PRIMESEP COLUMN OPERATION AND STORAGE
Good column care requires little additional time and will extend the lifetime of your PRIMESEP HPLC columns. Column care for PRIMESEP brand of columns involves operating in a suitable mobile phase and storing PRIMESEP column in an appropriate solvent system.
Mobile Phases for Column Operation
- TRY TO AVOID USING ALCOHOLS WITH Primesep A, Primesep 100, Primesep 200, Primesep C and Primesep P columns as you can esterify carboxylic acid attached to the surface of silica. The esterification might significantly Affect reproducibility and retention time of some basic analytes
- Do not use or store SIELC HPLC columns in pH outside of recommended ranges. SIELC Columns require buffered or acidified mobile phases to be present in the column at all times
- Do not store PRIMESEP HPLC columns in mobile phases containing modifiers that degrade easily such as triethylamine (TEA), tetrahydrofuran (THF). Use HPLC-grade solvents and additives for your chromatography.
- To achieve reproducible results, please use a thermostat. Operating temperature for all SIELC columns is 0-50°C
- To achieve reproducible results, always equilibrate SIELC columns prior to injection. In some cases replacement of the mobile phase inside the column does not make the column sufficiently equilibrated. SIELC columns’ stationary phase retain components of the mobile phase as counter ions. To equilibrate the column in this case requires a replacement of all the ions adsorbed in the column stationary phase. Typically, 50 mM buffer would equilibrate a column in 1 hour. If only the concentration of the buffer changed, the 2-4 volumes of the column are sufficient to reach and equilibration state.
- It is important to operate column below the specified maximum pressure limits (5000 psi). If you exceed maximum pressure, the column can be destroyed. Leaking column is an indication of excessive pressure range.
- Limit the use of tetrahydrofuran to 10% with SIELC columns
- An arrow on the column label is used to specify direction of flow through the column. Typically you should operate the column in the direction marked. If column needs to be washed in the opposite direction use reduced flow rate in order not to create pressure which can disturb packing.
- Use only fingers to tighten PEEK fittings to the column connection.
To clean the column, use the mobile phase consisting of 60/40/0.3 ACN/H2O/H2SO4.
The column can also be run in reverse at a reduced flow rate 2-3 times.
If your sample matrix contains many compounds, especially multi-charged compounds like proteins, we recommend the use of a guard column and replace it often as soon as you notice degradation in performance, i.e. peak shape, shift in retention, etc.
If you run a high-throughput operation, we recommend the use of FlipLC, which eliminates the use of constantly changing guard columns.
Table 1. Operational pH ranges for SIELC Columns
|pH Limits/Recommended Operating Range
|Short Term Storage
|Long Term Storage
|Alcohol in mobile phase
|Primesep A, Primesep 100, Primesep 200, Primesep C, Primesep P, Primesep AB, Primesep B2, Obelisc R, Promix SP, Promix AP
|1.5-7.0 / 2.0-6.5
|Buffered or acidified mobile phases within recommended pH range
|ACN / water / formic or acetic
ACN / water / acetic acid
|Primesep B, Primesep D, Primesep B4, Primesep SB, Primesep N, Primesep PB, Primesep S, Promix MP, Obelisc N, BIST A, BIST AC, BIST A+
|1.5-5.0 / 2.0-4.5
|Mobile phases within recommended pH range
|ACN / water / formic acid or acetic
|ACN / water / formic acid or acetic
Preform the column performance test using conditions described in the Certificate of Analysis and compare with the result in the report. Due to variations in particular LC systems you may observe slightly different results.