Skip to Content
Merck
HomeProductsAnalytical ChemistryAnalytical ChromatographyHPLC Buffers

HPLC Buffers

Heap of buffer salt crystals

Buffers are solutions of a weak acid and its conjugate base, or a weak base and its conjugate acid. In analytical chemistry, buffers are typically used in reversed-phase high performance liquid chromatography (RP-HPLC), when the sample contains acidic or basic functional groups. Buffers mitigate the influence of hydrogen/hydronium and hydroxide ions, subsequently reducing pH fluctuation.   

Related Resources

  • Brochure: LC-MS Resource Guide

    A Comprehensive Portfolio for Consistent Results in Routine and Advanced LC-MS applications

  • HPLC Method Transfer Calculator

    Easily transfer a method between HPLC columns, (scale between microbore through preparative range), based on the 2 column dimensions and current method conditions.

Mobile Phase Buffers

Proper buffer choice, in terms of buffering species, ionic strength, and pH, is the most critical step in RP-HPLC method development for ionic analytes. Ionic analyte retention in RP-HPLC is fundamentally effected by the pH of the mobile phase. Thus, pH control of the mobile phase in RP-HPLC is one of the most important parameters for effective analyte separation.

The typical pH range for reversed-phase separations on silica-based columns is between 2 to 8. The buffer must have a pKa close to the desired pH, since buffers control pH best at their pKa. As a rule of thumb, it is ideal to choose a buffer with a pKa value <2 units of the desired mobile phase pH. We offer HPLC grade buffers for a wide pH range for all your lab chromatographic requirements.

Factors to be considered while choosing a HPLC Buffer

Chemical Purity

The quality of mobile phase additives (buffers, salts, acids and bases) along with organic solvents utilized in an HPLC experiment must be adapted to the detector sensitivity and elution protocol. High-quality solvents and reagents are recommended for optimal chromatography.

Chemical Compatibility

Buffer composition along with mobile phase pH must be chosen in agreement with column housing material and nature of the stationary phase. This prevents damage to the column hardware and bed.

Buffer Solubility

Ideally, the buffer should be completely water soluble and should not precipitate. Buffer concentration must be carefully chosen to avoid precipitation at higher concentrations, when the buffer is blended with the organic solvent. If neglected, it can create operational problems in the pumps and instigate HPLC column blockage.

Buffer Strength

An eluent showing weak interaction with the stationary phase is only capable of eluting weakly bonded analytes from the column, whereas a strong interaction causes elution of strongly bonded sample molecules. The elution or solvent strength of various solvents depends on the type of stationary phase used. After the elution strength, the viscosity and UV absorbance of the buffer plays an important role in terms of their suitability for use in HPLC analyses. 

Related Product Categories
HPLC Columns
Achieve precise separations with our extensive HPLC column collection. Enhance retention, resolution, and selectivity. Order today.
Analytical Vials
Ensure precise analysis: Inert, certified vials prevent extractables, leachables. Reduce risk with Supelco® autosampler, general-purpose vials for HPLC, GC/MS, LC/MS.
Analytical Reagents
Analytical reagents and solvents for wet chemistry, chromatography, spectroscopy, titration, microbiology, microscopy, gravimetry, water analysis, and ion sensors.
Solvents
Your Solvent Source: Find the right fit with Supelco®, SigmaAldrich®, & SAFC® brands, covering analytical, lab, & biopharmaceutical uses. Order online.
Salts
Explore a comprehensive range of salts offered under three portfolios for a variety of applications: Supelco® for analytical methods and applications, Sigma-Aldrich® for classic research and production, and SAFC® for biopharmaceutical and pharmaceutical applications. Order now.
Acids
Explore a comprehensive range of acids offered under three portfolios for a variety of applications: Supelco® products for analytical chemistry, Sigma-Aldrich® materials for lab and production, SAFC® products for biopharmaceutical and pharmaceutical development and manufacturing.
Caustic Alkalis & Bases
Our three portfolio brands offer caustic alkalis and bases to meet the various requirements of your specific applications. Supelco® products for analytical chemistry, Sigma-Aldrich® materials for lab and production, SAFC® products for biopharmaceutical and pharmaceutical development and manufacturing.
Related Applications
Small Molecule HPLC
Small molecules are ions and compounds of molecular weight typically less than 900 daltons. These compounds can be effectively separated and analyzed by HPLC, UHPLC and LC-MS using mainly silica particles or monolithic stationary phases with a broad range of column chemistries (modifications).
Large Molecule HPLC
High performance liquid chromatography (HPLC) can be used to separate and identify different large biomolecules such as protein and peptides in a sample. It is based on the pumping of a sample with a solvent (mobile phase) through a column packed with sorbent material (stationary phase) at a high pressure.
Wet Chemical Analysis
The wet chemical analysis involves identifying and quantifying the desired elements present in a liquid sample using several measuring techniques. With chemical reagents, the analyte converts a dye proportionally, which can be read-out visually or photometrically.
Mass Spectrometry
Mass spectrometry (MS) is an analytical tool used to identify compounds, determine chemical structure, and assess isotopic abundance. In MS, samples are ionized, and the resulting ions are identified based on their mass-to-charge (m/z) ratios.
Sample Preparation by Filtration
Filtration separates substances based on physical or chemical properties. Lab samples are routinely filtered before analysis to reduce sample complexity and increase analyte purity. This allows scientists to generate higher quality analytical results.
Sample Purification
A range of purification methods may be used to prepare a sample prior to downstream analysis. Purification reduces sample complexity, removes interferences, and concentrates an analyte before analysis.
QuEChERS Sample Preparation Method
QuEChERS is a ‘Quick, Easy, Cheap, Effective, Rugged and Safe’ sample cleanup method used to prepare food and agricultural samples for pesticides analysis. QuEChERS is based on the Solid Phase Extraction (SPE) technique of sample preparation.
Sign In To Continue

To continue reading please sign in or create an account.

Don't Have An Account?