This the following series of our Q&A about Liquid Chromatography that was published before. This part will cover some more Q&A, which will help you to know more about liquid Chromatography.


Q: What are the column techniques? For example, endcapped and so on, where are these technologies reflected in the application?

A: Chromatographic column technology includes packing materials preparation, end-capping technology and packing technology etc. It goes without saying that packing technology has a decisive influence on the separation performance and selectivity of the chromatographic column, and the difference in the density of the bonded phase of the chromatographic packing will also affect how much silanol is exposed on the surface of the packing materials , which in turn affects the selectivity of the packing materials. The difference in the end-capping reagents used in the end-capping technology will also have a great impact on the properties of the chromatographic column, such as the pH tolerance range of the chromatographic packing, the water phase tolerance range, and the polarity etc. The packing technology is not as simple as imagined. Different stationary phases, different particle diameters, different inner diameters and lengths of the column tubes have different packing techniques. Pack a compact, stable and uniform column bed requires accumulation of experience.


Q: Under what circumstances does the column need to replace the frit? Will replacing the frit reduce the service life of the chromatographic column?

 A: If the chromatographic column frit or the packing materials at the column end is polluted by the sample, it is necessary to replace the frit and the packing materials at the column head end. Welch Materials Inc does not recommend that customers disassemble the column head screws by themselves. It is best to send them back to the manufacturer for maintenance. The replacement of frit and column head packing of chromatographic column will not affect the service life of HPLC column.


Q:If the column is taken down and placed for a period of time, what protection needs to be done?

A: For the general reversed-phase column, that is, after washing, put it in pure methanol (acetonitrile) or about 80% methanol (acetonitrile), and then plug both ends of the column with plugs to avoid volatilization of the preservation solvent. Take it out and reactivate it before next use.


Q: Is it better to use a guard column for the experiment?

A: It should be said that adding a guard column will definitely help protect the chromatographic column from blockage by some particulate matter. If the guard column is connected well and its compatibility is controlled as much as possible and replaced frequently, it will not affect the seperation and column efficiency. Just be careful when choosing a guard column cartridge, choose a guard column cartridge that is consistent with the analytical column packing material, otherwise it is very likely to affect the analysis of the compound.


Q: A quaternary gradient pump is used: A: 50% methanol; B: 50% water. Why does it often stop or enter bubbles?

A: When the water/methanol ratio is 55/45, the viscosity and column pressure have a maximum value. 50/50 is close to this extreme value, and the column pressure is relatively high. But the biggest influence on column pressure is the particle size of the packing material and the inner diameter of the chromatographic column. The system pressure is high, and bubbles may enter the system because the filter head in the solvent pump cannot keep up with the liquid supply speed. The shutdown should also be due to the drop in bubble entry pressure. Consider replacing the filter head with a larger liquid flux.


Q: Can you give a brief description of the four filling methods?

A: The data shows that under normal conditions, when the filler particle size is > 20 µ m, the dry packing preparation column is more appropriate; When the particles are less than 20 µ m, wet packing is ideal. There are generally four packing methods:

① High pressure homogenization method is mostly used for the packing of analytical columns and small-scale preparation columns;

② Radial pressure method.

③ Axial compression method is mainly used for packing large diameter columns, such as DAC;

④ Dry column filling is highly technical, and most laboratories use this packing for commercial columns.


Q: If use peek fitting instead of stainless steel joint, can you solve the joint matching problem completely?

A: Most of the column joints are not produced by the column manufacturers themselves. There are many suppliers, such as Valco, Parker, etc. Their standards are not unified with each other, so the standard of the column joints can not be unified. But generally this problem is not difficult to solve, it is ok to change the joint, and now there is a universal joint, can match all different types of stigma, do not leak, connection dead volume is very small.


Q: When making polypeptide drugs, mobile phase A: 0.1% or 1%TFA aqueous solution, mobile phase B: 0.1% or 1%TFA acetonitrile solution, the baseline fluctuation is large, and no main peak can be seen in the mobile phase without TFA, the baseline is good.

A: This is clearly the baseline fluctuation caused by the addition of TFA to the mobile phase and the gradient. TFA is superior to other ionic modifiers because it is volatile and can be easily removed from prepared samples. On the other hand, THE MAXIMUM uv absorption peak of TFA is less than 200nm, so it is prone to baseline interference at low wavelengths. The solution may be suggested by adding a solution of the other mobile phase in a medium proportion to one of the mobile phases.


Q: How to flush the column after using TFA mobile phase?

A: The use of TFA as an ion-pair reagent in the separation of peptides and proteins by reverse phase chromatography with the addition of mobile phase is a common method. TFA in the mobile phase interacts with hydrophobic bonded phases and residual polar surfaces in a variety of modes to improve peak shape and overcome peak broadening and tailing problems. Because TFA is also an ion-pair reagent, it is best to also flush the column using the ion-pair reagent flushing procedure, i.e. back flush the column with 50% methanol water overnight at a low flow rate. For analyzing protein samples, back flush overnight at a low flow rate of 10% methanol-acetonitrile/water /TFA (50/50/0.1) is recommended.


Q: According to the pharmacopoeia, the pore size of column packing materials is 300Å for samples with molecular weight greater than 2000. What is the difference between 300Å and 100Å for the results?

A: When doing polypeptide samples, the 300Å pore size of the packing materials is certain to have a better selectivity than 100Å pore size, that is, the separation degree is relatively good. Because the molecular weight of protein > 2000, it will be difficult to enter the hole of 120Å packing, so there is no retention, only remian on the surface of the packing and come out with the solvent. When selecting packing materials, we generally require the pore size to be at least three times the molecular diameter to ensure that molecules can enter the pore.

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