On chromatographic column, we have to say C18
High performance liquid chromatography (HPLC) has the advantages of high selectivity, high sensitivity, high analysis speed, and most of the soluble drugs can be analyzed by this method, so this technology has become the first choice of drug analysis. Chromatographic column is the most important part of high performance liquid separation system. In the industry, chromatographic column is often compared to the heart of liquid chromatography, because the type of chromatographic column determines the nature of chromatographic system, and the size and length of chromatographic column affect the analysis time and efficiency. Amino column, phenyl column, cyano column and C18, C8 reversed-phase column are the two most common columns in drug analysis and evaluation.
The reversed-phase chromatography of C18 or ODS, which is a chemically bonded silica gel stationary phase, has accomplished the task of liquid chromatography excellently. Compared with other chromatography, C18 reversed-phase chromatography has obvious advantages. It can directly inject and analyze water-soluble samples, so it has a wide range of application, and is known as the "standard stationary phase separation mode". Among all kinds of Pharmacopoeia, C18 column is the most widely used stationary phase. At present, there are more than 600 kinds of C18 chromatographic columns on the market, and new chromatographic columns are emerging. When different C18 columns are used to analyze specific samples, the retention time, the separation of solutes, and the order of peak generation may be different. The specifications of chromatographic column in USP, EP and JPA are detailed. In addition to indicating the large category of packing, the inner diameter, length, particle size, specific surface area and other information of chromatographic column are usually given. Some varieties will also directly indicate the manufacturer of chromatographic column. The USP recommended column brand and model can be found on the USP website. However, China Pharmacopoeia only specifies the packing of chromatographic column, such as octadecylsilane bonded silica gel. Due to the lack of detailed guidance on column selection in Chinese Pharmacopoeia, it is difficult to implement the recommended methods. When a HPLC method is implemented in the reference, when the column used in the reference cannot be obtained, it is necessary to seek a column with similar selectivity instead.
What is the difference between C18 columns?
Octadecylsilane (often referred to as ODS or C18) is one of the earliest bonded stationary phases. C18 is a bonded phase formed by the reaction of silane with alkyl chain and silanol group on the surface of silica gel. Because the covalent bond and method are used to fix the alkyl chain tightly on the surface of silica gel, the alkyl chain will not be washed off by the mobile phase, and it will play a stable role as the chromatographic stationary phase. The selectivity of C18 column is different because of the different bonding mode, particle size, length and inner diameter. In this way, hundreds of C18 columns with different separation and selection characteristics have appeared on the market.
The inspection of related substances and components of melatonin a was added to the Chinese Pharmacopoeia, but the model and brand of C18 column were not specified. Wu Qun et al. [7] investigated the analytical effect of 8 kinds of C18 chromatographic columns on the A-component and related substances of maibaimycin tablets. Among them, only five C18 columns can separate the components of melatonin a well. The resolution of different C18 columns is different, which affects the determination of related substances and components.
Because of different manufacturers, C18 columns of the same specification will also have large analysis differences. Five kinds of C18 chromatographic columns with the same specifications and different manufacturers were used to determine the new related substances of thymus method. The results showed that the resolution and retention time of five kinds of C18 chromatographic columns were very different. The separation effect of some chromatographic columns does not even meet the requirements of Pharmacopoeia. The separation of hydrocortisone sodium succinate for injection and its related substances by three kinds of C18 columns of the same specification and different brands were studied. The peak sequence of two C18 columns is not in accordance with the Chinese Pharmacopoeia. It can be seen that sodium hydrocortisone succinate has a strong selectivity for column stationary phase. The main reason for the above phenomenon is the difference of particle size, pore size, bonding amount, filling technology and so on.
Selection of C18 column?
In analytical chemistry, the suitable C18 column can be selected according to pH environment, mobile phase polarity, column classification system, drug polarity, etc. Because in drug analysis, the polarity of drugs is generally unchangeable, so the following mainly introduces the selection of C18 chromatographic column according to pH environment, mobile phase polarity and chromatographic column classification system.
One
PH environment?
For the traditional C18 column, the range of pH value is usually 2-7, because the C18 stationary phase will be limited when pH value is higher than 8. Because of the existence of residual silicon hydroxyl, it is easy to cause irreversible adsorption on alkaline compounds, leading to peak tailing and affecting separation and analysis. If the pH value is too low, the bonded alkyl group will fall off, and the separation of components will not meet the requirements.
1. In order to improve the stability of C18 column at low pH, a steric hindrance method was proposed. Steric hindrance refers to the steric hindrance of the solute group to attack the siloxane group, which enables the regeneration of siloxane and improves the anti hydrolysis ability of the chemically bonded stationary phase. Others have prepared C18 chemically bonded stationary phase containing isopropyl or isobutyl in the side chain. Due to the introduction of large side chain groups, the solid interaction between the residual silanol group and solute is hindered, and it is stable at low pH value. This chemical bonded stationary phase is more suitable for the separation of acid substances. Zorbax stable bond C18 chromatographic column uses steric hindrance technology, which uses single functional group silane with large side chain group of diisobutyl (sb-c18). The key siloxane of steric hindrance is bonded to the surface of silica gel to avoid hydrolysis damage under low pH value and provide good stability and reproducibility under acid mobile phase conditions. Therefore, the stable bond column is the first choice for the development of low pH method. In the early stage of the method development, if the mobile phase is acid, this kind of column can be used as the first choice.
2. The selection of C18 under alkaline condition to produce chemical bonded phase for the rapid separation of basic compounds has been a research hotspot. Each silanized molecule of the bidentate bonded stationary phase contains two silicon atoms, which are connected by groups such as - O - or - ch2-ch2. Each silicon atom contains one C18 functional group. Its ring structure shows high stability and satisfactory column efficiency at low pH and high pH. Compared with the traditional single tooth chemical bonded phase, the most outstanding advantage of this chemical bonded phase is that it has good stability at low, medium and high pH values. Zorbax extend-c18 chromatographic column is used as the commercial column of C18 bidentate chemically bonded stationary phase. Its tolerance pH value is up to 11.5, which is especially suitable for the effective chromatographic separation of strong alkaline compounds under the condition of high pH mobile phase. In the development of drug analysis methods for procaine related substances, eight C18 columns were compared, and finally Zorbax extend C18 column with a wide range of pH value was selected. The analytical samples and impurities are all alkaline. Zorbax extend C18 is used to make the separation of impurities and components the best. If the separation effect between the basic substances is not good, the C18 column packed with the above-mentioned bonding phase can be considered.
Two
Mobile phase polarity
The technology of embedding polar groups into chemical bonded phase is a method to separate polar substances, which can be used for a long time under the condition of strong polar mobile phase. The symmetry shield TM RP C18 column is a reversed-phase column embedded with carbamate group. The HPLC analysis of berberine hydrochloride was carried out on the column (250 m m × 4.6 mm, 5 μ m). The mobile phase was strong polar water and berberine hydrochloride was basic compound. Compared with other chromatographic columns, the theoretical plate number of the above chromatographic columns is the highest and there is no tailing phenomenon. The bonding phase embedded with carbamate group not only prevents the interaction between residual silanol group and basic compounds, but also improves the wettability of water, which is especially suitable for the separation of basic substances when polar mobile phase is used.
Zorbax bonus RP C18 column was used to determine 5 nucleosides and 5 free bases in Fritillaria mollissima. In the experiment, the polarity of the mobile phase is large, and the acid-base degree of the separated components is quite different. The use of other C18 columns results in poor peak symmetry, short service life and tailing. Zorbaxbonus RP C18 column has polar amide group, side chain and diisopropyl group, which can provide space protection and avoid acid hydrolysis. Under the condition of polar mobile phase, it can separate not only basic compounds, but also acid compounds.
The components of Chinese herbal medicine are more complex and contain more polar non-polar components. To study the HPLC analysis of total saponins in xuechangning formula, the fusion-rp C18 column was used. Under the condition of strong polar mobile phase, the best separation of saponins with different polarity was achieved. The fusion-rp C18 column can be operated for a long time under the condition of 100% aqueous mobile phase because of its polar group and hydrophobic chain. It is especially suitable for the analysis of mixtures containing polar and non-polar compounds under the condition of strong polarity.
Three
Column classification system?
When a method is repeated, but the target column cannot be obtained for some reason, it is necessary to select a column with similar retention behavior to the target column to carry out the experiment. By characterizing the retention behavior of the column, the similar column can be selected quickly. The method of characterizing RP-HPLC has been widely studied since 1970s. Five parameters were used to characterize the column, four parameters were used, six parameters were used, and five parameters were used for USP. The closer the parameters of the two columns are, the more similar their selectivity is, indicating that they can be replaced. The chromatographic column classification system established by Luhan university is characterized by four parameters. The similarity of more than 100 columns (mostly C18) can be compared by the column selection system, which provides great convenience for the selection of C18 columns.
Under the Chinese Pharmacopoeia, octadecylsilane was used as the stationary phase for spiramycin, but no detailed information such as brand and specification was given. By using the chromatographic column classification system established by Luhan University, a suitable chromatographic column was selected, and the scientific and practical of the chromatographic column classification system established by Luhan University was verified by practical experiments. The column classification system established by Luhan University was evaluated by analyzing the related substances of afzosin. The separation conditions of 36 kinds of columns were compared in the experiment, and then 36 kinds of columns were judged by the column classification system. The results show that the column classification system can characterize the retention behavior of the column, and then can be used for column selection and comparison.
Epilogue?
The retention behavior of different C18 columns is very different, so choosing the right C18 column is the focus of drug analysis. A scientific method is needed to characterize and classify the chromatographic column, and then rapid selection can be carried out. At present, all kinds of new C18 bonding phases emerge in endlessly, so it is difficult to classify them from the type of bonding phase. Moreover, the column selection behavior has little to do with the type of bonded phase. Therefore, this paper only discusses how to choose the C18 bonding phase with good separation effect under the condition of acid and alkali and how to choose the durable C18 bonding phase under the condition of strong polarity.