Reversed phase chromatographic column -- physical and chemic

1、 Definition
C18: the general term for the reversed phase stationary phase connected with Octadecyl carbon chain. C18 also includes fillers of other substrates, such as polymer matrix, alumina and zirconia as matrix, which are formed by bonding C18 chain. These can be called "C18".
ODS: octadecyl silane is the abbreviation of Octadecyl silane. It is a C18 filler bonded with silica gel.
RP18: RP here means "reverse phase", which means "reverse phase".
2、 Physicochemical properties of inverse filling material
Matrix: generally, silica gel is used for small molecule separation due to its high rigidity and low pH value. There are also polymer matrices with low rigidity, complex pore structure and uneven pore size, resulting in low column efficiency and strong hydrophobicity. The whole pH range is stable. Organic solvents may cause swelling and damage of polymer matrix, which is mostly used for macromolecular separation. There are also alumina and zirconia matrix.
The bonding phase (stationary phase, functional group): alkyl (C1 ~ C18, C30), with equal polar intercalation. Characteristics of AQ column: polarity sealing, polar embedding, low carbon loading, retention of silicon hydroxyl group, increase of polar embedding bonding phase, no hydrophobic collapse phenomenon (retention time drops sharply), 100% aqueous phase and low pH conditions can be used. Isopropyl siloxane and other alkylating agents can increase the space position of side chain, resist hydrolysis and increase the stability of bonding phase, pH 1-9.
Column shape and column shape can be reduced. For analysis, spherical packing is the best choice for reproducibility and high stability. The stability of the column bed is worse than that of the spherical particles. However, the particle size and specific surface area of the fillers are large and the price is low, which makes the amorphous particles attractive in large-scale preparation.
Purity: it is related to the concentration of residual metal ions, type a silica gel (with low purity) and type B Silica Gel (with high purity and weak acidity). Advantages of high purity silica gel column: good peak shape, high reproducibility and long service life.
Pore size: unit Å, 60-1000 Å. The packing particles with large pores can prolong the retention time of solute macromolecules on the surface of fillers, achieve full separation and improve the peak shape. The smaller pore size can provide higher specific surface area, which can be used for larger loading capacity and keep small organic compounds. The pore size of sample MW < 4000 should be about 80 μ and that of sample MW > 4000 should be about 300 μ.
Particle size: in μ m, smaller particles have higher column efficiency, but higher column pressure will be caused. The analytical semi preparation is generally 3-10 μ m, and the preparation is generally more than 20 μ M.
Specific pore volume (specific pore volume): the unit is ml / g. the specific pore volume is a measure of particle voids. The larger the specific pore volume, the smaller the proportion of the actual material in the particles. Therefore, the strength of the particles with larger specific pore volume is smaller. The specific pore volume of silica gel filler is 0.5ml/g or less, such as waters spherisorb, which is extremely durable in mechanical aspect. The specific pore volume of most HPLC grade silica gel fillers is 1 ml / g.
Specific surface area: unit M 2 / g, 200-450 M 2 / g. the packing with high specific surface area has stronger adsorption, longer sample retention time and higher sample loading; the packing with low specific surface area can reach equilibrium rapidly, which is important for gradient elution.
PH range: silica gel is easy to dissolve under alkaline conditions (resulting in the formation of a cavity at the front end of the column, resulting in abnormal and bifurcated chromatographic peaks and decreased column efficiency), and the bonding phase is easy to hydrolyze under acidic conditions. The results showed that the non bonding ph1-8, the traditional silica gel ph2-7, the modern high-purity silica gel ph2-8, the biphasic or triphasic bonding ph2-9 (generally resistant to 100% water), and the three-phase bonding can obtain better stability at low pH. Wide pH column (pH 1-12): cross-linking technology, beh hybrid particle technology, embedded silicone modification technology, polymer coating technology. The wide pH column has good stability, good peak shape and long life under high temperature.
Temperature range: the chromatographic column with reversed-phase silica gel bonded packing is generally at 60 ℃, and the bonded packing of some reversed-phase chromatographic column will not hydrolyze obviously at 120 ℃, and the performance of chromatographic column will not change too much.
Compared with the low temperature (0 ~ 15 ℃), the higher column temperature (35 ~ 50 ℃) can significantly reduce the column pressure, accelerate the mass transfer rate and improve the column efficiency.
Mechanical stability and pressure resistance: unit bar, silica gel has high mechanical strength. Silica gel with 5 μ m particle size and 60-100 μ m pore diameter can withstand the operating pressure of 60-70mpa; with the increase of pore size and pore size, the tolerable pressure will decrease. When the silica gel with 300-1000 μ m pore diameter is used, it can still withstand the operating pressure of 30MPa without damage. Back pressure: confirm the initial pressure of the new column, and judge whether the chromatographic back pressure is normal according to the standard in the later stage.
Separation flow rate: the maximum flow rate depends on the maximum withstand pressure of the liquid phase. Without considering the separation effect (number of plates and resolution), only the column pressure is considered. As long as the instrument and chromatographic column can tolerate, the flow rate can be increased. The optimal flow rate mainly depends on the separation effect of chromatographic peak (number of plates and resolution), which depends on the inner diameter of chromatographic column and the size of packing particles. In addition, the performance of instrument pump (some pumps have poor performance when the flow rate is low), the performance of detector and the separation time of single needle injection are also considered. According to vandermt's equation, for the column with 5um packing, the optimal flow rate corresponds to about 6cm / min, 1ml / min corresponds to the optimal flow rate of 4.6mm inner diameter column, and 1.18ml/min corresponds to the optimal flow rate of 5mm inner diameter chromatographic column. When inputting the flow rate on the chromatograph, it is more convenient to input 1ml / min than to input 1.18ml/min, so the inner diameter is designed as 4.6mm instead of 5mm. 3.0 mm inner diameter corresponds to 0.4 ml / min optimal flow rate, 2.1 mm inner diameter corresponds to 0.2 ml / min optimal flow rate, 1.0 mm inner diameter corresponds to 0.05 ml / min optimal flow rate. The optimum velocity range is used in practical application.
Chromatographic column specifications: 20 * 150 / 250 and 10 * 150 / 250 (semi preparative and multi-purpose), 8 * 50 / 300 and 6 * 150 / 250 (multi-purpose with ruler), 4.6 * 75 / 100 / 150 (reversed phase multi-purpose), 4.6 * 250 / 300 (multi-purpose column with ruler), 3.0 * 10 (protective column) - 250 (useful for Showa HILIC), 2.1 * 50 (for UPLC).
Bonding methods: single phase bonding, biphasic bonding, three-phase bonding, etc., single-phase bonding improves the mass transfer rate and accelerates the column balance; biphasic bonding increases the stability of chromatographic column and increases the sample load of chromatographic column.
Tail sealing method: the active silicon hydroxyl group and the basic group will have the ion exchange reaction, which increases the retention and leads to the tail shape of the peak. After the bonding step, the exposed silicon hydroxyl group is bound with a short chain, which has a great impact on small molecule compounds.
In general, the ratio of C to C in the reaction is small.
Note: chemical properties of 13-1 column are 13.
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