Most of the time, the laboratory partners carry out experiments according to the standards, but the sentence “using octadecyl silane bonded silica gel as filler” on the standard is enough for the partners to choose difficult patients, let alone do the experiment of method development by themselves. Indeed, chromatography is an experimental discipline, and no one can predict which chromatographic column can complete the separation task without doing experiments, especially when the standard does not clearly recommend the brand and model of chromatographic column, which also leads to many partners often have so many os:c18 columns in their hearts, which can be divided best?
Don’t worry, although we can’t accurately predict the experimental results, we can still do it by grasping the basic chromatographic column performance parameters. Now let’s listen to welch.
01 Carbon load
As we all know, reversed-phase C18 still undertakes a large proportion of separation tasks in today’s chromatographic separation field, because its nature is suitable for the chromatographic separation of most compounds. When selecting chromatographic columns, we must pay attention to the applicability of the chromatographic columns themselves, and choose chromatographic columns with strong universality and wide application as far as possible, and the parameter “carbon load” of C18 column is one of the particularly important parameters.
For a C18 chromatographic column with strong universality, the best carbon load parameter is between 10% and 20%, especially around 20% (16% – 22%), because such a slightly higher carbon load will reduce the number of active sites and the occurrence of residual adsorption on the premise of ensuring the effective retention of compounds. In addition, the bonding density slightly higher than that of ordinary C18 also helps to improve the selectivity of chromatographic bonding phase.
02 Silica gel purity
Except for special cases, the more suitable the filler is, the more B-type silica gel should be selected. The purity of type B silica gel is more than 99.999%, the content of metal ions is less than 10 ppm, and the pore size and particle size distribution are more uniform. In this way, the chromatographic peak broadening is smaller and the peak shape is more symmetrical.
03 Tail sealing
Different brands of chromatographic columns have different tailing processes and tailing reagents, which is one of the reasons why they are all C18, but their properties are quite different. Unless otherwise specified in the standard, the unsealed chromatographic column is used. Generally, the end capped chromatographic column, especially the double capped chromatographic column, is preferred for experiments. As for the sealing of chromatographic column, the young partner who has questions quickly went to make up a missed lesson: “sealing the end”, my concubine really said that she was tired!
04 Specific surface area
Many partners may intentionally or unintentionally ignore the specific surface area of the chromatographic column, which is also a very important parameter affecting the performance of the chromatographic column. The size of the specific surface area will affect the coverage of the bonding phase and the probability of the interaction between the target and the bonding phase. The size of specific surface area will also affect the length of gradient equilibrium time. Then, when choosing a column with strong universality, try to choose a specific surface area of 180m ²/ g~450m ²/ G chromatographic column, like the common 320m ²/ g，350m ²/ g，420m ²/ g，450m ²/ g. Are applicable to a wide range of areas.
The choice of pore size depends on the molecular weight. Generally speaking, the pore diameter is required to be more than three times the molecular diameter, so that the molecules can enter the pore smoothly and retain. Taking the molecular weight 2000 as the boundary, if the molecular weight is less than 2000, the pore diameter of 100 Å, 120 Å, 150 Å can be selected; Molecular weight > 2000, for the separation of macromolecules such as polypeptides and nucleic acids, chromatographic fillers with 300 μ m or even larger pore size can be selected.
06 Specification of chromatographic column
If there is a specific specification of chromatographic column specified in the standard, there is no need to say more. However, when we are doing method development, we can’t know what specification is the most suitable, and the difference of column length, inner diameter and particle size will affect the system applicability values such as column efficiency, resolution and retention time. Improper selection may also cause too high column pressure or pollution. Therefore, to be on the safe side, partners can choose 4.6 at the beginning × 250mm，5 μ M chromatographic filler, so 5 μ The column pressure of M particle size will not be very high, which is also convenient for washing and maintenance; The column length is long enough, and the multicomponent sample also has a certain separation efficiency; The inner diameter of the column is not too small to cause overload.
07 Special group
Special groups generally refer to special functional group reagents added in the bonding process or tail sealing process of chromatographic columns in order to make chromatographic fillers have unique characteristics and different selectivity. The addition of these reagents will certainly help to separate some components that are not easy to separate, but it is not recommended to choose chromatographic columns with special groups during routine experiments or the initial stage of method establishment, otherwise the reproducibility of chromatographic separation may become a potential problem in subsequent experiments.
How about, through the above introduction, does the little partner now have a more comprehensive understanding of the parameters of the chromatographic column? In the future, when choosing the chromatographic column, he will also have a more sense of direction, and he won’t know how to choose like “seeing flowers in the fog”.