Use Of HPLC Column
Basic structure: chromatographic column including HPLC column tube, pressure cap/sleeve, the screen plate (filter), joint, protection column, protection column sleeve, protection column core.
4mm conventional analysis HPLC column, 3.9mm conventional analysis column, 4.6mm conventional analysis column, 1.0mm narrow diameter column, 2.1mm narrow diameter column, 0.2-0.5mm capillary column.
The preparation of type
Semi-prepared column (5mm, 7.8mm, 9.8mm, 10mm), prepared column (21.2mm, 30mm, 50mm).
1. Silica gel matrix – pH 2-8 — at high or low pH, silica gel dissolves
2. Al2O3·nH2O — ph1-14 — chemical modification is difficult
Polymer matrix – ph1-14 — the pore structure is complex, the pore size is not uniform, the column effect is not high enough, the organic solvent may cause the polymer matrix swelling and damage.
A: physical factors
1. Silica gel purity — the purity and residual metal ion concentration of silica gel filler.
2. Column size — packed bed length and inside diameter.
3. Particle shape — irregular shape and round shape.
4. Particle size — average particle diameter, usually 3-10 m.
5. Surface area — the sum of the outside surface and the inside surface of the particle, expressed as m2/gram.
6. Aperture — the average size of a particle’s hole or cavity, range 80-300a.
B: chemical properties
1. Bond type — monomer bond — bond phase molecule is connected to the matrix at a single point.
2. Carbon coverage — the amount of bonding phase connected to the substrate.
3. End seal — after the bonding step, use a short chain to seal the exposed silica hydroxyl group.
Before choosing the chromatographic column, you should know more about your samples and impurities, their types, structures, polarity, acids and bases, molecular weight, etc.
1. The sample is polar and slightly acidic; Therefore, C18 HPLC column can be selected for detection in 100% acidic aqueous solution, that is, it should be selected to withstand 100% pure water and retain a good chromatographic column for polar compounds.
2. If the sample polarity is too strong, or the acidity is too strong, can choose CN, NH2, or silica gel column, HILIC (hydrophilic chromatography), there are also using C18 + strong anion of reagents or strong anion exchange chromatography column (defect is ion pair reagent balance time is long, the mobile phase pH requirements more precise, whether it is hard to repeat the experiment, it is difficult to wash down other ion-pairing reagent makes it, basically, with ion pair chromatography column) cannot be used for other experiments.
3. If the sample is alkaline; Can choose high purity silica gel column (high purity silicon lack of metal impurity, and silica gel base sealing) or a modified C18 column some (such as polarity or alkali to live embedded technology, etc.), they will reduce the trailing alkaline compound, usually choose neutral or alkali condition, because it can increase the alkaline reserved sample.
4. If the polarity of alkaline compounds is too strong, or too basic; Can choose wide pH C18 chromatographic column at high pH value detection (advantage is simple method development, the disadvantage is that be difficult to implement this technology at present, the price is high also) or choose HILIC chromatographic column (silica gel column under the condition of reverse phase is used, it is also a method to detect alkaline samples of the classic) choose strong ion exchange column, the disadvantage is that can’t be used to analyze other samples, the mobile phase pH requirements more precise, it’s hard to repeat the experiment) are using C18 + strong anion of reagents or strong anion exchange chromatography column.
How does the presence of bubbles in column chromatography affect the separation of samples? If there is a bubble, will reduce the separation ability, because the presence of the bubble phase to reduce the action site, reduce the force between the packing and the material. Therefore, in order to avoid the occurrence of this situation, when loading the tomographic column, the column wall should be continuously knocked and interference should be avoided.