ExcsepTM HPH-C18 HPLC Column User Manual

Introduction to Stationary Phase

OneTwo ExcsepTM HPH-C18 is a chromatographic stationary phase made of ultra-high purity spherical chromatographic bare silica gel prepared using OneTwo Chromatography Technology's unique organic-inorganic heterogeneous bonding technology and a special fully capped process for applications in extreme pH, especially alkaline environments, and is suitable for the separation of acidic, neutral and basic compounds.

Column Activation

ExcsepTM HPH-C18 columns are activated by first rinsing the column with 100% acetonitrile or methanol, typically 5 to 10 column volumes (approximately 20-40 min), and then equilibrating the column using the analytical mobile phase in the method. Note that if buffer salts or ion-pairing reagents are used 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

Column Pressure

ExcsepTM HPH-C18 columns have upper pressure tolerance 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, may cause to the column.

Storage of Columns

ExcsepTM HPH-C18 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, be sure to transition with the same proportion of aqueous phase.

Guard Columns

The use of guard columns is highly recommended. We recommend using the correct type of ExcsepTM HPH-C18 guard columns, which are packed with chromatographic stationary phase identical to the analytical columns, and replacing the guard columns when the column pressure increases or the column efficiency decreases. For the commonly used 4.6×250 mm column, it is recommended to use a 4.6×10 mm guard column.

Solutions for Column Efficiency Decrease or Column Pressure Increase

1. When particulate issue accumulates 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 the column back pressure is indeed from particulate contamination of the column (sample, mobile phase and system). In this case, 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.