Non-aqueous reverse phase chromatography (NARP) is a special mode of reverse phase chromatography primarily used to separate highly hydrophobic samples that are insoluble in water, such as lipids and synthetic polymers. These samples have strong retention capabilities, and the mobile phase consists entirely of organic solvents without the use of strongly polar aqueous solutions. Non-aqueous reverse phase high-performance liquid chromatography (HPLC) can be used as a routine analysis method for lipophilic drugs, nonpolar nitrogen-containing organic compounds, and their salt forms [1]. A large class of active ingredients in traditional Chinese medicine are lipophilic and insoluble in water, and they may even have limited solubility in methanol. Therefore, non-aqueous reverse phase chromatography is also commonly applied in the quality control of traditional Chinese medicine [2].
An example of the application of Welch Materials, Inc. chromatographic column in the analysis of glyceride samples in a traditional Chinese medicine formulation granule is provided below:
| Chromatographic Column | Welch Ultisil® XB-C18（4.6×250mm，5μm） |
| Mobile Phase | Acetonitrile: Dichloromethane = 65:35 |
| Column Temperature | 25℃ |
| ELSD Detector Temperature | 60℃ |
| Flow Rate | 1.2 mL/min |
| Injection Volume | 40μL |
In non-aqueous reverse phase chromatography, the mobile phase consists of a strong polar organic solvent A and a weak polar organic solvent B. Solvent A is commonly acetonitrile or methanol, while solvent B is typically tetrahydrofuran, isopropanol, dichloromethane, methyl tert-butyl ether, or other weak polar organic solvents.
(Mobile phase: Acetonitrile: Dichloromethane = 60:40)
The optimization of non-aqueous reverse phase chromatography method is similar to reverse phase chromatography, mainly achieved by changing the polarity of solvent B or the B solvent. Isocratic or gradient elution can both be employed, and the column temperature also affects sample separation. Typically, a mixture of ACN (A) and THF (B) is chosen as the initial mobile phase. If the sample retention is still too strong with 100% THF, a weaker polar solvent B (such as dichloromethane or chloroform) can be used as a replacement, but consideration should be given to the detection wavelength when using dichloromethane or chloroform.
It is important to note that when using non-aqueous reverse phase chromatography, the entire system and the aqueous phase in the column should be thoroughly flushed. The instrument system should be ensured to have been switched to the normal (non-reverse) phase to avoid hardware damage.
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