HPLC Mobile Phase
In Hplc mobile phase refers to a liquid solvent (it may be a single solvent or a mixture of solvents) that flows through the Hplc column carrying the sample with it. Compared to samples, the mobile phase solvent can easily move through the column . The selection of the mobile phase is important to get a perfect and a good chromagram.
The composition therefore is an essential part in method development. Depending on the sample to be used and type of Hplc method we decide mobile phase, Thus Hplc mobile phase is more than just the carrier, lets get deep into its details
HPLC Mobile Phase Solvents
Solvents used in the mobile phase play an important role in successful Hplc analysis; they play a major role in determining the separation efficiency, peak shape, retention time and sensitivity.the strength and composition of solvent determines how fast or slow the analytes moves through the column. There is a good number of solvent types to select from.During solvent selection another thing to keep in mind is about detection methods for normal detection like PDA. The detection can be achieved using non-volatile solvent but while using detectors like mass spectroscopy it is necessary to use violalite solvent . This is because the solvent will go through an ionization source like electrospray ionization. For this step to work efficiently it is necessary that mobile phase must evaporate easily and allow the analyte ion efficiently transferred to the gas phase
HPLC Mobile Phase Buffer
Buffer is used in most of the mobile phase preparation, buffer in general is used to prevent the fluctuation of pH in any solution.the stable ph is essential for controlling analyte ionization,improving separation and enhancing peak shape It plays a crucial role in achieving accurate, stable and reproducible results.
Importance of Buffers in HPLC
The pH of the mobile phase is an important factor for the interaction between the analyte and the particle present in the stationary phase Especially, if the analytes are weak acids or base. their charge state changes with pH. A buffer stabilizes the pH and ensures the analytes remain in their ionic or neutral form throughout the analysis process, this helps in formation of better peak shape and improved retention time.
Commonly used Buffer in HPLC
Phosphate Buffer
- Components: Monobasic and dibasic sodium or potassium phosphate (NaH₂PO₄ / Na₂HPO₄)
- pH Range: 2.1 to 8.0
- Advantages: Strong buffering capacity and chemical stability
- Limitations: Not volatile—not compatible with mass spectrometry (MS) detection
Acetate Buffer
- Components: Acetic acid and sodium acetate
- pH Range: 3.8 to 5.8
- Advantages: Moderate buffering strength; better MS compatibility than phosphate
- Limitations: May not be strong enough at high or low pH extremes
Formate Buffer
- Components: Formic acid and ammonium formate
- pH Range: 2.8 to 4.8
- Advantages: Volatile—ideal for LC-MS; good solubility in organic solvents
- Limitations: Lower buffering strength compared to phosphate
Ammonium Bicarbonate
- pH Range: ~7–8.5
- Advantages: Volatile; suitable for MS
- Limitations: Decomposes at high temperatures
HPLC mobile phase preparation
Mobile phase preparation is an important part for achieving accurate and reproducible results. Any errors in the preparation can cause poor separation of peaks, baseline noise, pressure fluctuations and might also cause damage to pump and column. Let go through some points important to keep in mind while preparing.
- Selecting the Right Solvents and Buffers
As discussed before the solvent selection must always be done depending on various factors like the nature of Hplc type that we use (reverse-phase, normal-phase or ion-exchange), sample analyte nature and detector being used. - Using High-Purity Reagents
During preparation it is always important to keep everything clean and free from impurities as for reagent and other components any impurities present in the solvent can cause fluctuation in peak formation. This can lead to formation of ghost peaks, high background noise, or damage to sensitive detectors - Accurate Measurement and Mixing
Use proper measuring beakers during preparation and weighing using calibrated weighing analytical balance, for dissolving we can use various methods like magnetic sitter, rotary shaker or sonicator. - Filtration and Degassing
Normally filtration of the mobile phase is done using 0.22 µm or 0.45 µm membrane filter to remove particulate matter that can clog the column or damage the pump. After filtration is it always advisable to also use Degassing which helps remove dissolved gases that can form in air bubbles causing noise in signal. Although most of the moderate Hplc come with online degasser it is always advisable to use an external sonicator for a worry free result . some of instrument used for sonication includes. Vacuum degassing,Ultrasonication,Helium sparging etc. while sonication it is advisable to remove the container caps
Types of HPLC Mobile Phases
1) Aqueous Mobile Phase (Water-Based)
- water or buffer solutions as the main component.
- used in reverse-phase HPLC along with an organic solvent.
- May include buffer salts (e.g., phosphate, acetate) to maintain pH.
- Suitable for polar or ionizable compounds.
2) Organic Mobile Phase (Non-Aqueous)
- Made up of pure organic solvents such as Methanol, Acetonitrile, Tetrahydrofuran(THF), Isopropanol.
3) Mixed Mobile Phase (Water + Organic Solvent)
- Most common in reverse-phase HPLC.
- Composition can be isocratic (fixed ratio) or gradient (ratio changes during run).
4) Buffer-Based Mobile Phase
- Contains buffer salts (e.g., phosphate, acetate, formate) to maintain a stable pH.
- For analyzing weak acids or bases, peptides, or proteins.
- Used for both ion-exchange and reverse-phase HPLC.
5) Volatile Mobile Phases
- Designed for use with LC-MS (liquid chromatography-mass spectrometry).
Example are Formic acid,Acetic acid and Ammonium formate etc
HPLC Mobile Phase Bottles
The primary function of mobile phase bottles is to store the mobile phase but it also functions as an important part of Hplc. First let’s talk about the material used , mostly made using glass or high-grade plastic. They must be inert in nature. These bottles come in different volumes from 250ml to 2L and more as required, also they can be
Amber-colored: Protects light-sensitive mobile phases
Borosilicate glass: Chemically resistant
Wide-necked: For easier filling and cleaning
Key features that you find in the mobile phase bottles
Bottle Caps with Multiple Ports
This is to provide access for multiple solvent inlet tubing and also used for helium sparging line.
Solvent Inlet Filters (“Sinkers”)
Small PTFE or stainless-steel filters at the end of the tubing prevent particles from entering the pump
Labels and Color Coding
Proper labeling (with date, contents, and preparer’s name) and color-coded caps or labels help avoid mix-ups