Amino NH2 HPLC Column: Usage, Protection, Cleaning, Storage, and Regeneration

Amino NH2 HPLC Column: Usage, Protection, Cleaning, Storage, and Regeneration

NH2(Amino) HPLC column can be used in both normal-phase and reversed-phase chromatography conditions. It is important to note that the normal-phase solvents and the reversed-phase solvents are not mutually compatible.

For a newly purchased column, first of all, take care to open the analytical test manual to know the storage solvent of the column. If the storage solvent and the mobile phase have different polarities and are not miscible, please use isopropyl alcohol for the transition first. During the transition process, the high viscosity of isopropyl alcohol will lead to high column pressure, so please reduce the flow rate appropriately.

If the mobile phase contains buffer salts, it is recommended to use the same proportion of the mobile phase without buffer salts before using the analytical mobile phase, so as to avoid the precipitation of buffer salts in the analytical column.

Column Efficiency Testing

The following method is very general, you can also use the same standard solution and mobile phase to test silica gel columns and cyanide-based columns.

Mobile phase: 10% ethyl acetate + 90% n-hexane
Flow rate: 1.0ml/min
Standard solution: ethylbenzene (or toluene) + methyl benzoate
Detection wavelength: 254nm

This method is particularly suitable for testing a new column (new columns are often kept in normal phase solvent), and also for testing as a record after the column is fully cleaned before it is ready to be placed for a considerable period.

In normal use, if the sample is fixed and repeated, we can certainly make a judgment from the separation of the sample. However, if the sample and the analysis conditions are different, regular testing of the column efficiency can avoid the process of wasting labor and material resources when the column efficiency decreases. It is important to note that when an NH2 column (or cyanide column) is used in reversed-phase conditions, please pay attention to the transition of the mobile phase when using this method.

The Usage of NH2 Column

The bonding functional group amino of the NH2 column is easier to hydrolyze than the C18 and C8 groups. First of all, you should know the short service life for the NH2 columns, especially in reversed-phase conditions. The lower the pH value, the more likely hydrolysis will occur. The higher the proportion of water in the mobile phase, the more likely hydrolysis will occur. Therefore, it is important to clean and keep the NH2 column in organic solvent after use or when preparing for long storage.

When using the NH2 column for acid analysis (sugar analysis), the presence of acid implies the presence of protons. Protons may protonate the slightly negatively charged amino functional groups, which may cause a change in retention properties or a decrease in column efficiency for some analytes after the column has been used for some time. It is recommended that the column be rinsed with a 50:50 v/v acetonitrile-water solution containing 0.5-1.0% NH3 at 5-10 times the column volume. After rinsing, wash off the excess ammonia with a mobile phase that does not contain base. A slight addition of ammonia (e.g., 0.1%) to the mobile phase is recommended for the analysis of such acidic analytes.

The Cleaning of NH2 Column

Briefly, for NH2 columns used under normal phase conditions, refer to the cleaning method for silica columns. For amine columns used in reversed-phase conditions, refer to the cleaning method for C18.

Normal-phase condition

Firstly, clean the column with 50 times the volume of isopropanol, and lower the flow rate due to the high viscosity of isopropanol. Then clean with 50 times the volume of methanol. Then Transite back to the normal phase with isopropanol.

Reverse-phase conditions

Rinse the buffer salt promptly, not directly with pure methanol. Rinse with 50 times pure methanol. After that, rinse the column with methylene chloride after the transition with isopropanol. After that, transition back to methanol conditions with isopropyl alcohol.

These cleaning methods, mainly for when the sample has impurities gradually adsorbed to accumulate on the packing method, the column performance behavior such as column pressure increases column efficiency decreases.

Storage of NH2 Column

In normal-phase conditions, the column should be rinsed well and stored in hexane-acetonitrile (99:1).
In reversed-phase conditions, for short period, it can be stored in methanol or acetonitrile; for long period, methanol should be replaced with isopropanol and chloroform in order and stored in n-hexane finally.

Regeneration of NH2 Column

Scheme 1

Sequentially flush the column with methanol, isopropanol, chloroform, n-hexane, chloroform, isopropanol, and methanol (each at a flow rate of 0.5 ml/min, 20 times the column volume), and then change the mobile phase. These cleaning methods, mainly for when the sample has impurities gradually adsorbed to accumulate on the packing method, the column performance behavior such as column pressure increased column efficiency decreased.

Scheme 2

NH2 column for reversed-phase conditions, NH2 bond will be hydrolyzed, especially outside the pH range of the column. In extreme acidic and alkaline conditions, the column life will decline very quickly. If it is used under these conditions needs to be cleaned, it needs to be flushed with 10 times the column volume solution, as follows: 95% water / 5% acetonitrile, THF tetrahydrofuran, 95% acetonitrile / 5% water, and keep 95% acetonitrile / 5 % water to continue rinsing at a low flow rate of 0.2-0.5 mL/min overnight.

Scheme 3

After using the column for a certain period of time, the column efficiency decreases, and aging, you could also clean the column to restore the column performance. You could clean it with 10 times the volume of the following solutions in turn: 95% water/5% acetonitrile, THF tetrahydrofuran, 95% acetonitrile/5% water. Then the mobile phase can be removed.

Note: The test conditions in this article are only according to common experience. They need to be adjusted based on specified conditions. We are not responsible for any adverse factors arising from this article.

GALAK ChromatographyAuthor
Tian Jing

Manager & Engineer in GALAK Chromatography. Master of Chemical Engineering.
During my college study, I found liquid chromatography to be a profound subject. I know the painful struggle a novice needs to go through to get started. I share this article to help you solve your problems quickly.

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