When use the amino((NH2) column for a period of time, have you ever encounter a decrease in column efficiency and an increase in column pressure？Or abnormal interference peaks and the retention time drifts? Or method can’t be reproduced under certain chromatographic conditions? Or the resolution, plates and tailing factor can’t meet the detection requirements? The uniqueness of the NH2 column can caused a lot of issues, so today we will discuss about the NH2 column.
The bonding functional group of the NH2 column is aminopropyl which is more easily to hydrolysis than conventional C18, C8 and C4. Therefore, before using the NH2 column, we must be mentally prepared that the life of NH2 column is much shorter than the conventional column.
The NH2 column can be used either as normal phase or reversed phase. As the solvents used in two phases are immiscible, so a transition with isopropanol is needed to switch between normal and reversed phase. The use and maintenance of NH2 column in reversed phase and normal phase will be discussed respectively below.
If the mobile phase doesn’t contain any acid, alkali or salts, NH2 column should be flushed and stored with 100% acetonitrile after sample test. If mobile phase contains acid, alkali or salts, firstly flush the column with 60% acetonitrile for 40 min, then flush and store with 100% acetonitrile.
When use at reversed phase, pay attention to pH range of the mobile phase. The lower the pH is, the easier the hydrolysis is. The ideal pH range is 3-7. In addition, the higher the proportion of water in the mobile phase is, the easier hydrolysis is.
Note: After use, it is recommended to flush and store the column in a pure organic phase to obtain good preservation effect and prolong the lifetime. In addition, if the column has abnormal circumstances of high pressure, abnormal peak shape, low column efficiency or low resolution, firstly use transition mobile phase to flush off the salts, then flush as following: 100% acetonitrile → 100% methanol → 100% isopropanol→ 100% acetonitrile at analysis flow rate for 30 minutes each step, while isopropanol needs a lower flow rate due to its high viscosity
The column should be flushed with 100% n-hexane and stored with n-hexane/isopropanol (90/10).
In normal-phase mode, the water content of the stationary phase is a key parameter to affect selectivity, while the water content of the mobile phase affects the retention time and resolution, so the retention time may easily shift when water content between stationary phase and mobile phase changes. It is recommended to remove the water from the amino column.
1. Remove water from stationary phase:
Flush the column with 30 column volumes of n-hexane containing 2.5% dimethoxy propane and 2.5% glacial acetic acid.
2. Use mobile phase with controlled water content
Half-saturation mobile phase method: divide the anhydrous non-polar mobile phase in half. Add a certain amount of water into one half, then mix and stir for about 1 hour. After static stratification, remove the excess water phase, remix two halves together to form a “half-saturation” mobile phase
Note: If the column has abnormal circumstances of high pressure, abnormal peak shape, low column efficiency or low resolution, flush the column as following: 100% isopropanol → 100% methanol → 100% isopropanol at analysis flow rate for 40 minutes each step, while isopropanol needs a lower flow rate due to its high viscosity.
Special column for Lactose
A special amino column is also introduced by Welch–Xtimate® Lactose-NH2. 100% acetonitrile is recommended for flushing and storage. If abnormal circumstances of high pressure, abnormal peak shape, low column efficiency or low resolution appear, flush the column with 100% methanol → 100% acetonitrile → 100% isopropanol → 100% acetonitrile for 3 hours each step.
If there are abnormalities such as increased noise, increased drift, insufficient resolution, or insufficient plates, the following points should be noted:
1. Mix the mobile phase and shake well, use vacuum filter and ultrasonic degassing for 10 minutes, use one channel and do not use inline filter.
2. Injection volume needs to be strictly in accordance with the requirements of the Pharmacopoeia. It’s better to use 10 μL loop if injection volume is 10 uL in order to reduce the influence of dead volume outside the column. The following chromatograms below are comparison between 20 μL injection volume (the left image) and 10 μL injection volume (the right image).
3. Because RID is sensitive to temperature, it is recommended that the oven be filled with dry towel or cotton to act as a heat transfer medium for stable baseline and chromatographic effects.
It is the fact that NH2 column’s lifetime is shorter than conventional columns due to unique polarity of the amino functional groups. Hope the above methods will help to extend the life of NH2 column greatly.
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