# Selected Chromatographic Methods for Determining the Biological Activity of Substances
## 2 Biomimetic Chromatographic System
### Stationary Phases in Biomimetic Chromatographic Systems  
Substances that enter the body interact with biological membranes--cell membranes consisting of a phospholipid bilayer, with transport proteins in the blood and with membrane receptors. Until recently, the most popular biomimetic phases in chromatography were non-polar stationary RP phases with aliphatic ligands C-18 and C-8. Such phases mimic the non-polar interior of a biological membrane. These phases are used in reversed phase chromatography (RP) and micellar liquid chromatography (MLC) [30; 31; 32; 33]. Other stationary phases that mimic cell membranes include IAM (Immobilised Artificial Membrane) and phases called IAM.PC DDC, which were developed by [PERSON] [34]. These phases are manufactured on a silicon dioxide base with phosphatidylcholine, the main component of cell membranes. The matrix of the IAM phase is silicon dioxide with chemically bound phosphatidylcholine molecules, which form a structure on their surface that is identical to the monolayer of the cell membrane (Figure 1). The retention values of substances obtained on such columns illustrate the interactions of the molecules of the substance with phospholipids--the affinity to phospholipids is essential for the permeation of substances through biological membranes [35; 36]. High retention characterises molecules that have a high affinity for phospholipids--too high an affinity for phospholipids reduces the permeability of the compound, as it prefers to remain within the cell membrane. Such compounds are not only poorly absorbed and distributed in the body but can also disrupt the cell membrane or disrupt phospholipid metabolism in the cells (phospholipidosis). New stationary phases with phospholipids are those with immobilised sphingomyelin (SPH) [37] and phosphatylethanolamine. Stationary phases with phospholipid bilayers are immobilised--liposomes are stationary phases [38; 39]. Stationary phases with active cell membranes--cell membrane stationary phases (CMSP)--are used to study the interactions of substances with membrane receptors. Fragments of cell membranes are deposited on the surface of silica or a polymer by physical adsorption, whereby the structure of the cell membranes is preserved and the spatial structure of the membrane receptors, their environment, and the enzyme activity remain unchanged [40; 41]. Currently, CMSP phases are obtained from cell cultures with high expression of a specific membrane receptor. Cholesterol phases are another stationary phase that contains an important component of the cell membrane. Like the IAM phases, they are usually composed of a silica base with chemically bound cholesterol molecules (Figure 1). Chromatography with stationary membrane phases is known as biomimetic phospholipid membrane chromatography (BPMC) [42].  
In stationary phases with immobilised proteins, interaction with proteins plays an important role in the pharmacological activity of a substance. The binding of a drug to proteins influences the volume of distribution of the drug, the absorption, and the duration of action. The most common stationary phases are those with immunised albumin and globulins [41; 43; 44]. Studies have shown that proteins in such stationary phases retain their most important properties, including sorption properties that determine their interaction with substances, including biologically active substances. When using columns with immobilised proteins, it is important to check that the proteins are not deactivated during the experiment. Many researchers recommend injecting a racemic warfarin mixture before each experiment to check the availability of the main HSA binding site--if the racemates separate, it means that the warfarin binding sites are active.