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The lifetime of the column, in addition to the analysis of the sample and mobile phase and frequency of use, Zui is mainly related to daily maintenance. To extend the life of your column and to protect your interests, please read this section carefully.
The service life of a column is mainly measured by the efficiency of the column and the column pressure. If a column is too low or the column pressure is too high, it is generally considered that the column has ended. Therefore, the key to extending the life of the column is to eliminate the factors that cause the column efficiency to drop and the column pressure to rise. Here are the routine maintenance methods for your column:
First, the PH of the mobile phase should be within the range of use
In addition to the reversed-phase cyano column pH 1.5-9.0, the reversed-phase column has a pH range of 1.5-10. Due to the presence of Si-C and Si-O bonds in the filler, the mobile phase will exceed its pH range. The loss of the silica matrix and the carbon chain breakage of the bonded phase result in a decrease in column efficiency and a shortened service life. Due to damage to the column caused by improper pH control of the mobile phase, it is often difficult to recover the column, so care must be taken to strictly control the pH of the mobile phase.
Second, remove the solid particles in the sample and mobile phase1.5ml screw hplc vials
The solid particulate matter contained in the sample and mobile phase will block the chromatographic sieve plate. Blocking the sieve plate will not only cause the column pressure to rise, but also cause the column efficiency to drop, because the blockage of the sieve plate will cause uneven flow. , causing the chromatographic peak shape to be tailed and widened, thereby reducing the efficiency of the column. Therefore, it is recommended to use ultrapure water and chromatographically pure reagents to filter the sample before the sample is analyzed, and the mobile phase is passed through a 0.45 μm filter.
Third, use the guard column or online filter
After filtration of the sample and mobile phase, the solid particulate matter cannot be completely eliminated, because the wear of the pump, the aging of the seal ring and the pipeline will also produce solid particulate matter, which is carried by the mobile phase into the column and blocks the sieve plate, resulting in The column pressure is increased and the column efficiency is lowered. Both the guard column and the in-line filter have sieve plates with the same pore size as the column pores, thus preventing solid particles from reaching the column and preventing blockage of the column. Since the increase in column pressure is a large percentage of analytical failures, it is recommended that you add a guard column or inline filter to the column injection end, in addition to filtering the sample and mobile phase.
If you confirm that the column pressure rise is due to plugging of the injection end screen, you can choose to remedy the following:
1. Add a guard column or an in-line filter in front of the column, and then reverse-collapse the column with methanol and water = 20/80 ml/min for 180 min.
2. Add a guard column or inline filter to the column injection end and use it in reverse.
Fourth, the correct use of buffer salts
The buffer salt is usually soluble in water and is insoluble in organic solvents. Therefore, improper use of the buffer salt causes it to precipitate, blocking the pores between the filler matrix and the voids between the particles, causing the filler to be kneaded and the column pressure to rise; The bonded carbon chain is free to stretch, which reduces the retention capacity of the column and reduces the efficiency of the column. After the buffer salt is precipitated, removal is very difficult, so proper use of buffer salts is very important to extend the life of the column.
The purpose of proper use of the buffer is to prevent the buffer salt from precipitating, so the correct use of the buffer salt can be attributed to one sentence: filter before use and rinse after use. The specific method is as follows:
1. Isocratic conditions: Before and after using the buffer salt, the transition mobile phase should be washed at a flow rate of 1.0 ml/min for 60 min; another method of removing the buffer salt after use is to flush the column with a transient mobile phase at a flow rate of 0.2 ml/min. overnight.
2. Gradient conditions: Before running the gradient with the mobile phase containing the buffer salt, rinse with the same mobile phase with the initial mobile phase at a flow rate of 1.0 ml/min for 60 min, and then rinse the column with 1.0 ml/min with the transition mobile phase. 120min. The gradient setting of the buffered salt mobile phase should be as gentle as possible to avoid buffer salt precipitation during the gradient process.
Note: The transition mobile phase means that the composition of the organic phase and the aqueous phase is the same as that of the analytical mobile phase, except that the transition mobile phase does not contain buffer salts.
3. Remedy for buffer salt precipitation:
1) Scheme 1: Backwash the column for 120 min with methanol/20/80 at a flow rate of 1.0 ml/min at 35 °C.
2) Scheme 2: The column was backflushed back and forth at a flow rate of 0.2 ml/min with methanol/water = 20/80 overnight.
5. Prevent strong retained substances from remaining on the column
Strongly retained substances and macromolecular compounds accumulate in the column, creating additional retention behavior for the compounds in the sample, which not only causes peak broadening, tailing, but also decreases column efficiency, and also causes retention time changes, accumulating To a certain extent, it will also cause the column pressure to rise. Since the effect of strongly retained substances and macromolecular compounds on chromatographic separation is a cumulative effect, it takes a certain amount of time to be reflected, but for many drugs, especially complex samples, it is difficult to judge whether they contain strong retained substances, so To prevent the accumulation of strongly retained materials, the column needs to be cleaned with pure methanol during daily maintenance.
cleaning method:1.5ml screw hplc vials
1. Unused buffer salt: After the analysis is completed every day, the buffer salt is removed by the above method, and then the column is washed with methanol or backwash for 60 min.
2. Using buffered salt: After the analysis is completed, the buffer salt is removed by the above method, and then the column is washed back with pure methanol or acetonitrile for 60 min.
Water - acetonitrile - (or isopropanol) - acetonitrile - water
Each column was backfluipped back to the column at a flow rate of 1.0 ml/min for 60 min.
Selection, maintenance and repair of C18 columns
According to statistics, nearly 80% of organic and inorganic substances can be separated by high performance liquid chromatography. The C18 column in reversed-phase chromatography is a commonly used column in the high performance liquid chromatography. The following is an introduction to the selection, maintenance and repair of C18 columns.
1. Selection of C18 column
The choice of C18 mainly considers two issues, namely the effect of column packing and column size on the column.
1.1 Effect of C18 column packing on the column The physical properties of the column packing have an important influence on the chromatographic behavior of the packing. The main physical properties of the filler include the following: particle size, pore size, pore volume, bonded phase chemistry, carbon content, and alkylation.
(1) Particle size means the size of the particle diameter of the column packing. In fact, the particle size on the column is an average. For example, the particle size "5 μm" is not the filler in the column. All the particles have a diameter of 5 μm, and actually have a particle distribution. This degree of distribution plays an important role in column back pressure and column efficiency. In general, the smaller the average particle size, the smaller the particle distribution, the higher the column efficiency and the higher the back pressure. At present, the particle size of the C18 column packing is between 4 and 10 μm.
(2) The pore size refers to the pore gap between the filler particles. Generally speaking, the pore size refers to the average pore diameter of the filler. After the spherical packing is installed, the average pore size distribution is relatively narrow, the column bed structure is uniform, the column efficiency is high, and the reproducibility is good; the amorphous pores have a wide average pore size distribution, the column bed structure is not uniform, the mobile phase linear velocity is not uniform, and the band is expanded. width. The size of the average pore size has a great influence on the separation of macromolecular compounds. When separating samples containing larger molecules, there may be a molecular exclusion effect, or an adsorption effect may occur to affect the quantitative recovery and accuracy. Therefore, large-aperture (eg, 30 nm) reverse phase column packing should be considered when separating samples such as proteins or peptides by reversed-phase chromatography. The pore volume is used as a parameter for the porosity of silica gel. It can be used as a reference for the separation and analysis of larger molecular compounds. A larger pore volume reverse phase column packing is used.
(3) Chemically bonded phase fillers play an important role in high performance liquid chromatography. It can bond more polar organic groups and use less polar solvent as the mobile phase. It is also possible to bond organic groups having a relatively small polarity and use a solvent having a relatively high polarity as a mobile phase. C18 columns are based on silanized bonding type (Si-O-Si-C), and such bonding reactions are currently common. For example, an octadecyl group is reacted with a fully porous silica gel M-Porasil-C18 to form an alkyl chemically bonded phase, which is sold under the trade name M-Bondapak-C18.
(4) The carbon content is the carbon content in the filler. The traditional measurement technique is to heat the filler to a carbon-hydrogen bond 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 the carbon bond or increasing the bonding density. As the carbon content increases, the column retention increases. The chromatographic behavior of the bonded phase is related to the bonding density, and also to the density of the silica gel and the surface area of ??the filler. The higher the density of the filler, the higher the amount of silica gel required to fill the column, and the higher the carbon content of the column. If the column is packed with two fillers of the same density and the same carbon content, the retention behavior will be significantly different. Therefore, predicting chromatographic behavior by carbon content 1.5ml screw hplc vials alone is not enough.
(5) The C18 silylating agent is a macromolecule larger than 2 nm and thus produces severe steric hindrance with the C18 silylating agent that has been bonded to the adjacent silanol group. As a result, a large amount of residual silanol groups on the surface of the silica gel are not reacted with the silylating agent. These polar silanol groups interact with the basic compound under certain chromatographic conditions to cause peak tailing, which may affect quantitative analysis. result. These problems can be overcome to some extent by alkylation. The alkylation treatment is an independent reaction performed on the bonded phase to reduce silanol groups on the surface of the silica gel. The alkylation treatment uses a small molecule reagent with a steric hindrance that is much smaller than the C18 group. Most stationary phases have only 30% of the bondable positions that can be covered. According to reports, through some extremely active chemical reagents and special reaction conditions, Zui can cover up to 50%. A good understanding of the physical properties of the silica-bonded phase will help to select the right column for the HPLC reaction. On the surface, although the C18 column has the same chemical functional group, in fact, the performance of different brands of C18 columns may be very different, resulting in different separation results.
1.2 Influence of C18 column specifications on the column The choice of column packing is related to the possibility of chromatographic separation, and the choice of column size directly affects the analysis speed, separation ability, detection ability and solvent consumption per analysis. Column specifications include two aspects: column inner diameter and column length. The inner diameter of the column is generally 2 to 6 mm for analysis, 20 mm for preparation, and 80 mm for the larger one; the length of the column is 5 to 30 cm for analysis and 15 to 50 cm for preparation. In general, the column inner diameter does not affect the relationship between resolution and analysis time. Today, column technology has evolved to have the same performance for columns with different column internal diameters. Columns with different internal diameters have their own characteristics. For the same analysis time and resolution, the column with the larger inner diameter consumes more solvent than the column with the smaller inner diameter. On the other hand, a column with a smaller inner diameter requires less sample for the same detection signal. Therefore, when the amount of sample is limited, a small inner diameter column can be used. Although the increase in the length of the column improves the separation effect, the resistance also increases, and the inlet pressure must be increased. Column pressure is a major obstacle that affects both increased resolution and reduced analysis time. Separation, analysis time and column pressure are mutually constrained. If you choose two of them, the third factor will be selected. Long columns give high resolution, short columns provide fast separation, and we can choose the right column for the sample.
1.3 Basic Principles for Selection of C18 Columns (1) The use of a seal that is sealed by alkylation can prevent the tailing of alkaline compounds. (2) Select a column with a high carbon content to increase the retention value. (3) Use a shorter column (eg 15 cm, 7.5 cm). (4) Use a small particle size filler. (5) A column with a large pore size is selected for the component having a large molecular weight.
2, daily use and maintenance of C18 column
In the daily separation analysis work, the proper use of the column directly affects the life of the column. The following points should be noted in the daily use of the C18 column.
(1) When loading and unloading or replacing the column, the action should be light and the joint should be tightened properly. Strong mechanical vibration must be prevented to avoid voids in the bed.
(2) If the instrument is used for routine analysis, the sample type is limited, but the number of analyses is large, it is possible to configure a dedicated column for each type of routine analysis, which helps to extend the life of the column.
(3) When using the column temperature control device, it should be noted that the temperature can be raised after the mobile phase is introduced.
(4) The mobile phase needs to be degassed before use to avoid reducing the efficiency of the column and affecting the detection. The sample solution needs to be properly pretreated and filtered to reduce column contamination and blockage.
(5) When replacing the mobile phase type, attention should be paid to the mutual solubility of the solvent to prevent salting out.
(6) The actual operating pressure of the column should be lower than the high pressure of zui at the time of filling, and Zui is better than half of the high pressure of zui. Generally no more than 20 593.965 ~ 29 419.95 kPa. Use in low pressure (≤14 709.975 kPa) to maintain the column for a long period of time.
(7) The C18 column is a non-polar bonded phase column. The pH of the mobile phase should be strictly controlled between 2 and 7 to avoid damage to the column.
(8) After the separation and analysis work is completed, 1.5ml screw hplc vials it should not be stopped immediately. It is necessary to rinse the chromatographic analysis system in time, generally 0.5 h or more, to remove impurities in the column.
(9) If there are salts in the mobile phase, first wash them thoroughly with water. If an amine (such as trimethylamine or tetrabutylamine) is added to the mobile phase, rinse with a mixture of 50% methanol and 0.05% phosphoric acid. Do not rinse with water alone.
(10) C18 generally uses 100% methanol as a storage solvent to prevent the column from being cracked and damaged. Never allow water or buffer solution to remain in the chromatographic flow path for extended periods of time.
(11) Select a suitable C18 guard column to protect the analytical column from impurity particles and irreversible adsorption interferences. The particle size of the packing in the guard column should be as consistent as possible with the particle size of the analytical column packing.
(12) The shelf life of the column should not be too long. The C18 column that is not used for a short period of time is rinsed with methanol for 30 to 60 minutes, and then the ends of the column are sealed. For the longer-term unused columns, one is to take regular flushing and re-sealing; the other is to fill the two ends of the column with a certain volume of filled methanol container (only one end can also be installed) to supplement Evaporation of the solvent in the column during long storage.
3, C18 column maintenance
In the daily use of the column, although the protection is strict, the sample and the mobile phase are pre-treated, but after a long period of use, it is still difficult to completely avoid the column contamination, the loss of the stationary phase, the slab, the collapse of the column bed, and the decrease of the column efficiency. . Some can be repaired to restore some of the efficiency.
3.1 Column pollution regeneration technology After the column is contaminated, it can be washed with a suitable solvent to regenerate the column. The conventional regenerative washing method of C18 column is to use 60 mL of methanol, methanol/water, respectively, to pass through the column, and then equilibrate the column with 100% methanol and 60 mL, and the column efficiency will return to normal. If necessary, according to the nature of the column pollution (such as organic pollution, salt pollution, etc.), 1.5ml screw hplc vials use 0.05 mol
This is the end of the introduction of Ways to extend column life. I hope it can help you.