Top ten rumors of chromatographic column! You have to read t
Myth 10: air will completely destroy an HPLC column - false!
When the column is not connected to the chromatograph, the user needs to ensure that the column is tightly sealed. In fact, in practical application, even if a small amount of air enters the end of the column, it doesn't matter. Because when you connect the column to the chromatograph, the air will be washed away by the solvent in a very short time during the initial pressure stage of the system. Therefore, do not assume that the column has been damaged because the column has entered the air.
Myth 9: all C18 (L1) columns are the same - false!
USP has developed a classification system for each type of bonded phase column. Because C18 column is a widely used column, it is called "L1" in this system. Unfortunately, more than 800 kinds of L1 have entered the market. Therefore, the system has proved to be unreliable and confusing.
Because each commercial C18 chromatographic column, although silica gel is also selected as the matrix, has its own specific packing bonding process, so the chromatographic performance is not the same. For example, some manufacturers use octadecyl monochlorosilane bonding reagent and silica gel with low surface area (see Figure 1), while others use the same silane reagent but silica gel matrix with higher surface area. The chromatographic properties of the two C18 columns are different, and the binding ratio of the latter C18 stationary phase is larger than that of the former. With a lower fixed phase bonding ratio, there are more unreacted silanol groups on the surface, and sometimes the mixed retention mechanism plays an important role. Some column producers use dichlorosilane and trichlorosilane as bonding agents to polymerize the bonded phase to form a thick hydrophobic layer with different diffusion characteristics. In order to reduce the proportion of unreacted silanol group on the surface of silica gel after bonding, some small-molecule silane reagents (such as trimethylchlorosilane) are used for tailing. Silicone alcohol group is one of the reasons for tailing of chromatographic peak when determining alkaline substances under neutral conditions. Some manufacturers further adopt the second small molecule silane double tailing process to provide a more inert (hydrophobic) silica surface. In addition, some manufacturers use polymer matrix materials for C18 bonding to produce a completely different C18 filler, which is still classified as "L1". Other manufacturers use organoalkoxysilanes with different reaction properties to produce a C18 stationary phase which is different from that produced by chlorosilane reaction.
Myth 8: reversed phase chromatographic column can not use pure water phase - false!
This misunderstanding actually stems from the phenomenon of phase collapse when some users use low organic solvent content or pure water as the mobile phase of reversed-phase chromatographic column, so we think that reversed-phase chromatographic column cannot use pure water phase. Many chromatographic workers are troubled by the phenomenon of phase collapse and retention time displacement, and even have lost the patience to find solutions. Therefore, they think that the mobile phase with high water content should not be operated in the reversed-phase chromatographic column. However, in fact, the reverse phase chromatographic columns (such as polar embedded and polar capped columns) sold on the market are all water infiltrating, and their surface characteristics allow the use of pure water without causing collapse or displacement of retention time. For example, the c18-paq series of cosmosil can use 100% water as mobile phase, which is suitable for the separation of hydrophilic compounds. Compared with the traditional octadecyl bonded silica gel column, it has a strong acid resistance.
Myth 7: it takes at least 10 column volumes to rebalance the LC column - false!
Equilibrium time is very important for gradient chromatography because it is a limiting factor for the whole technique. There are two types of equilibrium: repeated equilibrium and complete equilibrium. Repeated balance also means the balance that can be achieved when complete balance cannot be achieved. In fact, if the repeatability of retention time is less than 0.002min in the subsequent operation, then for the non slow rinse remover of non ionized solute and basic compound with the commonly used trifluoroacetic acid and formic acid as additives, the repeated balance can be achieved within the range of two column volumes.
Myth 6: compared with pure porous particles, porous particles on the surface will significantly reduce the sample capacity - false!
The capacity of HPLC column packing to sample is directly proportional to its surface area, which is related to the amount of chemical bonding phase formed by the monomer bonding of silanol group. However, the results show that under the same experimental conditions, the capacity of porous particles on the surface is basically the same as that of porous particles on the sample.
Myth 5: UHPLC packing column is easier to block than common HPLC packing column - false!
With the decrease of the porous material particle size, the main hardware design of the column changes with it. For the blockage of sub-2 μ muhplc column, it is the result of the sample and mobile phase substance lodging at the column inlet. If you clean the sample before injection, such as using solid-phase extraction, filtration or centrifugation, you can avoid any pollution problems.
Myth 4: there is no need to protect the pillar - false!
There are many advantages to using protective posts. First of all, the protection column can prevent chemical substances or particles from damaging the analytical column. Second, it costs less to replace a 5mm protection column than to replace an expensive one. The modern protective column has the advantages of almost no dead volume, rapid replacement, and high pressure suitable for UHPLC.
Myth 3: you can't reverse the HPLC column to wash out the particles - false (but sometimes true)!
In fact, the filling pressure of HPLC column is much higher than the maximum use pressure (usually 2 times higher). If a proper homogenizer is used and a certain time is allocated to stabilize the column bed, a well packed column can be used in both directions.
Basically, there is a - > mark on the liquid chromatographic column, indicating the flow direction of the mobile phase, and some of them are printed with "nerver lot flow in the It is controversial whether the column can be recoiled or not. But in our work, using the column recoil technology to repair the column can improve the column efficiency and restore the column efficiency.
1、 The reaction efficiency of the ion exchange column for amino acid analysis decreased obviously after using for a period of time, but it could not meet the requirements when it was activated by NaOH, but the column efficiency could be recovered quickly when it was activated by NaOH
2、 After the chromatographic column has been used for a period of time, the column efficiency will decrease. At this time, the screw cap at the column inlet end can be removed, 1-2mm contaminated packing can be removed, and then the same packing can be used to fill up, and then the column efficiency can be restored by flushing with the opposite flow, because the backflushing can improve the dead space of the column head.
3、 Because the fine particles deposited on the transition plate can be washed away by recoil, sometimes the column pressure can be reduced by recoil technology.
An exception to reverse column use is when the manufacturer uses a sieve plate with a larger aperture at the injection end of the column, which may flush the packing out of the column bed. If the manufacturer uses high porosity glass material at the column inlet, the column packing may be washed off the packed bed if the column is recoiled. When the chromatographic column is filled in the factory, the sieve plate aperture at the outlet end must be smaller than the particle size of the smallest particle in the chromatographic column. For example, the average particle size of chromatographic packing is 5 μ m, the particle size distribution range is 3-7 μ m, and the outlet sieve plate aperture must be less than 3 μ m, which makes it impossible for the packing to run from the column bed to the outside of the chromatographic column sieve plate. The sieve plate aperture selected by most manufacturers is 2 μ M. As for the different apertures of the column sieve plates at both ends of some manufacturers, the injection end is generally larger and the output end is smaller. As a result, some manufacturers place an arrow at the column label indicating that it must only be used in one direction. It is certain that there is such a possibility, so a chromatographic worker should carefully read the column manual or instructions, or determine with the manufacturer whether the column can be recoiled.
Myth 2: the smaller the packing particle size and the higher the pressure, the better the separation effect - false!
Ultra small particle size and ultra-high column pressure are not necessarily the best choice for chromatographic workers!
The study of column characteristics of modern chromatographic columns has led to some new methods to evaluate the performance of a column. For example, the column with new porous surface material has the same column efficiency as sub-2 μ m UHPLC, but the column pressure is very low compared with the conventional LC packed column.
Myth 1: column pressure will not affect the chromatographic separation effect - false!
Many parameters of chromatography are affected by column pressure, including molar volume, stagnant volume, column porosity, retention factor, mobile phase density, dielectric constant, fixed phase structure, pH value and ionization constant of some solutes. Why has column pressure attracted more and more attention? The reason is that many of the commercial chromatographs are ultra-high pressure chromatographs and chromatographic columns. When the chromatographic column is operated at a pressure of about 2000 psi (13.789 MPa), even if there is a small difference in retention time, it will not attract our attention, especially if the repeatability is good and the quantification is not affected. However, when the column pressure is close to 2000 psi (13.789 MPa), the effect of column pressure may be quite obvious.
