The stationary phase in HPLC refers to the solid support contained within the column over which the mobile phase continuously flows. The sample solution is injected into the mobile phase of the assay through the injector port. As the sample solution flows with the mobile phase through the stationary phase, the components of that solution will migrate according to the non-covalent interactions of the compounds with the stationary phase. The chemical interactions of the stationary phase and the sample with the mobile phase, determines the degree of migration and separation of the components contained in the sample. For example, those samples which have stronger interactions with the stationary phase than with the mobile phase will elute from the column less quickly, and thus have a longer retention time, while the reverse is also true. Columns containing various types of stationary phases are commercially available. Some of the more common stationary phases include: Liquid-Liquid, Liquid-Solid (Adsorption), Size Exclusion, Normal Phase, Reverse Phase, Ion Exchange, and Affinity.
Liquid-Solid operates on the basis of polarity. Compounds that possess functional groups cabable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compoounds. Thus, less polar compounds will elute from the column faster than compounds that are highly polar.
Liquid-Liquid operates on the same basis as liquid-solid. However, this technique is better suited for samples of medium polarity that are soluble in weakly polar to polar organic solvents. The separation of non-electrolytes is achieved by matching the polarities of the sample and the stationary phase and using a mobile phase which possesses a markedly different polarity.
Size-Exclusion operates on the basis of the molecular size of compounds being analyzed. The stationary phase consists of porous beads. The larger compounds will be excluded from the interior of the bead and thus will elute first. The smaller compounds will be allowed to enter the beads and will elute according to their ability to exit from the same sized pores they were internalized through. The column can be either silica or non-silica based. However, there are some size-exclusion that are weakly anionic and slightly hydrophobic which give rise to non-ideal size-exclusion behavior.
Normal Phase operates on the basis of hydrophilicity and lipophilicity by using a polar stationary phase and a less polar mobile phase. Thus hydrophobic compounds elute more quickly than do hydrophilic compounds.
Reverse Phase operates on the basis of hydrophilicity and lipophilicity. The stationary phase consists of silica based packings with n-alkyl chains covalently bound. For example, C-8 signifies an octyl chain and C-18 an octadecyl ligand in the matrix. The more hydrophobic the matrix on each ligand, the greater is the tendancy of the column to retain hydrophobic moieties. Thus hydrophilic compounds elute more quickly than do hydrophobic compounds.
Ion-Exchange operates on the basis of selective exchange of ions in the sample with counterions in the stationary phase. IE is performed with columns containing charge-bearing functional groups attached to a polymer matrix. The functional ions are permanently bonded to the column and each has a counterion attached. The sample is retained by replacing the counterions of the stationary phase with its own ions. The sample is eluted from the column by changing the properties of the mobile phase do that the mobile phase will now displace the sample ions from the stationary phase, (ie. changing the pH).
Affinity operates by using immobilized biochemicals that have a specific affinity to the compound of interest. Separation occurs as the mobile phase and sample pass over the stationary phase. The sample compound or compounds of interest are retained as the rest of the impurities and mobile phase pass through. The compounds are then eluted by changing the mobile phase conditions.
Liquid-Solid operates on the basis of polarity. Compounds that possess functional groups cabable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compoounds. Thus, less polar compounds will elute from the column faster than compounds that are highly polar.
Liquid-Liquid operates on the same basis as liquid-solid. However, this technique is better suited for samples of medium polarity that are soluble in weakly polar to polar organic solvents. The separation of non-electrolytes is achieved by matching the polarities of the sample and the stationary phase and using a mobile phase which possesses a markedly different polarity.
Size-Exclusion operates on the basis of the molecular size of compounds being analyzed. The stationary phase consists of porous beads. The larger compounds will be excluded from the interior of the bead and thus will elute first. The smaller compounds will be allowed to enter the beads and will elute according to their ability to exit from the same sized pores they were internalized through. The column can be either silica or non-silica based. However, there are some size-exclusion that are weakly anionic and slightly hydrophobic which give rise to non-ideal size-exclusion behavior.
Normal Phase operates on the basis of hydrophilicity and lipophilicity by using a polar stationary phase and a less polar mobile phase. Thus hydrophobic compounds elute more quickly than do hydrophilic compounds.
Reverse Phase operates on the basis of hydrophilicity and lipophilicity. The stationary phase consists of silica based packings with n-alkyl chains covalently bound. For example, C-8 signifies an octyl chain and C-18 an octadecyl ligand in the matrix. The more hydrophobic the matrix on each ligand, the greater is the tendancy of the column to retain hydrophobic moieties. Thus hydrophilic compounds elute more quickly than do hydrophobic compounds.
Ion-Exchange operates on the basis of selective exchange of ions in the sample with counterions in the stationary phase. IE is performed with columns containing charge-bearing functional groups attached to a polymer matrix. The functional ions are permanently bonded to the column and each has a counterion attached. The sample is retained by replacing the counterions of the stationary phase with its own ions. The sample is eluted from the column by changing the properties of the mobile phase do that the mobile phase will now displace the sample ions from the stationary phase, (ie. changing the pH).
Affinity operates by using immobilized biochemicals that have a specific affinity to the compound of interest. Separation occurs as the mobile phase and sample pass over the stationary phase. The sample compound or compounds of interest are retained as the rest of the impurities and mobile phase pass through. The compounds are then eluted by changing the mobile phase conditions.
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