HPLC Mobile Phase: How to Make it Cost-Effective

Introduction and Outcome: HPLC Mobile Phase

HPLC Mobile Phase play a vital role in HPLC method development since it is one of the separation deciding factors. A minor error during mobile phase preparation can change the elution pattern. That is why I decided to share my expertise on this topic. In this article, you will learn the Mobile phase, mobile phase preparation steps, how to avoid common errors and how to make a cost-effective mobile phase with case studies and FAQs

HPLC Mobile Phase Chemistry

The mobile phase is a mixture of aqueous solvent and organic solvent or a mixture of buffer and organic solvent or a mixture of organic solvent in different compositions or an aqueous solvent or organic solvent depending on the mode of HPLC chromatography. It is responsible for the separation of the different components of the sample mixture.

Depending upon the chromatographic mode the HPLC Mobile Phase may be the following types:

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 optimizing 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 solvent) plays an important role in optimizing the separation. The following solvents are used in the Normal Phase HPLC Mobile phase:

Solvents	Polarity
n-Hexane	Non polar
n-Heptane	Nonpolar
Isooctane	Nonpolar
Chloroform	Proton acceptor
Dichloromethane	Large dipole
Ethyl acetate	Proton donor
THF	Proton acceptor
Propylamine	Proton acceptor
Acetonitrile	Dipole
Ethanol	Proton donor
Methanol	Proton donor

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 optimize the separation process, and it can significantly affect retention times, resolution, and selectivity in HPLC analysis.

Mobile Phase Selection Steps

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 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.

Solvent Mixing or Mobile Phase Composition

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

Factors affecting the quality of the Mobile phase

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 stationary phase
Sample components should be miscible fully 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 optimizing 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 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 Effective Mobile Phase?

Try to use less costly solvents like methanol during method development in place of acetonitrile
Prepare the mobile phase as per 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

Mobile Phase Elution Mode

The following HPLC mobile phase elution modes are used during analysis

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.
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 ratio of 70:30

Impact of Mobile phase on HPLC analysis

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

Change in retention time
Tailing or fronting in the peak
Failure in the system suitability
Increase in the analysis cost
Noise in the chromatogram
Ghost peak elution

Conclusion

I hope this article has helped you understand mobile phase chemistry and its importance. Now you can easily avoid common errors during mobile phase preparation.

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

HPLC principle,
HPLC method development
HPLC column
HPLC interview questions and
HPLC mobile phase adjustment
Capacity factor in HPLC how to understand?
What is the role of tailing factor in HPLC analysis?
What is the HETP (height equivalent to theoretical plate)

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FAQs

How to select a mobile phase in HPLC method development?

The mobile phase is selected based on the polarity of the sample components and mode of the chromatography

What is normal phase mobile phase in HPLC?

Normal phase mobile phase 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.

Which type of solvents are used in HPLC for mobile phase?

Solvents are decided based on the mode of chromatography. In normal phase chromatography mode nonpolar mobile phase is used and in the reverse phase mode polar mobile phase is used

What is the purpose of using a mobile phase in HPLC?

The purpose of using a mobile phase in HPLC is to separate the different components of the sample and optimise the retention time of the sample components

References

Mobile Phase composition :Science Direct.Com

Abbreviations

RPC: Reverse phase
NPC: Normal phase chromatography