8 points on how to care for HPLC columns

HPLC columns are a critical component of chromatographic systems for the effective separation and analysis of components across a wide range of applications. Each HPLC column has a limited lifespan, and to maximize its utility, proper care is essential, which can extend its usage time. It is recommended to regularly monitor the column's efficiency and separation capabilities using standards. If you plan to store the column for an extended period, use a mobile phase that inhibits microbial growth (such as mobile phases containing sodium azide or higher concentrations of methanol, acetonitrile, etc.).

To achieve optimal HPLC column performance and longevity, follow these recommendations:

• #1 Check the column‘s efficiency on a regular basis.
• #2 Always sure that both mobile phases are well miscible when changing them.
• #3 Avoid salts from precipitation in the column, e.g. high content of the organic component in the buffer.
• #4 Use only perfectly filtered and degassed mobile phases.
• #5 Keep the column from drying out. Close securely when not in use.
• #6 For long-term preservation, use a mobile phase to inhibit the growth of mold and bacteria, for example, by adding NaN₃.
• #7 Use the column according in direction on the label. Reverse flow of 5 - 10 column volumes can assist in clearing a clogged inlet frit when there is a significant column backpressure.
• #8 Use guard column to protect your analytical column from particulates, solvent impurities and retained sample components.

The right column choice is as essential for successful analysis as is its subsequent care. Watrex Praha offers a broad portfolio of HPLC columns, which they prepare in-house at their laboratory.

Types filling of and Applications for HPLC Columns

1. Reversed Phase

Watrex reversed-phase columns use high-quality silica-based stationary phases modified with C18 alkyl chains, with effective endcapping to deactivate residual silanol groups.

They are ideal for the separation of slightly polar to nonpolar compounds across a wide range of products, including components of medicines, food products and supplements, environmental pollutants or other natural products.

Watrex: Figure 1: Chromatogram 1 - Column: Watrex IonPlus CS19, 200x4mm; Mobile phase: 4mM HNO3; Flowrate: 1.0 ml/min; Detection: Conductivity without suppression. Chromatogram 2 - Column: Watrex IC Anion I; Mobile phase: 4mM Na 2 CO 3 + 1mM NaHCO 3 + 0.1mM NaSCN; Flowrate: 1,2 ml/min; Pressure: 54 bar; Detection: Conductivity with suppression; Injection: 20 μl.

• Column Dimensions: Depending on customer requirements – from standard analytical columns (2.1 - 4.6 mm diameter, typical lenght 100 mm to 250 mm) packed with strictly monodisperse particles sized 3 or 5 μm. Preparative columns suitable for the separation and purification of biomolecules (peptides, amino acids, nucleotides, etc.) with larger porosity are available in semi-preparative (8 mm diameter) and preparative (20 mm diameter) formats.
• Example Applications: Analysis of pharmaceuticals, environmental analyses, food composition.

2. Ion-Exchange Columns

Watrex ion-exchange columns are designed for the separation of ionic compounds, based on interactions between ions in the stationary phase and analytes. These columns are suitable for the separation of inorganic ions, amino acids, or proteins.

Watrex: Figure 2: Chromatogram 3 - Column: Watrex Deltasil 100 C18; Mobile phase: 85% ACN; Flowrate:0.65 ml/min; Pressure: 54 bar; Detection: 254 nm; Injection: 2 μl.

• Column Dimensions: Columns available in various dimensions.
• Example Applications: Analysis of ions, biomolecules, peptide separation.

3. Ion-Exclusion Columns

These Watrex Polymer IEX HPLC columns are suitable for the separation of weak organic acids and carbohydrates, based on ion exclusion principles. The basis is polymer monodisperse particles with a size of 8 μm modified by sulfonation and supplied in various ionic forms.

• The H-form is intended for the determination of weak organic acids in food or agricultural and veterinary products.
• The Ca- and Pb-forms are typically used for the determination of mono-, di- and oligosaccharides in the food industry.

Watrex: Figure 3: Chromatogram 4 - Column: Watrex Polymer IEX Ca 250x8mm; Eluent: H2O; Flow-rate: 0.5 ml/min; Pressure: 26 bar; Inj. Volume: 20 μl; Concentration: 20 ppm; Temperature: 90 °C; Detection: RI. Chromatogram 5 - Column: Watrex Polymer IEX H 250x8mm; Eluent: 9mM H2SO4; Flow-rate: 0.5 ml/min; Pressure: 45 bar; Inj. Volume: 20 μl; Concentration: 20 ppm; Temperature: ambient; Detection: UV 210 nm.

• Column Dimensions: 8 mm diameter and lengths of 250 or 300 mm.
• Example Applications: Analysis of organic acids in food products, fermentation processes.

4. Gel Permeation/Size Exclusion Chromatography (GPC/SEC) Columns

Watrex gel permeation chromatography (GPC) columns are used for separating highmolecular-weight compounds, typically synthetic or water-soluble polymers, based on molecule size. Smaller molecules penetrate more deeply into the porous structure of the sorbents and thus elute later than larger molecules.

According to the type of stationary phase, the columns are usually divided into Gel Permeation Chromatography (GPC) columns used in the separation of synthetic polymers with organic mobile phases (e.g. tetrahydrofuran, THF, DMF, toluene THF) on polystyrene - divinylbenzene sorbents with defined porosity.

In contrast, the term Size-Exclusion Chromatography (SEC) typically refers to columns for the separation of water-soluble polymers, biomacromolecules (e.g. peptides or proteins), which occur in buffered aqueous phases on porous hydrophilic sorbents (e.g. based on agarose or methacrylate particles modified with hydrophilic groups).

Watrex: Figure 4: Chromatogram 6 - DeltaGelTM Mixed C. Chromatogram 7 - DeltaGelTM Mixed D.

• Column dimensions: Watrex GPC/SEC columns are available in analytical (8x250 mm or 8x300 mm) and preparative configurations (20x300 mm).
• Examples of use: Separation of synthetic polymers, peptides, proteins, polysaccharides.

Advantages of Watrex HPLC columns

Columns manufactured directly in the Watrex laboratory provide several fundamental advantages:

• High quality at friendly prices. Thanks to the fact that they produce the columns themselves, they can offer top quality at very affordable prices.
• A wide range of sizes and types. It offers columns adapted to various chromatographic techniques, including analytical and preparative applications.
• Flexibility in applications. Watrex columns are compatible with a variety of mobile phases and can be used for the separation of ionic compounds, organic acids, polymers and biomolecules.

Conclusion

Watrex HPLC and GPC/SEC columns are a highly reliable solution for a wide range of chromatographic applications and combine high quality with a favorable price. Whether you need columns for reverse phase, ion exchange, ion exclusion, size exclusion chromatography (SEC) or gel permeation chromatography (GPC), Watrex products guarantee you excellent separation properties and reproducible results.

For more information, feel free to contact Watrex ([email protected]) or visit their website (www.watrex.com).