Introduction
Brief overview of HPLC and its importance in various fields of research
High-performance liquid chromatography (HPLC) is a powerful analytical technique used to separate, identify, and quantify the components of complex mixtures. It is widely used in various fields of research, including pharmaceuticals, environmental analysis, food and beverage analysis, forensic science, and more.
HPLC works by pumping a liquid sample through a column packed with a stationary phase material, which separates the components of the sample based on their physicochemical properties, such as size, charge, and hydrophobicity. The separated components are then detected and analyzed using various types of detectors, such as UV-Vis spectrophotometers, mass spectrometers, or fluorescence detectors.
The importance of HPLC in research cannot be overstated. In the pharmaceutical industry, HPLC is used to identify and quantify active pharmaceutical ingredients and impurities in drug formulations, ensuring their safety and efficacy. In environmental analysis, HPLC is used to detect and quantify various contaminants in water, soil, and air samples, helping to monitor and protect the environment. In food and beverage analysis, HPLC is used to detect and quantify nutrients, additives, and contaminants, ensuring the safety and quality of the food supply. In forensic science, HPLC is used to identify and quantify drugs of abuse and other compounds in biological samples, aiding in criminal investigations.
Explanation of HPLC columns and their role in separating complex mixtures
What is HPLC column?
An HPLC column is a critical component of a high-performance liquid chromatography (HPLC) system. It is a cylindrical tube packed with a stationary phase material that separates the components of a liquid sample as it flows through the column. The stationary phase material is typically made of a porous material, such as silica or polymer, which is coated with a thin layer of a specific chemical composition.
The separation of the sample occurs due to the interaction of the components with the stationary phase material. The components with stronger interactions with the stationary phase material will take longer to travel through the column, while the weaker interacting components will move through the column more quickly. This difference in travel time results in the separation of the components of the sample, which can be further detected and analyzed using a detector.
HPLC columns come in various sizes, shapes, and chemistries, allowing for the separation of a wide range of compounds. The selection of the appropriate column depends on the nature of the sample and the analytical goals. The correct choice of column is essential to achieve the desired separation and maximize the sensitivity and specificity of the analysis. The use of high-quality HPLC columns is critical to ensure the accuracy and precision of the analytical results in various fields, including pharmaceuticals, environmental analysis, food and beverage analysis, and more.
HPLC column and how it separates complex Mixtures
High-performance liquid chromatography (HPLC) is a powerful analytical technique used to separate, identify, and quantify the components of complex mixtures. It is widely used in various fields of research, including pharmaceuticals, environmental analysis, food and beverage analysis, forensic science, and more.
HPLC works by pumping a liquid sample through a column packed with a stationary phase material, which separates the components of the sample based on their physicochemical properties, such as size, charge, and hydrophobicity. The separated components are then detected and analyzed using various types of detectors, such as UV-Vis spectrophotometers, mass spectrometers, or fluorescence detectors.
The importance of HPLC in research cannot be overstated. In the pharmaceutical industry, HPLC is used to identify and quantify active pharmaceutical ingredients and impurities in drug formulations, ensuring their safety and efficacy. In environmental analysis, HPLC is used to detect and quantify various contaminants in water, soil, and air samples, helping to monitor and protect the environment. In food and beverage analysis, HPLC is used to detect and quantify nutrients, additives, and contaminants, ensuring the safety and quality of the food supply. In forensic science, HPLC is used to identify and quantify drugs of abuse and other compounds in biological samples, aiding in criminal investigations.
Overall, HPLC is a critical tool for researchers across various fields, providing them with the ability to separate and analyze complex mixtures with high sensitivity and specificity.
Waters Corporation
Brief history and background of Waters Corporation
Waters Corporation is a leading provider of analytical instruments, software, and services for the life sciences and other industries. The company was founded in 1958 by James L. Waters, a chemist and entrepreneur who developed the first high-performance liquid chromatography (HPLC) system.
James Waters’ invention of the HPLC system revolutionized the field of analytical chemistry, allowing for the separation and analysis of complex mixtures with higher sensitivity and resolution than previously possible. The first HPLC system was developed in a garage in Framingham, Massachusetts, and was initially used by Waters and his colleagues for their own research.
As the demand for HPLC systems grew, Waters Corporation was officially founded in 1958, and the company began manufacturing and selling HPLC systems to researchers and scientists in various fields. In the 1970s, Waters Corporation introduced the first mass spectrometer compatible with HPLC, further expanding the company’s product line and capabilities.
Over the years, Waters Corporation has continued to innovate and develop new technologies, including ultraperformance liquid chromatography (UPLC), mass spectrometry, and informatics software. Today, the company serves customers in the pharmaceuticals, environmental, food and beverage, clinical, and academic markets, among others.
Waters Corporation is headquartered in Milford, Massachusetts, and has operations in more than 30 countries worldwide. The company has received numerous awards for its innovative products and services and continues to be a leader in the analytical instruments industry.
Waters HPLC columns and their features
Waters Corporation offers a range of HPLC columns designed to meet the specific analytical needs of various industries and applications. Here are some of the features and benefits of Waters HPLC columns:
- Broad selection: Waters offers a comprehensive selection of HPLC columns, including reverse-phase, normal-phase, ion-exchange, size exclusion, and mixed-mode columns. This allows customers to choose the most appropriate column for their specific application and analytical needs.
- High-quality materials: Waters HPLC columns are made of high-quality materials, such as silica or polymer-based resins, that are optimized for chromatographic separations. The columns are designed to withstand high pressure and flow rates, making them suitable for use with a variety of HPLC systems.
- Consistent performance: Waters HPLC columns are manufactured under strict quality control standards to ensure consistency and reproducibility of analytical results. Each column is rigorously tested for performance and batch-to-batch variation, ensuring consistent performance over time.
- Wide compatibility: Waters HPLC columns are compatible with a wide range of detection methods, including UV-Vis, mass spectrometry, fluorescence, and electrochemical detection. This allows customers to choose the most appropriate detection method for their specific application.
- Specialized chemistries: Waters offers a range of specialized chemistries, such as C18, C8, and phenyl phases, for reversed-phase chromatography. These chemistries allow for the separation of a wide range of compounds, from hydrophilic to hydrophobic, and can be used for a variety of applications, including pharmaceuticals, environmental analysis, and food and beverage analysis.
- Application-specific columns: Waters offers HPLC columns designed specifically for certain applications, such as the BEH Amide column for glycan analysis and the XSelect Peptide CSH column for peptide separations. These columns provide superior performance for specific applications and can help to streamline the analytical process.
Types of Waters HPLC Columns
Waters Corporation offers a wide range of HPLC columns designed for different applications and chromatographic separations. Some of the most commonly used types of Waters HPLC columns include:
- Reversed-phase columns: Reversed-phase columns are the most commonly used type of HPLC column and are designed to separate nonpolar or hydrophobic compounds. Waters offers a range of reversed-phase columns with different bonded phases, such as C18, C8, and phenyl, to achieve different selectivities and separation efficiencies.
- Normal-phase columns: Normal-phase columns are used to separate polar or hydrophilic compounds based on differences in their polarity. Waters offers normal-phase columns with silica or diol-based stationary phases, which can provide high resolution and selectivity for certain applications.
- Ion-exchange columns: Ion-exchange columns are designed to separate charged molecules, such as proteins or peptides, based on their net charge. Waters offers ion-exchange columns with both anion- and cation-exchange chemistries, which can provide high selectivity and resolution for these types of molecules.
- Size-exclusion columns: Size-exclusion columns are used to separate molecules based on their size or molecular weight. Waters offers size-exclusion columns with different pore sizes, which can be used to separate a wide range of molecules, from small peptides to large proteins and polymers.
- Chiral columns: Chiral columns are designed to separate enantiomers, which are stereoisomers that are mirror images of each other. Waters offers chiral columns with different chiral stationary phases, which can provide high selectivity and resolution for certain chiral compounds.
- Mixed-mode columns: Mixed-mode columns are designed to separate compounds based on both hydrophobic and ionic interactions. Waters offers mixed-mode columns with both reversed-phase and ion-exchange chemistries, which can provide high selectivity and resolution for certain complex samples.
Overall, the selection of the appropriate column depends on the nature of the sample, the analytical goals, and the desired separation mechanism. The wide range of Waters HPLC columns allows customers to choose the most appropriate column for their specific application and analytical needs.
Waters Column type,Selectivity and Application
| olumn Name | Particle Size (µm) | Pore Size (Å) | Bonded Phase | Selectivity | Applications |
|---|---|---|---|---|---|
| Symmetry C18 | 3.5 | 100 | C18 | Reversed-phase | Pharmaceuticals, natural products, environmental analysis |
| SymmetryShield RP18 | 3.5 | 100 | C18 | Reversed-phase | Pharmaceuticals, natural products, food and beverage analysis |
| XBridge BEH C18 | 3.5 | 130 | C18 | Reversed-phase | Pharmaceuticals, peptides, proteins, natural products, environmental analysis |
| XBridge Phenyl | 3.5 | 130 | Phenyl | Reversed-phase | Pharmaceuticals, peptides, proteins, natural products |
| Atlantis dC18 | 3 | 100 | C18 | Reversed-phase | Pharmaceuticals, natural products, environmental analysis |
| Atlantis T3 | 3 | 100 | C18 | Reversed-phase | Polar and nonpolar analytes, natural products |
| Acquity BEH C18 | 1.7 | 130 | C18 | Reversed-phase | Pharmaceuticals, peptides, proteins, natural products, environmental analysis |
| Acquity HSS T3 | 1.8 | 100 | C18 | Reversed-phase | Polar and nonpolar analytes, natural products |
| XSelect CSH C18 | 3.5 | 130 | C18 | Reversed-phase | Peptides, proteins |
| XSelect HSS T3 | 3.5 | 100 | C18 | Reversed-phase | Polar and nonpolar analytes |
| Ion-Pac AS11-HC | 4 | N/A | Weak anion-exchange | Ion-exchange | Organic acids, amino acids, nucleotides |
| Ion-Pac AS7 | 9 | N/A | Strong anion-exchange | Ion-exchange | Inorganic anions, oxyhalides |
| Nova-Pak Silica | 4 | 100 | Silica | Normal-phase | Polar and hydrophilic compounds |
| Nova-Pak C18 | 4 | 80 | C18 | Reversed-phase/normal-phase | Pharmaceuticals, environmental analysis, natural products |
It’s worth noting that this is just a small sample of the many columns available from Waters, and the suitability of a particular column for a specific application depends on the specific sample and separation requirements.
xbridge c18 column
XBridge C18 is a reversed-phase HPLC column offered by Waters Corporation. It is a high-performance column designed for the separation of a wide range of compounds, including small molecules, peptides, and proteins. The XBridge C18 column features a 100% aqueous-stable hybrid silica-based material that provides a high surface area, high efficiency, and low bleed. The particle size is available in 3.5 µm, 2.5 µm, and 5 µm, with a pore size of 130 Å.
The XBridge C18 column provides excellent retention and resolution for a wide range of compounds, making it a popular choice for many applications. Its high stability and low column bleed make it ideal for sensitive applications such as proteomics and metabolomics. It can be used for a variety of applications such as pharmaceuticals, natural products, environmental analysis, and food and beverage analysis.
Overall, the XBridge C18 column is a reliable and versatile column that can provide high-quality separations for a wide range of analytes, making it a popular choice for many HPLC applications.
Selectivity,Application details of XBridge C18 Column
| XBridge C18 Column | Selectivity | Applications |
|---|---|---|
| XBridge C18 3.5 μm | High hydrophobic selectivity | Pharmaceuticals, natural products, environmental analysis, food and beverage analysis |
| XBridge C18 2.5 μm | High efficiency and high hydrophobic selectivity | Peptides, proteins, and small molecules |
| XBridge C18 5 μm | General purpose column with moderate hydrophobic selectivity | Pharmaceuticals, natural products, environmental analysis, food and beverage analysis |
The XBridge C18 column is a high-performance reversed-phase HPLC column designed for the separation of a wide range of compounds. Like the SunFire C18 column, it is also available in 3.5 µm, 2.5 µm, and 5 µm particle sizes, each with unique selectivity and applications.
The XBridge C18 3.5 µm column offers high hydrophobic selectivity and is suitable for the separation of pharmaceuticals, natural products, environmental analysis, and food and beverage analysis. It is a reliable and versatile column that can provide high-quality separations for a wide range of analytes.
The XBridge C18 2.5 µm column offers both high efficiency and high hydrophobic selectivity, making it suitable for the separation of peptides, proteins, and small molecules. Its high efficiency makes it ideal for high-throughput applications, while its selectivity provides excellent peak shape and retention for hydrophobic compounds.
The XBridge C18 5 µm column is a general-purpose column with moderate hydrophobic selectivity, suitable for a wide range of applications, including pharmaceuticals, natural products, environmental analysis, and food and beverage analysis. Its moderate selectivity provides good retention and peak shape for a wide range of analytes.
Overall, the XBridge C18 column is a reliable and high-performance column that can provide high-quality separations for a wide range of analytes, making it a popular choice for many HPLC applications.
Spherisorb
Spherisorb is a brand of HPLC columns offered by Waters Corporation. Spherisorb columns are known for their high efficiency and excellent peak shapes, making them popular for a variety of HPLC applications. The Spherisorb columns use a silica-based material with a unique bonding technology that provides high stability and reproducibility.
Spherisorb columns are available in a range of bonded phases, including C18, C8, Phenyl, NH2, and Cyano. The particle size options include 3 µm, 5 µm, and 10 µm, with a pore size of 80 Å or 100 Å. The Spherisorb columns are compatible with both aqueous and organic solvents, making them ideal for both normal and reversed-phase chromatography.
The Spherisorb columns are widely used for a variety of applications, such as pharmaceuticals, natural products, environmental analysis, and food and beverage analysis. The C18 and C8 phases are the most commonly used for reversed-phase HPLC separations of small molecules, peptides, and proteins. The Phenyl and Cyano phases are often used for separations of aromatic compounds or polar analytes, respectively. The NH2 phase is useful for separation of polar compounds that may not retain well on traditional reversed-phase columns.
Overall, Spherisorb columns are reliable, high-performance HPLC columns that can provide excellent separations for a variety of analytes. The wide range of bonded phases and particle sizes make them a versatile choice for many applications.
sunfire c18 column
SunFire C18 is a reversed-phase HPLC column offered by Waters Corporation. It is a high-performance column designed for the separation of a wide range of compounds, including small molecules, peptides, and proteins. The SunFire C18 column features a silica-based material with a low level of metal impurities, which ensures high column stability and reproducibility. The particle size is available in 3.5 µm, 2.5 µm, and 5 µm, with a pore size of 130 Å.
The SunFire C18 column provides excellent retention and resolution for a wide range of compounds, making it a popular choice for many applications. Its high stability and reproducibility make it ideal for use in method development and validation, as well as routine analysis. The SunFire C18 column can be used for a variety of applications such as pharmaceuticals, natural products, environmental analysis, and food and beverage analysis.
One of the unique features of the SunFire C18 column is its use of Waters’ patented BEH (bridged ethyl hybrid) particle technology. This technology provides a more stable and reproducible silica-based material with a higher surface area and lower pore size distribution. This results in higher efficiency and better peak shape compared to traditional C18 columns.
Overall, the SunFire C18 column is a reliable and high-performance column that can provide high-quality separations for a wide range of analytes, making it a popular choice for many HPLC applications.
Selectivity,Application details of SunFire C18 Column
| SunFire C18 Column | Selectivity | Applications |
|---|---|---|
| SunFire C18 3.5 μm | High hydrophobic selectivity | Pharmaceuticals, natural products, environmental analysis, food and beverage analysis |
| SunFire C18 2.5 μm | High efficiency and high hydrophobic selectivity | Peptides, proteins, and small molecules |
| SunFire C18 5 μm | General purpose column with moderate hydrophobic selectivity | Pharmaceuticals, natural products, environmental analysis, food and beverage analysis |
The SunFire C18 column is a high-performance reversed-phase HPLC column designed for the separation of a wide range of compounds. The column is available in 3.5 µm, 2.5 µm, and 5 µm particle sizes, each with unique selectivity and applications.
The SunFire C18 3.5 µm column offers high hydrophobic selectivity and is suitable for the separation of pharmaceuticals, natural products, environmental analysis, and food and beverage analysis. It is a reliable and versatile column that can provide high-quality separations for a wide range of analytes.
The SunFire C18 2.5 µm column offers both high efficiency and high hydrophobic selectivity, making it suitable for the separation of peptides, proteins, and small molecules. Its high efficiency makes it ideal for high-throughput applications, while its selectivity provides excellent peak shape and retention for hydrophobic compounds.
The SunFire C18 5 µm column is a general-purpose column with moderate hydrophobic selectivity, suitable for a wide range of applications, including pharmaceuticals, natural products, environmental analysis, and food and beverage analysis. Its moderate selectivity provides good retention and peak shape for a wide range of analytes.
Overall, the SunFire C18 column is a reliable and high-performance column that can provide high-quality separations for a wide range of analytes, making it a popular choice for many HPLC applications.
Symmetry Columns
Symmetry columns are another line of HPLC columns manufactured by Waters Corporation. These columns are designed to provide high-resolution separations with excellent peak shape and efficiency. The Symmetry column family includes reversed-phase, normal-phase, and HILIC columns, each with unique selectivity and applications.
Reversed-phase Symmetry columns are based on a hybrid packing material that combines the properties of silica and polymer. The SymmetryShield RP18 column is a popular choice for the analysis of small molecules in pharmaceuticals, chemicals, and environmental samples. It provides excellent peak shape and resolution for a wide range of analytes. Other reversed-phase Symmetry columns include Symmetry C18 and Symmetry C8.
Normal-phase Symmetry columns use bare silica as the stationary phase and are used for the separation of non-polar and moderately polar compounds. The SymmetrySilica column is a popular choice for the separation of polar and non-polar analytes, including fatty acids, carbohydrates, and steroids. It provides high-resolution separations with excellent peak shape.
HILIC Symmetry columns use hydrophilic interaction chromatography (HILIC) to separate polar and hydrophilic compounds. The SymmetryHydrophilic Interaction Chromatography (HILIC) column is a popular choice for the analysis of polar compounds in biological and environmental samples. It provides excellent peak shape and retention for a wide range of analytes.
Overall, the Symmetry column family offers a range of selectivities and applications, making it a versatile choice for a variety of HPLC separations. Whether analyzing small molecules or biomolecules, there is likely a Symmetry column that can provide the resolution and peak shape required for the analysis.
Symetry column details,selectivity and applications
| Symmetry Column | Selectivity | Applications |
|---|---|---|
| SymmetryShield RP18 | High hydrophobic selectivity | Pharmaceuticals, chemicals, environmental samples |
| Symmetry C18 | Moderate hydrophobic selectivity | Pharmaceuticals, chemicals, environmental samples |
| Symmetry C8 | Lower hydrophobic selectivity than C18 | Polar analytes |
| SymmetrySilica | Normal-phase separation | Fatty acids, carbohydrates, steroids |
| SymmetryHydrophilic Interaction Chromatography (HILIC) | HILIC separation | Polar compounds, biological and environmental samples |
As mentioned earlier, the Symmetry column family includes reversed-phase, normal-phase, and HILIC columns, each with unique selectivity and applications.
The reversed-phase SymmetryShield RP18 column offers high hydrophobic selectivity and is suitable for the separation of pharmaceuticals, chemicals, and environmental samples. It provides excellent peak shape and resolution for a wide range of analytes.
The reversed-phase Symmetry C18 column offers moderate hydrophobic selectivity and is also suitable for the separation of pharmaceuticals, chemicals, and environmental samples. It provides good retention and peak shape for a wide range of analytes.
The reversed-phase Symmetry C8 column has lower hydrophobic selectivity than C18 and is more suitable for polar analytes. It provides good retention and peak shape for polar analytes and is a popular choice for the separation of peptides and proteins.
The normal-phase SymmetrySilica column uses bare silica as the stationary phase and is used for the separation of non-polar and moderately polar compounds. It provides high-resolution separations with excellent peak shape and is a popular choice for the analysis of fatty acids, carbohydrates, and steroids.
The HILIC SymmetryHydrophilic Interaction Chromatography (HILIC) column uses HILIC to separate polar and hydrophilic compounds. It provides excellent peak shape and retention for a wide range of polar compounds and is a popular choice for the analysis of polar compounds in biological and environmental samples.
Atlantis Columns
Atlantis columns are a line of HPLC columns manufactured by Waters Corporation. They are designed to provide high-resolution separations with excellent peak shape and efficiency for a wide range of applications. The Atlantis column family includes reversed-phase, normal-phase, and HILIC columns, each with unique selectivity and applications.
Reversed-phase Atlantis columns are based on a hybrid packing material that combines the properties of silica and polymer. The Atlantis T3 column is a popular choice for the analysis of small molecules in pharmaceuticals, chemicals, and environmental samples. It provides excellent peak shape and resolution for a wide range of analytes, including basic compounds. Other reversed-phase Atlantis columns include Atlantis dC18, Atlantis HILIC Silica, and Atlantis Silica.
Normal-phase Atlantis columns use bare silica as the stationary phase and are used for the separation of non-polar and moderately polar compounds. The Atlantis Silica column is a popular choice for the separation of fatty acids, natural products, and complex mixtures. It provides high-resolution separations with excellent peak shape.
HILIC Atlantis columns use hydrophilic interaction chromatography (HILIC) to separate polar and hydrophilic compounds. The Atlantis HILIC column is a popular choice for the analysis of polar compounds in biological and environmental samples. It provides excellent peak shape and retention for a wide range of analytes.
Overall, the Atlantis column family offers a range of selectivities and applications, making it a versatile choice for a variety of HPLC separations. Whether analyzing small molecules or biomolecules, there is likely an Atlantis column that can provide the resolution and peak shape required for the analysis.
Selectivity,details and Application of Atlantis Column
| Atlantis Column | Selectivity | Applications |
|---|---|---|
| Atlantis T3 | C18-like, with enhanced selectivity for basic compounds | Pharmaceuticals, chemicals, environmental samples |
| Atlantis dC18 | Enhanced retention for polar compounds | Pharmaceuticals, chemicals, environmental samples |
| Atlantis HILIC Silica | HILIC separation with bare silica stationary phase | Polar compounds, biological and environmental samples |
| Atlantis Silica | Normal-phase separation with bare silica stationary phase | Fatty acids, natural products, complex mixtures |
The Atlantis column family includes reversed-phase, normal-phase, and HILIC columns, each with unique selectivity and applications.
The reversed-phase Atlantis T3 column provides C18-like selectivity, but with enhanced selectivity for basic compounds. It is a popular choice for the analysis of pharmaceuticals, chemicals, and environmental samples, providing excellent peak shape and resolution for a wide range of analytes.
The reversed-phase Atlantis dC18 column provides enhanced retention for polar compounds, making it a good choice for the analysis of compounds that are difficult to retain on traditional C18 columns. It is also a popular choice for the analysis of pharmaceuticals, chemicals, and environmental samples.
The HILIC Atlantis HILIC Silica column uses hydrophilic interaction chromatography (HILIC) to separate polar and hydrophilic compounds. It provides excellent peak shape and retention for a wide range of polar compounds, making it a popular choice for the analysis of polar compounds in biological and environmental samples.
The normal-phase Atlantis Silica column uses bare silica as the stationary phase and is used for the separation of non-polar and moderately polar compounds. It provides high-resolution separations with excellent peak shape and is a popular choice for the analysis of fatty acids, natural products, and complex mixtures.
Acquity UPLC Columns
Acquity UPLC (Ultra Performance Liquid Chromatography) is a line of columns and instruments manufactured by Waters Corporation. These columns are designed to operate at higher pressures and provide faster, higher-resolution separations than traditional HPLC columns. The Acquity UPLC column family includes reversed-phase, normal-phase, and HILIC columns, each with unique selectivity and applications.
Reversed-phase Acquity UPLC columns are designed for the separation of small molecules, peptides, and proteins. They are based on a hybrid packing material that combines the properties of silica and polymer. The C18 column is the most popular choice for reversed-phase separations, offering high resolution and reproducibility for a wide range of analytes. Other reversed-phase Acquity UPLC columns include C8, CSH C18, and BEH Shield RP18.
Normal-phase Acquity UPLC columns are used for the separation of non-polar and moderately polar compounds. They are based on a bare silica stationary phase and offer high resolution and selectivity for the separation of positional isomers and structural analogs. The HSS T3 column is a popular choice for the analysis of natural products, lipids, and metabolites. Other normal-phase Acquity UPLC columns include HSS C18, HSS PFP, and HSS Cyano.
HILIC Acquity UPLC columns use hydrophilic interaction chromatography (HILIC) to separate polar and hydrophilic compounds. They offer high-resolution separations with excellent peak shape and retention for polar compounds. The BEH Amide column is a popular choice for the analysis of peptides, oligosaccharides, and glycopeptides. Other HILIC Acquity UPLC columns include BEH HILIC, BEH Amide, and HILIC Silica.
Overall, the Acquity UPLC column family offers a range of selectivities and applications, making it a versatile choice for a variety of HPLC separations. The high performance and resolution of these columns make them particularly well-suited for challenging applications, such as the analysis of complex mixtures and low-abundance compounds.
Selectivity,details and Application of Acquity UPLC column
| Column Type | Selectivity | Applications |
|---|---|---|
| Reversed-Phase C18 | Hydrophobicity | Small molecules, peptides, and proteins |
| Reversed-Phase C8 | Lower hydrophobicity than C18 | Small molecules, peptides, and proteins |
| Reversed-Phase CSH C18 | Hybrid of silica and polymer | High resolution and selectivity for small molecules, peptides, and proteins |
| Reversed-Phase BEH Shield RP18 | Unique bonding chemistry | High resolution and selectivity for small molecules, peptides, and proteins |
| Normal-Phase HSS T3 | Bare silica stationary phase | Non-polar and moderately polar compounds, including natural products, lipids, and metabolites |
| Normal-Phase HSS C18 | C18 bonded phase | Non-polar and moderately polar compounds |
| Normal-Phase HSS PFP | Pentafluorophenyl stationary phase | Non-polar and moderately polar compounds |
| Normal-Phase HSS Cyano | Cyanopropyl stationary phase | Non-polar and moderately polar compounds |
| HILIC BEH Amide | Amide-bonded stationary phase | Polar and hydrophilic compounds, including peptides, oligosaccharides, and glycopeptides |
| HILIC BEH HILIC | Zwitterionic stationary phase | Polar and hydrophilic compounds |
| HILIC HILIC Silica | Bare silica stationary phase | Polar and hydrophilic compounds |
It’s important to note that these columns are not limited to the applications listed above and can be used for a wide range of separations. The selectivity and applications listed in the table are meant to provide a general overview of the capabilities of each column type.
Choosing the Right Waters HPLC Column
Choosing the right Waters HPLC column is essential to achieving optimal separation and analysis of your target compounds. There are several factors to consider when selecting a column, including the sample type and properties, analyte polarity and hydrophobicity, column dimensions and packing material, and mobile phase composition.
Sample type and properties:
The first step in selecting a column is to consider the sample type and properties. Is it a complex matrix or a simple mixture? Is it a small molecule or a large biomolecule? The sample matrix can contain impurities and interferents that can negatively impact separation and detection. It’s important to choose a column that can effectively separate the target analyte from the matrix components.
Analyte polarity and hydrophobicity:
The polarity and hydrophobicity of the analyte can also impact column selection. Polar compounds tend to interact strongly with polar stationary phases, while non-polar compounds tend to interact with non-polar stationary phases. The degree of hydrophobicity can also affect retention time and peak shape. It’s important to choose a column with the appropriate selectivity and retention characteristics for the target analyte.
Column dimensions and packing material:
Column dimensions and packing material can also play a role in column selection. Column length and diameter can impact resolution, retention, and flow rate. Smaller particle size packing material can improve efficiency and resolution but may require higher pressures to operate. It’s important to choose a column that meets your specific separation requirements while taking into account the limitations of your chromatography system.
Mobile phase composition:
The mobile phase composition can also influence column selection. The mobile phase should be chosen based on the analyte’s properties, including polarity and hydrophobicity. The pH of the mobile phase can also affect separation and should be chosen based on the pKa of the analyte. The choice of solvent can impact the solubility and retention of the analyte, and the addition of additives such as salts or organic modifiers can improve separation.
In summary, selecting the right Waters HPLC column requires careful consideration of several factors, including the sample type and properties, analyte polarity and hydrophobicity, column dimensions and packing material, and mobile phase composition. By taking into account these factors, you can optimize your separation and achieve accurate and precise results.
Maintenance and Care of Waters HPLC Columns
Proper maintenance and care of Waters HPLC columns is essential for achieving consistent and reliable performance. Here are some key practices to follow:
- Proper storage: Store columns in a cool and dry place to prevent degradation of the stationary phase. If possible, store columns in their original packaging to protect them from dust and contamination.
- Column equilibration: Before use, equilibrate the column with the mobile phase to ensure that it is ready for analysis. The amount of time required for equilibration will depend on the column type and mobile phase composition.
- Column flushing: After each use, flush the column with a solvent to remove any remaining sample or mobile phase components. This will help prevent buildup of contaminants and extend column lifetime.
- Mobile phase preparation: Use high-quality solvents and water for mobile phase preparation to prevent contamination of the column. Filter mobile phase solutions to remove any particulates.
- Backflushing: For some columns, backflushing with an appropriate solvent can help remove any retained contaminants and extend column lifetime.
- Column regeneration: If the column performance begins to degrade, regeneration may be possible. Consult the column manual for specific instructions on how to regenerate the column.
- Regular column replacement: HPLC columns have a limited lifespan, and regular replacement is necessary to ensure optimal performance. The frequency of replacement will depend on factors such as sample matrix complexity and column usage.
Applications of Waters HPLC Columns
Waters HPLC columns have a wide range of applications in various fields of research, including pharmaceuticals, biotechnology, environmental analysis, food and beverage analysis, and more. In this article, we will explore some of the most common applications of Waters HPLC columns and the specific columns that are best suited for each application.
- Pharmaceuticals:
In the pharmaceutical industry, Waters HPLC columns are widely used for drug discovery and development, quality control, and regulatory compliance. The main applications of HPLC in the pharmaceutical industry are quantification of active pharmaceutical ingredients (APIs), impurity profiling, and formulation analysis. Some of the commonly used Waters HPLC columns for pharmaceutical applications are the Symmetry and XBridge C18 columns. These columns offer high selectivity and resolution for a wide range of pharmaceutical compounds.
- Biotechnology:
HPLC is widely used in biotechnology for the analysis of proteins, peptides, and other biomolecules. Waters HPLC columns such as the BEH C18 and BEH Shield RP18 are commonly used for protein and peptide analysis. These columns provide high resolution, reproducibility, and stability for the analysis of complex biomolecules.
- Environmental analysis:
Waters HPLC columns are also widely used for the analysis of environmental samples, such as water, soil, and air. The main applications of HPLC in environmental analysis are the determination of organic pollutants, such as pesticides, herbicides, and industrial chemicals. The SunFire C18 and XSelect CSH C18 columns are commonly used for environmental analysis due to their high sensitivity and selectivity for a wide range of environmental contaminants.
- Food and beverage analysis:
HPLC is widely used in the food and beverage industry for the analysis of food additives, flavorings, and contaminants. Waters HPLC columns such as the Atlantis T3 and XBridge BEH Amide columns are commonly used for food and beverage analysis due to their high selectivity, sensitivity, and reproducibility.
- Forensic analysis:
HPLC is also widely used in forensic analysis for the analysis of drugs of abuse, toxicological analysis, and trace analysis. Waters HPLC columns such as the Atlantis dC18 and XBridge BEH Phenyl columns are commonly used for forensic analysis due to their high sensitivity, selectivity, and reproducibility.
In conclusion, Waters HPLC columns have a wide range of applications in various fields of research, including pharmaceuticals, biotechnology, environmental analysis, food and beverage analysis, and forensic analysis. By choosing the right column for your specific application, you can achieve optimal separation, resolution, and sensitivity for your analytical needs.
FAQs of Waters HPLC columns
- Q: What is the recommended flow rate for Waters HPLC columns?
- A: The recommended flow rate for Waters HPLC columns depends on the specific column and application. It is generally in the range of 0.1-5 mL/min.
- Q: How should I store my Waters HPLC column?
- A: Waters HPLC columns should be stored in a cool and dry place, away from direct sunlight and sources of heat. They should be stored in their original packaging or in a column storage box.
- Q: Can I reuse my Waters HPLC column?
- A: In general, Waters HPLC columns are designed for single use only. Reusing a column can lead to decreased performance and unpredictable results.
- Q: How often should I replace my Waters HPLC column?
- A: The frequency of column replacement depends on the sample matrix and the number of samples analyzed. Generally, columns are replaced after 100-200 injections or when a decrease in performance is observed.
- Q: What is the difference between a C18 and C8 column?
- A: C18 columns have longer hydrocarbon chains than C8 columns, which leads to stronger hydrophobic interactions. C8 columns are less retentive than C18 columns and are better suited for separating moderately hydrophobic compounds.
- Q: Can I use a different brand of mobile phase with my Waters HPLC column?
- A: Yes, as long as the mobile phase meets the specifications provided by Waters and is compatible with the column material.
- Q: How do I choose the right Waters HPLC column for my application?
- A: The choice of column depends on the sample type and properties, analyte polarity and hydrophobicity, column dimensions and packing material, and mobile phase composition.
- Q: How do I prepare my Waters HPLC column for first-time use?
- A: Waters HPLC columns should be flushed with the mobile phase for at least 1 hour prior to first-time use to remove any residual manufacturing solvents.
- Q: Can I use organic solvents with my Waters HPLC column?
- A: Yes, but the solvent compatibility depends on the specific column material. Check the column specifications or contact Waters for guidance.
- Q: How should I clean my Waters HPLC column?
- A: Cleaning procedures vary depending on the type of contamination. Waters provides detailed instructions for column cleaning in their user manuals.
- Q: Can I use a guard column with my Waters HPLC column?
- A: Yes, a guard column can protect the analytical column from contamination and prolong its lifespan.
- Q: What is the temperature range for Waters HPLC columns?
- A: Waters HPLC columns can be used at temperatures ranging from 5-80°C, depending on the specific column and application.
- Q: Can I use a different particle size with my Waters HPLC column than the one specified?
- A: No, the particle size specified by Waters is critical for the column’s performance and should not be changed.
- Q: Can I use a Waters HPLC column for preparative chromatography?
- A: Yes, some Waters HPLC columns are suitable for preparative chromatography. Check the column specifications for details.
- Q: How do I troubleshoot my Waters HPLC column? A: Check the column manual and verify that the column is being used correctly. If the problem persists, contact Waters technical support.
- Can I reuse a Waters HPLC column?
- Answer: It is generally not recommended to reuse HPLC columns as they may become contaminated or damaged during use. It is more efficient and accurate to use a new column for each analysis.
- How do I properly store a Waters HPLC column?
- Answer: Store the Waters HPLC column in a clean, dry environment at a temperature recommended by the manufacturer. It is also important to protect the column from any physical damage or exposure to direct sunlight.
- How can I increase the lifespan of my Waters HPLC column?
- Answer: To increase the lifespan of your Waters HPLC column, follow proper maintenance procedures, use appropriate mobile phase conditions, and avoid exposing the column to harsh chemicals or extreme temperatures.
- How do I select the appropriate mobile phase for my Waters HPLC column?
- Answer: The appropriate mobile phase for a Waters HPLC column depends on the sample properties and the desired separation. Factors such as pH, buffer concentration, and solvent composition should be considered when selecting a mobile phase.
- Can I use a Waters HPLC column for reversed-phase chromatography?
- Answer: Yes, many Waters HPLC columns are suitable for reversed-phase chromatography. Check the product specifications to confirm the column’s suitability for this type of analysis.
- Can I use a Waters HPLC column for size exclusion chromatography?
- Answer: Yes, some Waters HPLC columns are specifically designed for size exclusion chromatography. Check the product specifications to confirm the column’s suitability for this type of analysis.
- How can I troubleshoot poor chromatography on my Waters HPLC column?
- Answer: Poor chromatography on a Waters HPLC column can be caused by a variety of factors, including sample preparation, column age, or mobile phase conditions. Refer to the product manual or contact Waters technical support for troubleshooting guidance.
- How do I properly dispose of a used Waters HPLC column?
- Answer: Used Waters HPLC columns should be disposed of according to local regulations for hazardous waste. Consult with your institution’s environmental health and safety department for specific disposal instructions.
- Can I modify a Waters HPLC column to change its selectivity?
- Answer: It is generally not recommended to modify Waters HPLC columns, as this can damage the column and affect its performance. Instead, select a column with the desired selectivity for your analysis.
- How can I determine the particle size of a Waters HPLC column?
- Answer: The particle size of a Waters HPLC column is typically specified in the product manual or on the product label. If this information is not readily available, contact Waters technical support for assistance.
- What are the common issues that can affect the performance of Waters HPLC columns?
- There are several issues that can impact the performance of HPLC columns, including: column degradation, insufficient equilibration, column contamination, mobile phase issues (e.g. pH changes, impurities), improper column storage, and sample overload.
- How can I troubleshoot peak splitting issues with my Waters HPLC column?
- Peak splitting can be caused by several factors, including: column contamination, insufficient equilibration, high column backpressure, and mobile phase issues. To troubleshoot, try performing a column wash and conditioning procedure, and check the mobile phase composition for any issues. It may also be helpful to check the column’s backpressure and ensure it is within the recommended range.
- How often should I replace my Waters HPLC column?
- The frequency of column replacement will depend on several factors, including the type of column, the sample matrix, and the frequency of use. However, a general rule of thumb is to replace the column after 100-200 injections or when a significant decrease in performance is observed.
- Can I reuse my Waters HPLC column?
- In general, it is not recommended to reuse HPLC columns as it can result in decreased performance and potential contamination. However, some columns may be suitable for regeneration, which involves removing the contaminants and refreshing the stationary phase.
- How should I store my Waters HPLC column?
- Columns should be stored in a cool, dry place to prevent degradation or contamination. It is recommended to store columns in the mobile phase or with a suitable storage solution to maintain the integrity of the stationary phase.
- How can I prevent contamination of my Waters HPLC column?
- Contamination can be prevented by properly preparing the sample and ensuring that all equipment and mobile phases are clean and free of impurities. It is also recommended to use guard columns and replace them regularly to prevent contamination from reaching the analytical column.
- Can I perform reversed-phase chromatography with a polar analyte using a Waters HPLC column?
- Yes, reversed-phase chromatography can be used for polar analytes by selecting a column with a polar stationary phase or modifying the mobile phase to include a polar component.
- How can I troubleshoot issues with peak tailing in my chromatogram?
- Peak tailing can be caused by several factors, including: column contamination, pH changes in the mobile phase, or an improperly packed column. To troubleshoot, try performing a column wash and conditioning procedure, and check the mobile phase composition for any issues. It may also be helpful to check the column’s backpressure and ensure it is within the recommended range.
- How do I determine which Waters HPLC column is best for my application?
- The selection of the appropriate HPLC column will depend on several factors, including the analyte’s properties (e.g. size, polarity, hydrophobicity), the sample matrix, and the separation goal. Waters offers a wide range of columns with various selectivities and properties to meet different analytical needs.
- How can I extend the lifetime of my Waters HPLC column?
- To extend the lifetime of the column, it is important to properly maintain and care for the column, including performing regular column wash and conditioning procedures, avoiding sample overload, and using a guard column to prevent contamination. It is also important to ensure the mobile phase composition and column storage conditions are suitable for the column.