Separation Methods Technologies (SMT) is an Original Equipment Manufacturer (OEM), therefore, we have a complete control over quality and ensuring reproducibility of your chromatography methods. As an OEM, we utilize proven procedures at every stage of manufacturing to guarantee consistency in our silica purity, pore size, particle size, as well as chemical bonding. Each HPLC column is tested to meet our stringent specifications.
The following information should be reviewed and applied to guarantee optimum lifetime and reliability of your HPLC column.
For optimum peak shape and sensitivity, it is recommended to prepare the sample in the operating mobile phase or to use a mobile phase, that is a weaker solvent than the mobile phase. If possible, filter samples through a 0.2–0.45 μm syringe filter before injection.
To extend column lifetime and prevent contamination, the use of a guard column or pre-column filter is recommended. Guard columns will protect the column from sample contaminants and highly retained solutes. Pre-column filter will protect the column from small, insoluble particles. For maximum column lifetime guard columns or pre-column filter should be replaced at regular intervals, depending on the level of sample contamination.
Solvents and Mobile Phase
Use only HPLC grade solvents, filtered through a 0.2–0.45 μm membrane and degassed. Use only solvent additives (e.g. Ion-pairing reagents or buffer salts) of high purity (HPLC grade) or filter all aqueous buffers prior to use. The use of freshly prepared aqueous buffer solutions is recommended in order to minimize bacterial growth. Do not use mobile phases or compounds that chemically attack the bonded phase or silica. Never pump immiscible solvents sequentially through the column. The use of an online degassing unit is also recommended. For SMT Silica, AP (NH2), CP (CN), Diol 1 & Diol 2, Ion-exchange and the AquSep1 & AquSep2 columns the use of 100% aqueous mobile phase is possible. Other stationary phases are not compatible with 100% aqueous eluents and the water content should not exceed 95%. For reversed phase separations typical eluents are acetonitrile, methanol or tetrahydrofurane in mixture with pure water or buffer. For normal phase separations typical eluents are n-hexane or n-heptane in mixture with an aliphatic alcohol (e.g. 2-propanol or ethanol).
Prior to measurement of samples the column must be rinsed with the eluent at the same flow rate and temperature as the method to be applied. Column equilibration is finished, when the baseline of the detector no longer shows a drift (generally after 10 column volumes).
The recommended pH range limit for most columns from SMT is pH = 1.0 to 12.0 depending on the phase and application.
SMT HPLC columns can tolerate pressure of up to 6000 psi. In order to maximize column lifetime, operation at pressure higher than 4400 psi should be avoided. Exposure of a column to rapid changes in back pressure may reduce column lifetime, especially for preparative columns.
The maximum operating temperature is 95°C. However, any temperature above ambient may have negative effect on column lifetime, which will vary depending on the pH and buffer conditions used. Variation of the temperature influences retention time and especially peak shape of analytes. Optimum temperature for a successful separation should be determined empirically.
The flow-rate is limited by the maximum column back pressure, which should not exceed the limit specified above. The flow-rate recommended for analytical columns is 0.2–2.0 mL/min. Typical flow rates for different column internal diameters (I.D.) are shown in the table on the right:
- I.D. 2 mm – ~0.2 mL/min
- I.D. 3 mm – ~0.4 to ~0.5 mL/min
- I.D. 4 mm – ~0.7 to ~0.8 mL/min
- I.D. 4.6 mm – ~1.0 mL/min
- I.D. 8 mm – ~3.0 to ~4.0 mL/min
- I.D. 10 mm – ~4.0 to ~5.0 mL/min
- I.D. 20 mm – ~9 to ~20 mL/min
- I.D. 30 mm – ~20 to ~42 mL/min
- I.D. 40 mm – ~30 to ~75 mL/min
All HPLC columns should to be stored in an appropriate solvent and capped tightly to prevent draining. Store column in a cool area, free from vibration using a special column storage cabinet or the shipping box. Flush columns after use with pH-neutral solvent to remove all possible contaminants and tag the column with the used storage solvent for later use (even for short-term storage). The original eluent shown on the initial column test chromatogram (see HPLC Column Certificate of Analysis) is recommended as storage solvent. In general, the storage solvent should not contain more than 50% water to prevent bacterial and fungal growth. For SMT AP (NH2) and Diol 1 and 2 stationary phases we recommend 2-propanol, which is compatible with both Normal Phase and Reversed Phase Chromatography.
Regeneration and Cleaning Procedure
Contamination of the column by impurities from the sample or mobile phase can cause changes in peak shape, peak splitting, shifts in retention or high backpressure. In some cases, these contaminants may be removed using a standard rinsing procedure employing different solvents, as shown in the following regeneration schemes.
Regeneration scheme for reversed-phase columns:
- 20 column volumes of water/acetonitrile (95:5 v/v) or 20 column volumes of acetonitrile
- 5 column volumes of 2-propanol
- 20 column volumes of n-heptane or n-hexane 5 column volumes of 2-propanol
- 20 column volumes of acetonitrile
- Re-equilibrate column with desired mobile phase
Regeneration scheme for normal-phase columns:
- 20 column volumes of n-heptane, 5 column volumes of 2-propanol, 20 column volumes of acetonitrile, 20 column volumes of water
- 20 column volumes of acetonitrile, 5 column volumes of 2-propanol, 20 column volumes of n-heptane
If an analytical HPLC column has dried out during storage, regeneration may be achieved via rinsing the column with about 10 column volumes of the storage solvent at a flow rate of 0.1 to 0.2 mL/min. Reflushing of the column in opposite flow direction is furthermore permitted but should be carried out carefully at low flow rates.