HPLC Column Requirements for Mobile

The pH of the mobile phase should be controlled between 2 and 8. When the pH value is greater than 8, the carrier silica gel can be dissolved; when the pH value is less than 2, the chemically bonded phase connected to the silica gel is easily hydrolyzed and peeled off. When a mobile phase with a pH value greater than 8 is used in the chromatography system, an alkali-resistant filler should be used, such as a bonded silica filler with high surface coverage and a high surface coverage, polymer filler Organic-inorganic hybrid fillers or non-silica gel fillers; when a mobile phase with a pH value less than 2 is required, an acid-resistant filler should be used, such as diisopropyl with a large volume side chain that can provide steric protection Based or diisobutyl substituted octadecylsilane bonded silica filler, or organic-inorganic hybrid filler.

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In High-Performance Liquid Chromatography (HPLC), the choice of an appropriate column is crucial for achieving optimal separation and analysis of compounds. The mobile phase, which is the liquid that carries the sample through the column, interacts with the stationary phase in the column to separate the components of the sample. Here are some key requirements for an HPLC column regarding the mobile phase:

1. Compatibility with Mobile Phase:

  • The column material must be compatible with the selected mobile phase. Common mobile phases include aqueous solutions, organic solvents, or a combination of both. Ensure that the column material is chemically inert and does not react with or degrade in the presence of the mobile phase.

2. Stationary Phase Type:

  • Choose a column with a stationary phase that is suitable for the separation you need. The stationary phase can be reversed-phase, normal-phase, ion-exchange, size-exclusion, or affinity, depending on the nature of the compounds in your sample.

3. Particle Size:

  • The particle size of the stationary phase in the column affects the efficiency of the separation. Smaller particle sizes generally provide higher resolution but may result in higher backpressure. The choice of particle size depends on the nature of the sample and the desired separation.

4. Column Length:

  • The length of the column influences the separation efficiency. Longer columns generally provide better resolution but may result in longer analysis times. The choice of column length depends on the complexity of the sample and the desired chromatographic performance.

5. Column Diameter:

  • The diameter of the column also affects the separation efficiency and backpressure. Smaller diameter columns usually result in higher backpressure but can offer higher resolution. Larger diameter columns are suitable for higher flow rates and may be preferred for fast separations.

6. Temperature Stability:

  • Some analyses may require temperature control. Ensure that the column material and design can withstand the temperature range required for your analysis without compromising performance or stability.

7. Column Chemistry:

  • Consider the chemical properties of the stationary phase. For example, in reversed-phase chromatography, the hydrophobicity of the stationary phase is crucial. In ion-exchange chromatography, the type and charge of the ion-exchange groups play a significant role.

8. pH Range Compatibility:

  • The pH range of the mobile phase should be within the specified limits of the column. Some columns have pH limitations, especially those with bonded phases or specific coatings.

9. Mobile Phase Additives:

  • If you plan to use additives or modifiers in the mobile phase (such as buffers, salts, or acids), ensure that the column is compatible with these additives and that they do not negatively impact the column performance.

Cleaning and storage of HPLC column

Have a good understanding of the sample made, and wash it with the mobile phase that has the strongest eluting capacity for the sample. For silica gel HPLC column, first wash away the polar impurities with methanol, then use dry dichloromethane and n-heptane 100 — 20OraL is activated in sequence. For the bonds and phase columns, it is generally washed with methanol-chloroform-methanol-water in order. Each column has a specific cleaning method, which must be read in accordance with its instructions.

Remove impurities and salts before storage, use a suitable storage solvent to avoid drying out of the column bed, avoid mechanical vibration, prevent bacterial growth, and pay attention to the storage temperature.

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The HPLC column system consists of a reservoir, a pump, an injector, a chromatographic column, a detector, and a recorder. The mobile phase in the reservoir is driven into the system by a high-pressure pump, and the sample solution enters the mobile phase through the sampler. Different distribution coefficients, when making relative movement in the two phases, after repeated repeated adsorption-desorption distribution processes, the components have a large difference in moving speed, and are separated into individual components and flow out from the column in turn. When passing the detector, the sample concentration is converted into an electrical signal and transmitted to the recorder, and the data is printed in the form of a map.

The temperature of the ordinary bonded stationary phase of HPLC column with silica gel as the carrier usually does not exceed 35 ° C. In order to improve the separation effect, the temperature of the column can be appropriately increased, but it cannot exceed 60 ° C. If you want to know more information about HPLC column don’t hesitate to contact us.

When you get a new HPLC column, be sure to read its instructions first, then measure the HPLC column efficiency, periodically check the column efficiency, keep the chromatogram obtained on the new HPLC column, and record the conditions, and regularly detect the instrument band broadening. If it is used for on-line monitoring, it should provide online physical and chemical protection for the chromatographic column, such as the installation of online filters, self-packed guard columns and pre-packed guard columns. The function of the online filter is to prevent particles from accumulating on the HPLC column head.

The advantage is that it does not affect the column efficiency. It is often used when high column efficiency is needed. The consumables that are often replaced are the filter element and the gasket to protect the column from chemical pollution. The disadvantage is that most guard columns affect the efficiency of the chromatographic column, especially when using microcolumns, the common self-packed guard columns are commonly used.

Some recently introduced guard columns, such as Sentry-guard guard columns, are characterized by high quality and efficient packing, increase the efficiency of analytical columns, large load capacity for dirty samples, and extend the life of guard columns. Hand-tighten the joints during installation. No tools, time saving, replaceable core design, can use a variety of different fillers, mainly suitable for very dirty and complex sample backgrounds, such as biochemical samples, environmental samples and food, or do not want to do too In the case of phase extraction, we do not want to reduce the column effect.