimize your chromatographic methods and achieve your analytical goals. By considering the polarity, solvent strength, viscosity, UV cutoff, detector compatibility, and other relevant factors, you can choose the mobile phase that is best suited for your specific application. Moreover, knowledge of isocratic and gradient elution techniques, mobile phase additives, and carrier gas considerations will further enhance your ability to fine-tune your separations and achieve optimal performance. Remember that maintaining mobile phase purity and addressing any mobile phase-related issues promptly are essential for ensuring the accuracy and reliability of your chromatographic analyses.