Cleaning and regeneration of the hottest HPLC colu

Cleaning and regeneration of HPLC column

cleaning and regeneration of reversed-phase HPLC column in high performance liquid chromatography

reversed phase chromatography is the most widely used technology in high performance liquid chromatography so far, mainly because it is suitable for the analysis of a large number of non-polar substances and many ionizable and ionic compounds. Most stationary phases used in reversed-phase chromatography are natural hydrophobic substances. Therefore, analytes are separated according to the size of their hydrophobic interaction with the stationary phase, and the hydrophobic mechanism can also be separated in the same retention manner

there are also a few substances on the stationary phase, such as mixed phase (such as phenyl hexyl), end closed and non end closed species and polar embedded phase, which also exist on these bonded silica gel. There are also many fillers used in reversed-phase chromatography, including polymers, polymer surfaces coated with silica gel and alumina, inorganic organic mixtures, coated zirconia, and graphitized carbon. Different kinds of stationary phases have their own advantages and disadvantages

reverse phase chromatographic columns can be used in many applications by using various mobile phases and additives. Some technologies use additives to change or modify the surface of fillers. Sometimes these additives may contaminate the bonding phase surface

the surface of silica gel has some other chemical properties because of the hydrophobic bonding phase. Residual silanol exists in all silica gel bonded fillers. These silane alcohols are weakly acidic, so they can combine with some matrix components, especially alkaline components, to be analyzed. Because the pKa value of silane alcohol is about 4.5, ionization can occur at neutral pH, so electrostatic interaction with cations is possible. The older A-type silica gel can accommodate high concentrations of metal ions (sometimes 100ppm or more), which can make the surface of silica gel more acidic, and even cause metal chelation or remove some compounds. Residual silanol is more annoying on non terminally closed silica bonded short chain bonded phases such as C2 or C4

users must be aware of the special properties of the surface of the stationary phase they use and the possible analyte stationary phase surface interactions, so that when they use the reverse phase method, they can consider the possible matrix interactions. For example, very hydrophobic sample matrices such as corn oil, highly aromatic substances, and wax can adhere to the surface of the stationary phase and change their properties. Biological fluids containing protein substances can also be adsorbed on the loading surface. Although analysts try their best to protect the HPLC column, some analyte matrix contamination can adversely affect the stationary phase

when the column is polluted, its chromatographic behavior will be slightly different from that of the uncontaminated column. Contaminated columns can cause back pressure problems. The contaminated reversed-phase column must be cleaned and regenerated to restore the original operating conditions. This part of "column observation" will discuss feasible methods to restore the column to its original or almost original state

what causes inverse column pollution

usually, the sample contains something that is not of interest to the analyst. Salts, lipids, fatty substances, humic acids, hydrophobic proteins and other biological substances are substances that may interact with HPLC columns during use. These substances have less or greater retention values than the analyst's target. Substances with small retention values, such as salts, are generally washed out of the chromatographic column when the volume is empty. The interference of these non target products can be detected by the detector and can form chromatographic peaks, bubbles, baseline up shifts or negative peaks. If the components of the sample are strongly retained in the column and the components of the mobile phase solution are not enough to elute these substances, these substances adsorbed on the surface of the column will usually accumulate in the column head after multiple loading. These behaviors can usually be found only through parallel experiments. Samples with medium retention values can be washed out slowly and show wide peaks, baseline disturbances, or baseline drift

sometimes these adsorbed sample components accumulate to a certain extent enough to make them start to form a new stationary phase. The analyte can interact with these impurities to form a certain separation mechanism. Retention time will fluctuate and tailing will occur. If enough pollution occurs, the back pressure of the column can exceed the maximum pressure that the pump can bear, making the column unable to work and generating empty volume at the blockage

cleaning silica gel bonded columns

the key to regenerating contaminated HPLC columns is to know the nature of pollutants and find appropriate solvents to remove them. If the pollution is caused by the accumulation of strong retention substances during repeated injection, simple steps to remove these pollutants can often restore their chromatographic behavior. Sometimes, after several operations, the chromatographic column can be cleaned by washing 20 volumes with 90 ~ 100% solvent B (the stronger solvent in the dual solvent reverse phase system). For example, non-aqueous solvents such as methanol, acetonitrile and tetrahydrofuran can be used for the residual lipids in the column. If you are using a buffer system, do not switch directly to strong solvents. Sudden switching to high concentration organic solvents may precipitate the buffer in the HPLC flow system, which will lead to greater problems such as column head blockage, pipe blockage, pump leakage, piston damage or rotating shaft failure of the sampling valve. The unbuffered mobile phase should be used first (i.e. replace the buffer with water). Replace the strong solvent after washing 5 ~ 10 volumes

sometimes, strong solvents cannot wash away the pollutants left on the chromatographic column. Then stronger solvents or a series of solvents are necessary to clean the column. If the pollutants are non biological substances, users can skip one or more other organic solvents to remove the pollutants. There are many combinations of solvents. The recommended solvent system can be found on the page of the column manufacturer

generally speaking, all cleaning methods have similar forms. The solvents used increase with the strength of the solvent. Often the last solvent is very hydrophobic (such as ethyl acetate or even hydrocarbons), which can be used to dissolve non-polar substances such as lipids and oils. We must ensure that each solvent in a series of solvents can be mixed with the next solvent. At the end of the cleaning process, it is necessary to return to the original solvent system with the help of a medium strength and miscible solvent. For example, isopropanol is a very good solvent as an intermediate step, because it can be miscible with n-hexane or dichloromethane and with aqueous solvents. However, the viscosity of isopropanol is very high, so a low flow rate must be ensured to avoid excessive pump pressure. Of course, if the UV detector is used, avoid the absorption of the solvent in the UV region, otherwise a large amount of solvent washing is required to make the baseline stable

for a typical silica gel bonded column, if there is no buffer solution, the following solvent series is recommended:

100% methanol

100% acetonitrile

75% acetonitrile - 25% isopropanol

100% isopropanol

100% dichloromethane

100% n-hexane

after using dichloromethane or n-hexane, At the same time, it also greatly improves the shopping experience of consumers. The solvent compatibility of the column must be washed with isopropyl alcohol before using the original aqueous solvent. Wash at least 10 column volumes of each solvent. Such as 250mm × For the 4.6mmhplc analytical column, the analyst can flush it with a flow rate of 1 ~ 2ml/min. To restore the original solvent system, it is not necessary to flush every step, and the intermediate step can be skipped. It is recommended to use isopropanol in the intermediate step, then use the mobile phase without buffer, and finally return to the initial mobile phase configuration. Tetrahydrofuran is another popular solvent for removing pollution. If the user suspects that the column is seriously polluted, dimethyl sulfoxide (DMSO) or dimethylformyl ammonium can be mixed with water in the ratio of 50:50 and flow through the chromatographic column at a flow rate of less than 0.5ml/min. The successful regeneration of the reversed-phase column is a very time-consuming process, and the solvent flushing can be operated overnight by using the gradient system

ask: when using HPLC for analysis, the retention time sometimes drifts, sometimes changes rapidly. What is the reason? How to solve it

answer: about the drift problem:

1. The temperature control is not good. The solution is to use the constant temperature device to keep the column temperature constant

2. The mobile phase changes. The solution is to prevent the evaporation and reaction of the mobile phase.

3. The column is not well balanced, and the column needs to be balanced for a longer time

about the problem of rapid change

1. The solution is to reset the flow rate, To keep it stable, the axle weight will be gradually increased from the existing 23t to 30t

2. There are bubbles in the pump, which can be driven out by exhaust and other operations

3. The mobile phase is not suitable, solution ----- Professor Zhang Yajun/Deputy Dean of the school of electrical engineering, Beijing University of chemical technology decided to change the mobile phase or make the mobile phase mix properly in the control room

ask: what is the reason for the tailing or double peaks in the peak of liquid chromatography

answer: 1. If the sieve plate is blocked or the column fails, the solution is to backwash the column, replace the sieve plate or replace the column

2. There are interference peaks, and the solution is to use a longer column, change the mobile phase or replace the column with good selectivity

Q: the main reason for insufficient sensitivity of HPLC and the solution

answer: 1. Insufficient sample size: the solution is to increase the sample size

2. The sample does not flow out of the column: the mobile phase or column can be changed according to the chemical properties of the sample

3 The sample does not match the detector: adjust the wavelength or change the detector according to the chemical properties of the sample

4. The detector has too much attenuation: adjust the attenuation

5. The detector time constant is too large: the solution is to reduce the time parameter

6. The detector tank window is polluted: the solution is to clean the tank window

7. There are bubbles in the detection pool: the solution is to exhaust

8. The pressure measuring range of the recorder is improper: adjust the voltage range

9. Inappropriate flow rate of mobile phase: adjust the flow rate

10. The detector and recorder exceed the correction curve: the solution is to check the recorder and detector and redo the correction curve

ask: why is the column pressure unstable when doing HPLC analysis? How to solve it

answer: the reasons may be:

1. There is air in the pump. The solution is to remove the air in the pump and degass the solvent

2. If the proportional valve fails, replace it

3. If the pump gasket is damaged, replace the gasket

4. For the bubbles in the solvent, the solution is to degass the solvent and change the degassing if necessary The pneumatic method of reducer is composed of synchronous toothed belt and precision ball screw pair

5. System leak detection, find out the leak point, and then seal it

6, gradient elution, at this time, the pressure fluctuation is normal

Q: I recently replaced another brand of ODS column. Although the separation is still OK, the retention time cannot be reproduced. Why

answer: This is because the analyte may have the ability to form hydrogen bonds. Although the manufacturing technology of fillers has been greatly improved in the past few years, the concentration of silanol groups on the surface of ODS fillers from different manufacturers is different. It is these silanol groups that may interact with the sample. Therefore, the relative retention time of each component in the same analyte on ODS columns of different brands may be different. Adding a small amount of competitors, such as triethylamine (tea), to the mobile phase will saturate the bonding ability of silanol group, so as to ensure that no