3.1.3 Mixed mode column
In order to satisfy the separation of complex samples, some emerging chromatographic columns have been developed and utilized. Mixed mode chromatographic columns combine different fillers and bonded functional groups on the same chromatographic column to separate compounds with large differences. At present, there are many applications of mixed mode chromatography columns in impurity separation. 45-47Size Exclusion Chromatography (SEC) is an analytical method that realizes separation based on the relative relationship between pore size and molecular weight. 48 It is suitable for the separation of substances with different molecular weights, especially for biological products. With the increase of repeating units, high molecular weight compounds also have great differences in their physical and chemical properties, such as solubility, ionic bonding capacity. When reverse chromatography is used to separate compounds with large molecular weights, these differences may cause precipitation and adsorption, which may cause the chromatographic peak to broaden and fall below the detection line, or it may not be eluted. 49Lin et al. 50 proposed a size-exclusion chromatography on a reversed-phase column that separated trace high molecular weight species with a variety of functional groups from high concentration of small molecule substance and solve a majority of high-MW impurity cases in active pharmaceutical ingredient. reversed-phase chromatography-size-exclusion chromatography is the stationary phase of reverse chromatography in the mode of size exclusion chromatography.51 Because of its high sensitivity and wide solvent compatibility, it can potentially become a method to detect the high molecular weight impurity.
There are many liquid chromatography method for separating impurities in drugs. Haddad’s team studied the impact of Ion Chromatography (IC) with universal detectors 52 and organic solvent53 on the analysis of pharmaceutical impurities. In addition, the application of two-Dimensional Liquid Chromatography54 (2D-LC) and Ultrahigh Performance Liquid Chromatography 55 (UPLC) facilitates the separation of complex samples and improves the efficiency of impurity analysis. Of course, these impurities separation methods can only be combined with suitable detectors to achieve greater benefits.

3.2 Thin layer chromatography

Thin-layer chromatography (TLC) also plays a key role in the separation and determination of impurities because of economic benefits.56-58 After the sample is separated, the TLC image can be obtained by a flatbed scanner, and then processed by image analysis software to calculate the area or gray value of the characteristic spots on the TLC thin-layer plate for quantitative analysis. Bagcinele et al.59 developed a manual Thin Layer Chromatography (TLC) to separate a mixture of all lipopeptide families with other peptide and lipid impurities and validated for the semi quantitative determination of the surfactin lipopeptide family. Precision would be comparable to that of estimation via HPTLC, and the analytical method reduces the measurement cost, time and consumption of organic reagents. Eglal et al.60 is the first use of TLC method for the determination of aspirin, omeprazole and the impurity of aspirin (salicylic acid). TLC method is eco-friendly and greener when compared to the already reported method.

3.3 Other separation methods

There are many methods for separating impurities in drugs. In the separation of chiral impurities, Supercritical Fluid Chromatography (SFC) is also a good choice. Kalíková et al. 61 used SFC to analyze 21 chiral compounds with different physical and chemical properties on a cellulose 3s-(3,5-dimethylphenylcarbamate)-based chiral stationary phase. It has the best separation effect on β - receptor blockers, and achieve better separation in a shorter time than HPLC. Henrik et al. 62 used Capillary Electrophoresis (CE) method to determine dapoxetine hydrochloride and its enantiomeric impurities and the result was comparable to the data of an enantioselective HPLC method. Enea et al. 63 also gave a detailed review of structurally related compounds in Gas Chromatography (GC) separation of medicines.