Overview: HPLC/FPLC Buffers
Chromatography is the separation and purification of biomolecules/components in a mixture based on their unique molecular characteristics. This separation occurs based on size, shape, charge, presence of hydrophobic groups on the surface, and their binding affinity with matrices comprising the stationary phase. This process analyzes differential interactions of each component in a fluid solvent (liquid or gaseous) called the mobile phase. The components are then carried in this phase through a system, typically a column, capillary tube, or sheet, on a material comprising a stationary phase. Components are separated based on their affinities for each of the mobile and stationary phases, resulting in the separation and purification of components of mixtures based on their physical characteristics. Chromatography can be used in a variety of different scientific applications, and the type of chromatography used generally corresponds directly to the analyte in question
High Performance Liquid Chromatography (HPLC) and Fast Protein Liquid Chromatography (FPLC) are two very prevalent types of liquid chromatography techniques used within the biological sciences and each have different applications and requirements.
Image 1: The mechanism of high-performance liquid chromatography is a series of steps involving a mobile phase and stationary phase to separate analytes of interest.
High Performance Liquid Chromatography (HPLC) and Fast Protein Liquid Chromatography (FPLC) are two very prevalent types of liquid chromatography techniques used within the biological sciences and each have different applications and requirements.
HPLC is often used for separation, identification, and quantification of components of complex organic samples. HPLC Â analyzes small molecules up to 3,000 Daltons, and works with high pressure, high temperature, and organic solvents [1]. It also often uses silica beads with small particle and pore sizes in its stationary phase, aiming to identify, characterize, and quantify analytes.
Reversed-Phase HPLC (RP-HPLC) has become the common form of HPLC, which employs partition equilibrium between the non-polar (hydrophobic) stationary phase and the polar mobile phase, with the higher polarity of mobile phase than the stationary phase. It is used for the purification and analysis of peptides.
FPLC on the other hand operates at low pressure, ambient temperature and uses aqueous buffers to preserve the structure and function of biomolecules [2]. This chromatography type also uses polymer materials such as agarose to produce larger pore sizes in the stationary phase that are specialized for purifying larger biomolecules such as nucleotides, proteins and peptides of thousands of Daltons in size. FPLC can also be used to separate and analyze carbohydrates, from complex mixtures, and for isolating and purifying enzymes, thus enabling researchers to study their structures, biological functions, and applications. FPLC aims to obtain native organic product with a high purity.
Applications of HPLC & FPLC
HPLC & FPLC are both very prevalent liquid chromatography techniques in biological sciences and each support several application types. Such application types of HPLC & FPLC and include:
| HPLC | FPLC |
|---|---|
| Drug development in pharma/biopharma | Protein purification in biotechnology and biological sciences research |
| Precision medicine and gene therapy in proteomics/genomic research | Purification of antibodies for diagnostic and therapeutic purposes |
| Characterizing and sequencing peptides, proteins, and nucleic acids | Separation of plasmid DNA for gene delivery and cloning |
| Monitoring quality control of biopharmaceutical products | Isolation and purification of recombinant proteins for enzyme replacement therapy |
| Analyzing biomarkers for disease diagnosis and prognosis | Fractionation of cell organelles for academic research |
Importance of Buffers in HPLC & FPLC
By regulating the pH of mobile phases, buffers help in controlling the retention and separation of analytes within chromatography workflows. pH plays a vital role in biological processes and can ensure both the stability and degradation of many biomolecules. Maintaining pH also maintains the ionic strength and polarity of the mobile phase, which in turn plays a role in influencing interactions between analytes and column materials.
Buffers and reagents made with high purity materials are critical for chromatography columns and must be correctly formulated according to robust requirements to yield reproducible chromatography results. High purity reagents minimize the risk of particulates that could clog chromatography columns, and ruin results. Some buffers are also preferred over others based on their compatibility with Mass Spectroscopy, another analytical technique that can be used following chromatography to analyze the mass of compounds. Although buffers can vary based on the column, analyte, chromatography practice, and further analytics, some of the most common buffers used in HPLC & FPLC can be seen below.
| Buffer | Description |
|---|---|
| Ammonium Bicarbonate Buffer | A weak base buffer with a pKa of 6.4, suitable for neutral to slightly alkaline pH applications and for separating basic analytes. It can be used with polymer columns. It is also compatible with MS detection. |
| Acetate Buffer | A weak acid buffer with a pKa of 4.7, suitable for low pH applications and for separating acidic analytes. It can be used with both silica and polymer columns. |
| Bis-Tris Propane Buffer | A zwitterionic buffer with a pKa around 6.8, ideal for biological and biochemical applications. Known for its stability and minimal interference with biological processes. |
| Borate Buffer | A buffer containing boric acid and its conjugate base, suitable for electrophoresis and enzymatic reactions. Borate buffers are often used in the separation of negatively charged molecules. |
| Citrate Buffer | A weak acid buffer with a pKa around 3.1, commonly used in molecular biology and biochemistry. It is effective for antigen retrieval in immunohistochemistry and as a buffering agent in enzymatic reactions. |
| Formate Buffer | A weak acid buffer with a pKa of 3.8, suitable for low pH applications and for separating acidic analytes. It can be used with both silica and polymer columns. It is also compatible with mass spectrometry (MS) detection. |
| HEPES Buffer | A zwitterionic buffer with a pKa of approximately 7.5 that ensures stable pH conditions during chromatographic separations, enhancing precision in bioanalytical and pharmaceutical applications. |
| MOPS Buffer | A zwitterionic buffer with a pKa around 7.2, MOPS Buffer excels in delivering constant pH levels, optimizing separation, and resolution in chromatographic processes. It provides stable pH conditions and is suitable for various biological experiments. |
| Phosphate Buffer | A weak base buffer with a pKa of 7.2, suitable for neutral to slightly alkaline pH applications and for separating basic analytes. It can be used with both silica and polymer columns. |
| Triethylammonium Acetate Buffers | A volatile buffer suitable for high-performance liquid chromatography (HPLC) and mass spectrometry. Used for the separation of various analytes, particularly in the field of proteomics. |
| Triethylammonium Bicarbonate Buffers | A buffer often employed in protein chemistry and mass spectrometry, with a pKa around 8.4. It is volatile and compatible with downstream analyses. |
| Triethylammonium Phosphate Buffers | A buffer suitable for ion-exchange chromatography and protein purification. It is particularly useful for applications involving proteins and peptides. |
| Tris Buffer | A weak base buffer with a pKa of 8.1, suitable for alkaline pH applications and for separating proteins and nucleic acids. It can be used with polymer columns. |
HPLC/FPLC Buffers at Boston BioProducts
Every HPLC and FPLC buffer is unique to the analyte used and the experimental application. Select the appropriate HPLC or FPLC Buffer from the catalog or design your optimal formulation with custom manufacturing options at Boston BioProducts.
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References:
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- Hamilton, R. J., & Sewell, P. A. (1982). Introduction to high performance liquid chromatography. Springer.
- Madadlou, A., O’Sullivan, S., & Sheehan, D. (2016). Fast protein liquid chromatography. In R. J. Beynon & J. S. Bond (Eds.), Protein chromatography: Principles and practice(pp. 327-338). Springer.