The liquid chromatograph consists of a high-pressure liquid pump, a detector and a liquid chromatography column. The correct installation and use of the liquid chromatography column is the key to the liquid chromatography work; it is also the correct and reliable liquid chromatography worker. The only way to pass experimental data.
First, the installation of liquid chromatography column:
1. Structure of liquid chromatography column:
a. The empty column is assembled from column joints, column tubes and filters.
The column joints are constructed with a low dead volume. The column joints are threaded assemblies at both ends, with 7/16-inch external threads on one end and 3/16-inch internal threads on the other end (standardized at home and abroad). The 7/16-inch external thread is connected to the 1/4-inch column tube (Φ6.35mm) and placed in the middle for sealing. The 3/16" internal thread is connected to the 1/16" (Φ 1.57mm) connecting tube, and a pressure ring is placed in the middle for the seal of the column joint. In order to minimize the dead volume outside the column, when installing the column, use a Φ1.57mm connecting tube to insert the hollow screw into the ring, and then tighten the hollow screw. The pressure ring is pressed and deformed by the hollow screw and then hung on the connecting pipe (the length of the pipe exposed after the connecting pipe passes through the pressure ring should be strictly controlled to 2.5 mm long or other fixed size).
In the column joints at both ends, a stainless steel filter (or strainer) is placed at each end of the column tube to block the column packing from being washed out of the column by the mobile phase. Each component of the empty column is made of 316# stainless steel, which can withstand the general solvent effect. However, since the chloride-containing solvent is corrosive to it, it should be noted that the solvent and the connecting tube cannot be stored for a long time to avoid corrosion.
b, column packing:
The separation of the liquid chromatography column is carried out between the filler and the mobile phase, and the classification of the column depends on the type of filler.
Normal phase column: mostly silica gel column packing. According to the appearance, it can be divided into two types, amorphous and spherical, and the particle diameter is in the range of 3-10 μm. Another type of normal phase filler is silica gel surface bonding - a functional group such as CN, -NH2, so-called bonded phase silica gel.
Reversed-phase column: A non-polar filler which is mainly based on silica gel and has an octadecyl functional group (ODS) bonded to its surface. There are also amorphous and spherical types. Other commonly used reverse phase packings are also bonded C8, C4, C2, phenyl, etc., and the particle size is between 3 and 10 μm.
2. Installation of the column:
a. Unpack the column and confirm the type, size, date of manufacture, and solvent stored in the column.
b. Unscrew the sealing plug from the joints at both ends of the column and put it back in the box for spare.
c. According to the flow direction indicated on the column tube, connect the inlet end of the column to the inlet of the injection valve through the connecting pipe (if the conditions permit, it is recommended to use the guard column in front of the column); the outlet of the column is connected with the detector. The connecting pipe is a stainless steel pipe having an outer diameter of 1.57 mm and an inner diameter of 0.1 to 0.3 mm. Both ends of the connecting tube have a hollow screw and a sealing ring for sealing. Always try to reduce the dead volume outside the column when taking over. After the connecting tube passes through the hollow screw and the pressure ring, insert it as far as possible into the end, then tighten the hollow screw clockwise until it stops, then use the wrench to continue to screw 1/4-1/2 turn clockwise, remember not to use too much force. If the column is leaking after being pressurized by the mobile phase, use a wrench to continue to turn 1/4 turn clockwise until it does not leak.
Second, the use of liquid chromatography column
Before using the column, it is best to test the performance of the column and save the results as a reference for future evaluation of column performance changes. However, it should be noted that the column performance may vary depending on the conditions of the sample, mobile phase, column temperature, etc.; in addition, the column performance test is performed according to the conditions in the column factory report (factory test) The conditions are the best conditions), and only then, the measured results are comparable.
1. Pretreatment of the sample:
a. It is best to use a mobile phase to dissolve the sample.
b. Use a pretreatment column to remove strong polarities in the sample or impurities that irreversibly adsorb with the column packing.
c. The particulate impurities were removed by filtration using a 0.45 μm filter membrane.
2. Preparation of mobile phase:
Liquid chromatography is the separation of sample components between the column packing and the mobile phase for mass exchange. Therefore, the mobile phase is required to have the following characteristics:
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 is inert and does not react chemically with the sample (except in special cases).
c. The viscosity of the mobile phase should be as small as possible so that a good separation effect can be obtained when using a longer analytical column; at the same time, the column pressure drop is reduced and the life of the liquid pump is prolonged (the temperature can be lowered to reduce the viscosity of the mobile phase). ).
d. The physical and chemical properties of the mobile phase should be compatible with the detector used. If a UV detector is used, it is best to use a solvent with a lower UV absorption. e. The boiling point of the mobile phase should not be too low, otherwise bubbles will be easily generated, which may make the experiment impossible.
f. After the mobile phase is prepared, it must be degassed. Removal of trace gases dissolved in the mobile phase facilitates both detection and prevention of trace oxygen in the mobile phase from interacting with the sample.
3. Selection of mobile phase flow rate:
Since the column efficiency is a function of the linear flow rate of the mobile phase in the column, different column rates can be obtained using different flow rates. For the best column performance for a particular column, it is best to use the optimum flow rate. For a column with an inner diameter of 4.6 mm, the flow rate is generally selected to be 1 ml/min, and for a column having an inner diameter of 4.0 mm, a flow rate of 0.8 ml/min is preferred.
When the optimum flow rate is selected, the analysis time may be extended. A method of changing the washing strength of the mobile phase can be employed to shorten the analysis time (for example, when a reverse phase column is used, the content of methanol or acetonitrile can be appropriately increased).
note:
a. Since methanol is inexpensive, it is recommended to use a methanol system for the reverse phase column (except where acetonitrile must be used).
b. For normal phase columns, it is recommended to use petroleum ether with a boiling range of 30-60 ° C or purified hexane as the mobile phase. Unpurified hexane should not be used. It is best to use ultrapure water (resistivity greater than 18 megohms) for water, and phenolic impurities in deionized water and double distilled water, which may affect the analysis results.
c. The aqueous mobile phase was most prepared before the experiment, especially in the summer when the buffer solution was used as the mobile phase. It is best to add sodium azide to prevent bacterial growth.
d. The mobile phase is filtered using a 0.45 μm filter to remove particulate impurities.
e. Use a HPLC grade solvent to formulate the mobile phase and use a suitable mobile phase to extend column life and column performance.
4, column performance test:
Start the liquid chromatograph:
a. The mobile phase flow rate was set to 1 ml/min.
b. The UV detector wavelength is set to 254 nm.
c. Use the mobile phase composition and test samples used in the factory test.
d. Record and calculate the test results.
Shanghai Jiapeng Technology Co., Ltd. specializes in: UV analyzer, three-use UV analyzer, dark box UV analyzer, black box UV analyzer, black box UV analyzer, portable UV analyzer, three-use UV analyzer, dark box , UV detectors, partial collectors, constant current pumps, peristaltic pumps, gel imaging systems, gel imaging analysis systems, chemiluminescence imaging analysis systems, photochemical reactors, vortex mixers, vortex mixers, glass chromatography columns, Gradient mixer, gradient mixer, nucleic acid protein detector, glass chromatography column, fluorescent whitening agent analyzer, fraction collector, rubber cutting instrument, blue gel cutting instrument, chromatography system and other products. Welcome to inquire.
Shanghai Jiapeng Technology Co., Ltd.
Telephone 36162366
400 free consultation phone
Mobile phone 18918199710 13501668369
Enterprise QQ, 2880150292, 2880150293, 2880150290, 2880150297
We are a professional Chinese manufacturer of Purgative herb Extract; we supply various products of Relaxing Bowels Plant Extract, and can providing product images and basic parameters with each Relaxing Bowels Plant Extract, Plant Purgative Material and Plant Aperients. Look forward to your cooperation!
Vegetable Fruit Enzyme Powder,Purgative Material,Senna Leaf Extract,Louts Leaf Extract,Semen Cassiae Extract
Xi'an Quanao Biotech Co., Ltd. , https://www.quanaobio.com