ExcsepTM Reversed Phase Columns User Manual

Introduction to Stationary Phases

Excsep^TM series reversed-phase stationary phases (including C18, HPH-C18, AQ-C18, SCB-C18, Polar-C18, C8, HPH-C8, AQ-C8, SCB-C8, C4, C1, Phenyl, Polar Phenyl, Phenylhexyl, Fluorophenyl, Biphenyl, etc.): Alkyl or Polar group-modified alkyl chromatography silica matrix stationary phases that are typically used for reversed-phase conditions with polar mobile phases.

Column Activation

Excsep^TM series reversed-phase columns are activated by first rinsing the column with 100% acetonitrile or methanol, typically 5-10 column volumes (~20-40 min), and then equilibrating the column with the analytical mobile phase from the method. Special attention should be paid to the fact that if buffer salts or ion-pairing reagents are used in the mobile phase, the transition must be made with the same proportion of the aqueous phase, or an aqueous solution with a lower proportion of the organic phase. For example, if the mobile phase is 20% methanol and 80% phosphate buffer, the column should be flushed with 20% methanol and 80% water for at least 5-10 column volumes before switching to this mobile phase, otherwise the buffer salt system may not be compatible with the pure organic phase, resulting in buffer salt precipitation problems. It should also be noted that the buffer salt flush should be performed at a low flow rate (0.2 mL/min) first, and then gradually increased to a normal flow rate to equilibrate the column.

Mobile Phase

*Excsep^TM HPH-C18, Excsep^TM HPH-C8 columns pH range can be widened to 2~11;

Excsep^TM SCB-C18, Excsep^TM SCB-C8 columns pH range can be broadened to 0.5~8;

Excsep^TM AQ series, Excsep^TM SCB series reversed-phase columns, and Excsep^TM C4 columns have a broadened range of aqueous phase compatibility to 100% aqueous phase;

Column Pressure

Excsep^TM series reversed-phase columns have pressure limits of 4500 psi (5μm), 7500 psi (3μm), and 13500 psi (1.8μm).

Whether it is the mobile phase or the sample itself, particulate matter must be prohibited from entering the column, otherwise, if particulate matter clogs the sieve plate of the column, it will lead to a significant increase in column pressure, which in turn will cause irreversible damage to the column.

Storage

Excsep^TM series reversed-phase columns are recommended to be stored in a high proportion of organic phase, which should not be less than 75%. If buffer salts are used in the mobile phase, ensure to transition using the same proportion of aqueous phase.

Guard Columns

The use of guard columns is highly recommended. We recommend the use of Excsep^TM guard columns of the correct type, which are packed with chromatographic stationary phase identical to that of the analytical column, and are recommended to be replaced when the column pressure increases or the column efficiency decreases. For commonly used 4.6 x 250 mm columns, it is recommended to use 4.6 x 10 mm guard columns.

Solutions for Column Efficiency Decrease or Column Pressure Increase

1. When particulate issueaccumulates on the sieve plate or column bed causing an increase in column pressure and a decrease in column efficiency, disconnect the column from the injector and start the pump to determine if the source of column backpressure is indeed from particulate contamination of the column (sample, mobile phase, and system). At this time, it is recommended to reverse the column connection and flush the column in the opposite direction with the mobile phase for 40~60min.
2. As the frequency of use increases, large molecules of proteins, or small molecules of fats, esters, sugars, organic acids and pigments, etc., tend to adhere to the column packing and contaminate the column, it is recommended that the column be reversed, and the column be flushed with a solvent that can dissolve the adherents or contaminants at a flow rate of 1/4 of the analytical flow rate for 2~4h.

*If a transition is required between the flushing solvent and the mobile phase, be sure to make a sufficient transition.

Regeneration

1. Reverse the column.
2. Rinse the column sequentially with: water - methanol - acetonitrile - isopropanol - dichloromethane - isopropanol - acetonitrile - methanol - water at a flow rate of 10.0 ml/min. water, rinse the column at a flow rate of 1.0 ml/min for about 45~60 minutes for each solvent .
3. Equilibrate the column with the desired mobile phase with the column oriented for normal use.

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