Selection, use, maintenance and troubleshooting of liquid ch

Selection of reversed phase chromatographic column
1. Range of pH value of column
The advantages of the reversed phase column are that the stationary phase is stable and widely used, and a variety of solvents can be used. However, the pH range of the mobile phase should be paid attention to when using silica gel as the filler. Generally, the pH range of C18 column is 2-8. When the pH value of mobile phase is less than 2, the bonding phase will be hydrolyzed; when the pH value is greater than 7, the silica gel is easy to dissolve; the stationary phase often uses buffer solution to degrade. Once this happens, the column will collapse. The chromatographic columns of different brands with the same packing are not the same. If the pH of mobile phase is high or buffer solution is often used, it is recommended to select columns with wide pH range, such as Daian's acclaim column pH 2-9 or Zorbax's pH 2-11.5 column.
2. End base sealing of packing
Sealing the residual silicon hydroxyl groups of fillers with sealing technology can improve the adsorption or tailing of polar compounds; the increase of carbon content is conducive to the separation of difficult retention compounds; the stability of fillers is good, and the retention time reproducibility of components is good. If the sample to be analyzed belongs to acidic or alkaline compounds, it is better to use the chromatographic column with the packing end capped.
Use of liquid chromatography column
Before using the chromatographic column, it is better to test the performance of the column and keep the results as a reference for evaluating the performance changes of the column in the future. The column performance test should be carried out according to the conditions in the delivery report of chromatographic column (the condition used in the factory test is the best condition). Only in this way can the measured results be comparable. However, it should be noted that the performance of the column may be different due to the different conditions of sample, mobile phase and column temperature.
1. Sample pretreatment
a. It is better to use the mobile phase to dissolve the sample.
b. The pretreatment column was used to remove the impurities with strong polarity or irreversible adsorption with column filler.
c. A 0.45 μ m membrane was used to remove particulate impurities.
2. Preparation of mobile phase
Liquid chromatography is the separation of sample components by mass exchange between column packing and mobile phase
a. The flow relative to the sample has a certain solubility to ensure that the sample components will not precipitate in the column (or remain in the column for a long time).
b. The mobile phase does not react with the sample.
c. The viscosity of the mobile phase should be as small as possible in order to get a good separation effect, reduce the column pressure drop and prolong the service life of the pump (the viscosity of the mobile phase can be reduced by increasing the temperature).
d. The physicochemical properties of the mobile phase should be compatible with the detector used. If UV detector is used, it is better to use solvent with low UV absorption.
e. The boiling point of the mobile phase should not be too low, otherwise it is easy to produce bubbles, leading to the experiment can not be carried out.
f. After the mobile phase is prepared, degassing must be carried out. Removing the trace gas dissolved in the mobile phase is not only beneficial to the detection, but also can prevent the interaction between the trace oxygen in the mobile phase and the sample.
3. Selection of flow rate of mobile phase
Because the column efficiency is a function of the linear velocity of the mobile phase in the column, different column efficiency can be obtained by using different velocity. For a specific column, the best column efficiency is to use the best flow rate. For a 4.6 mm inner diameter column, the flow rate is generally 1 ml / min, and for a 4.0 mm inner diameter column, the flow rate is 0.8 ml / min.
When the optimal flow rate is selected, the analysis time may be prolonged. The analysis time can be shortened by changing the washing strength of the mobile phase (for example, the content of methanol or acetonitrile can be appropriately increased when the reversed-phase column is used).
be careful:
a. It is better to prepare the mobile phase with water before the experiment, especially in summer, when using buffer solution as mobile phase, do not stay overnight. It is better to add sodium azide to prevent bacterial growth.
b. The mobile phase requires the use of 0.45 μ m membrane filtration to remove particulate impurities.
c. HPLC grade solvent was used to prepare mobile phase, and suitable mobile phase could prolong the service life of chromatographic column and improve its performance.
Maintenance of chromatographic column
1. Balance of chromatographic column
The reversed phase chromatographic column was stored in acetonitrile / water after factory test. The new column should be washed with methanol or acetonitrile of 10-20 times the volume of the column. Please make sure that the mobile phase you use to analyze the sample is miscible with acetonitrile / water. Use enough time every day to balance the column with the mobile phase, and you will get the maximum "compensation" in dealing with the problem, and your column life will be longer
a. At the beginning of equilibrium, slowly increase the flow rate and use the mobile phase to equilibrate the column until a stable baseline is obtained (if the buffer salt or ion pair reagent flow rate is low, it will take a long time to equilibrate)
b. If the mobile phase used contains buffer salt, attention should be paid to "transition" with pure water, that is, before the start of analysis every day, it must be washed with pure water for more than 30 minutes, and then the mobile phase with buffer salt should be balanced; after the analysis, the column must be washed with pure water for more than 30 minutes to remove the buffer salt, and then to wash the column with methyl alcohol for 30 minutes.
2. Regeneration of chromatographic column
For long-term use of chromatographic column, the column efficiency will decrease (the number of theoretical plates of the column will decrease). The chromatographic column can be regenerated, and a cheap pump should be used in the conditional laboratory.
Volume of solvent recommended for column flushing
Column size column volume volume volume of solvent used
125-4mm 1.6ml 30ml
250-4 mm 3.2ml 60ml
250-10mm 20ml 400ml
Selection of regeneration method:
Regeneration of polar stationary phases (such as Si, NH2 *, diol based chromophore fillers)
N-heptane → chloroform → ethyl acetate → acetone → ethanol → water**
The regeneration of nonpolar stationary phase (such as RP-18, RP-8, CN, etc.) is: water → acetonitrile → chloroform (or isopropanol) → acetonitrile → water
be careful:
a. In the regeneration of NH2 modified chromatographic column, NH2 may exist in the form of ammonium ion, so it should be washed with 0.1M ammonia water after water washing, and then washed with water until the alkali solution completely flows out.
b. 0.05m dilute sulfuric acid can be used to clean the contaminated chromatographic column. If the impurities adsorbed on the surface of silica gel can not be completely removed by simple treatment with organic solvent / water, it is very effective to add 0.05m dilute sulfuric acid after water washing.
3. Maintenance of chromatographic column
a. Use pre column to protect analytical column (silica gel has certain solubility in polar / ionic mobile phase)
b. The pH stability range of most RP-HPLC columns is 2-7.5, which should not exceed the pH range of the column
c. Avoid drastic changes in composition and polarity of mobile phase
d. The mobile phase must be degassed and filtered before use
e. If polar or ionic buffer solution is used as mobile phase, the column should be washed clean after the experiment and stored in methanol or acetonitrile
f. Chloride solvent has certain corrosivity to it, so it should be noted that the column and connecting pipe can not retain such solvent for a long time to avoid corrosion.
For more related technologies, please consult MS technologies technicians