According to statistics, recently80%Organic and inorganic compounds can be separated by high-performance liquid chromatography, with the reverse phase chromatography being used forC18Chromatography column is the most commonly used type of chromatography column in high-performance liquid chromatography. Below isC18Introduce the selection, maintenance, and repair of chromatographic columns.

1C18Selection of columns

C18The main considerations for selection are2The question is about the influence of column packing and column specifications on chromatographic columns.

The physical properties of column packing have a significant impact on the chromatographic behavior of packing. The main physical properties of fillers include particle size, pore size, pore volume, bonding phase chemistry, carbon content, and alkylation treatment.

(1)Particle size refers to the size of the particle diameter of the column packing. In fact, the particle size marked on the chromatographic column is an average value. Like particle size“5Mm”Not all particle diameters of the packing in the column are5MmActually, there is a particle distribution degree. This distribution degree plays an important role in column back pressure and column effect. Generally speaking, the smaller the average particle size, the smaller the particle distribution, the higher the chromatographic column efficiency, and the higher the back pressure. currentlyC18The particle size of column packing is4～10Mmbetween.

(2)The aperture refers to the pore gap between the filler particles. The commonly referred pore size refers to the average pore size of the filler. The average pore size distribution of spherical packing after column installation is relatively narrow, the column bed structure is uniform, the column efficiency is high, and the reproducibility is good; The average pore size distribution of amorphous fillers is wide, the column bed structure is uneven, the linear velocity of the mobile phase is uneven, and the spectral band is widened. The size of the average pore size has a significant impact on the separation of large molecule compounds. When separating samples containing larger molecules, there may be molecular exclusion effects or adsorption effects, which can affect the quantitative recovery rate and accuracy. Therefore, when using reverse phase chromatography to separate samples such as proteins or peptides, consideration should be given to selecting large pore sizes(as30 nm)Reverse phase column packing. As a parameter of the porosity of silica gel, pore volume can be used as a reference for the separation and analysis of larger molecular compounds, and larger pore volume reverse phase column packing can be selected.

(3)Chemical bonded phase fillers play an extremely important role in high-performance liquid chromatography. It can bond organic groups with higher polarity and use solvents with lower polarity as the mobile phase. Organic groups with lower polarity can also be bonded, and solvents with higher polarity can be used as the mobile phase.C18The chromatographic column is of the silane bonded type(Si-O-Si-C)Yes, this type of bonding reaction is currently the most commonly used. If using octadecyltrichlorosilane and fully porous silica gelM-Porasil-C18Reaction generates alkyl chemical bonding phase, trade name isM-Bondapak-C18.

(4)The carbon content refers to the carbon content in the filler. The traditional measurement technique is to heat the filler until the carbon hydrogen bond breaks, and then calculate the carbon content by measuring the weight lost or the carbon dioxide formed. The carbon content can be increased by increasing the length of carbon bonds or increasing the bond density. As the carbon content increases, the retention value of the column also increases. The chromatographic behavior of bonded phases is related to bond density, as well as the density of silica gel and the surface area of the filler. The higher the density of the filler, the more silica gel is required to fill the column, and the higher the carbon content of the column. If using2The retention behavior of columns filled with fillers of different densities and the same carbon content will be significantly different. Therefore, predicting chromatographic behavior solely based on carbon content is not sufficient.

(5)C18Silanization reagent is a greater than2 nmLarge molecules, therefore, will bond with adjacent silanol groupsC18Silanization reagents cause severe steric hindrance. As a result, there are a large number of residual silanol groups on the surface of silica gel that have not reacted with silanization reagents. These polar silanol groups can interact with alkaline compounds under certain chromatographic conditions, causing peak tailing and affecting quantitative analysis results. These problems can be overcome to some extent through alkylation treatment. Alkylation treatment is an independent reaction carried out on the bonded phase to reduce the silanol groups on the surface of silica gel. Alkylation treatment uses small molecules(Like trimethylsilane)The spatial hindrance of the reagent is much smaller thanC18Functional groups. Most stationary phases only have30%Covered key positions. According to reports, the highest coverage can be achieved through certain highly active chemical reagents and special reaction conditions50%A good understanding of the physical properties of silica gel bonded phases will help in selecting suitable chromatography columns in high-performance liquid chromatography reactions. On the surfaceC18Although the chemical functional groups of the column are the same, in reality, different brands haveC18The column performance may vary greatly, resulting in different separation results.

The choice of column packing is related to the possibility of chromatographic separation, while the selection of column specifications directly affects analysis speed, separation ability, detection ability, and solvent consumption for each analysis. Column specifications include two aspects: column inner diameter and column length. Column inner diameter, analytical type is generally2～6 mm,Preparation type20 mmThe larger one can be reached80 mmColumn length, analytical type5～30 cmPreparation type15～50 cmGenerally speaking, the inner diameter of the column does not affect the relationship between separation degree and analysis time. Today, column technology has developed to the point where columns with different inner diameters can have the same performance. Columns with different inner diameters have their own characteristics. For the same analysis time and separation degree, columns with larger inner diameters consume more solvent than those with smaller inner diameters. On the other hand, columns with smaller inner diameters require less sample size for the same detection signal. Therefore, when the sample size is *, a small inner diameter column can be used. Although increasing the length of the column can improve the separation effect, the resistance also increases accordingly, and it is necessary to increase the inlet pressure. Column pressure is the main obstacle that simultaneously affects increasing separation and reducing analysis time. Separation degree, analysis time, and column pressure are interdependent. If two of them are chosen well, then3One factor has already been chosen. Long columns can provide high resolution, while short columns can provide rapid separation. We can choose the appropriate chromatographic column based on the sample situation.

C18Basic principles for column selection:

(1)The use of alkylated sealing fillers can prevent the tailing phenomenon of alkaline compounds.

(2)Select columns with high carbon content to increase retention value.

(3)Choose shorter columns(as15 cm，7.5 cm).

(4)Choose fillers with small particle size.

(5)Choose columns with large pore size fillers for components with high molecular weight.

IIC18Daily use and maintenance of columns

In daily separation and analysis work, the proper use of chromatographic columns directly affects their lifespan. Below is an introductionC18Precautions for columns in daily use.

(1)When loading, unloading, and replacing columns, the action should be light and the joint tightening should be moderate. Strong mechanical vibrations must be prevented to prevent gaps from forming in the column bed.

(2)If the instrument is used for routine analysis and the sample types are limited but the analysis frequency is high, it may be advisable to configure each type of routine analysis1Root specific columns can help extend the lifespan of the columns.

(3)When using a column temperature control device, it should be noted that the temperature can only be raised after introducing the mobile phase.

(4)Before using the mobile phase, it is necessary to perform degassing treatment to avoid reducing column efficiency and affecting detection. The sample solution needs to undergo appropriate pretreatment and filtration to reduce column contamination and blockage.

(5)When changing the type of mobile phase, attention should be paid to the solubility of solvents to prevent salt precipitation.

(6)The actual operating pressure of the column should be lower than the maximum pressure during filling, preferably below half of the maximum pressure. Generally not exceeding20 593.965～29 419.95 kPaUnder low pressure(≤(14 709.975 kPa)The range of use can maintain high column efficiency for a long period of time.

(7)C18The chromatographic column belongs to non-polar bonded phase chromatographic column, and the mobile phase ispHThe value should be strictly controlled within2～7Between them, to avoid damaging the pillars.

(8)After completing the separation and analysis work, the machine should not be stopped immediately, and the chromatographic analysis system should be flushed in a timely manner. Generally0.5 hAbove, to remove impurities from the chromatographic column.

(9)If there are salts in the mobile phase, first rinse thoroughly with water. If it is an amine(Such as trimethylamine or tetrabutylamine)To add to the mobile phase, use50%Methanol and0.05%Wash with a mixture of phosphoric acid solution and solvent, not just with water.

(10)C18Methanol is generally used as a storage solvent to prevent the column from drying out and being damaged. It is strictly prohibited to retain water or buffer solution in the chromatographic flow path for a long time.

(11)Choose the appropriate oneC18Protect the column to prevent interference from impurity particles and irreversible adsorption. The particle size of the packing inside the protective column should be as consistent as possible with the particle size of the packing in the analytical column.

(12)The shelf life of the column should not be too long. Not needed in the short termC18Column, rinse with methanol30～60 minThen seal both ends of the column. For columns that are not used for a long time, one method is to regularly rinse and then seal them; Secondly, after flushing, install the columns at both ends1A container filled with methanol with only a certain capacity(Only installing one end is also acceptable)To supplement the evaporation of solvents inside the column during longer storage periods.

IIIC18Maintenance of pillars

In daily use, despite strict protection and pre-treatment of samples and mobile phases, it is still difficult to avoid column contamination, loss of stationary phase, compaction, column bed collapse, and decreased column efficiency after prolonged use. Some can be repaired to restore some column effects.

1,Column pollution regeneration technology

After the chromatographic column is contaminated, it can be washed with a suitable solvent to regenerate the column efficiency.C18The conventional regeneration washing method for columns is to use methanol and trichloromethane separatelyMethanol/Each water60 mLPass through the chromatography column sequentially, then use methanol60 mLAfter the equilibrium chromatography column is sealed, the column efficiency will return to normal. If necessary, based on the nature of column contamination(Such as organic pollution, salt pollution, etc), using0.05 mol/L H2SO4The0.5 mol/L H3PO4or0.1 mol/L EDTARinse with sodium salt, then rinse with water, and finally equilibrate the chromatography column with methanol before sealing. For severe pollutionC18Column can be washed with water, methanol, chloroform, and ethane in sequence, and then washed in reverse order1Next time, the solvent used each time60 mLDo not connect the detector, and finally seal it with a methanol equilibrium chromatography column.

2,Restoration of Column Pollution

When column pollution regeneration is ineffective or it is known that column pollution is severe, column repair can be used to solve it, but the depth of column pollution should not exceed5 mmThe method is to use a special shovel to dig out the contaminated part, then mix the fixed phase and mobile phase with the same column packing to make a slurry, and carefully fill the slurry fixed phase into the excavated part(Try to make the post filled fixation as close as possible to the original tightness)Just flatten the end face. If the aperture of the sieve plates at both ends of the repaired column head is consistent, the column can be reversed and used for a general period of time, with the aim of using the flushing effect of the mobile phase to restore the tightness of the column bed.

3,Restoration of Column Collapse

There are many reasons for column collapse. When the collapse is not too severe, if the sieve plate aperture at both ends of the column head is the same, the column can be reversed and used for a general time to restore performance. When the collapse is severe(5 mm左右)When repairing, the method of repairing column pollution can be used.