Instructions for installation and use of the hotte

2022-08-09
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Instructions for installation and use of liquid chromatographic column

instructions for installation and use of liquid chromatographic column

the liquid chromatograph is composed of high-pressure liquid pump, detector and liquid chromatographic column. The correct installation and use of liquid chromatographic column is the key to the work of liquid chromatography; It is also the only way for liquid chromatography workers to obtain correct and reliable experimental data

I. installation of liquid chromatographic column:

1. Structure of liquid chromatographic column:

a. the empty column is assembled by column joints, column tubes and filters

the column joint adopts a low dead volume structure. The column joint is a threaded component at both ends, with 7/16 inch external thread at one end and 3/16 inch internal thread at the other end (it has been standardized at home and abroad). 7/16 inch external thread is connected with 1/4 inch column pipe (6.35mm), and the middle is crushed for sealing. The 3/16 inch internal thread is connected with the 1/16 inch (1.57mm) connecting pipe, and a pressure ring is also placed in the middle for sealing the column joint. In order to minimize the dead volume outside the column, when installing the chromatographic column, use a 1.57mm connecting pipe to insert it to the bottom after passing through the hollow screw pressing ring, and then tighten the hollow screw. After the compression ring is extruded and deformed by the hollow screw, it is clamped on the connecting pipe (the length of the exposed pipe after the connecting pipe passes through the compression ring should be strictly controlled at 2.5mm long or other fixed size)

it is discharged into the end column joint in the environment without treatment or recovery after two or more times of synthesis, and a stainless steel filter (or filter) is placed at both ends of the column tube to block the column filler from being washed out of the column by the mobile phase. All components of the hollow column are made of 316# stainless steel, which can withstand the action of general solvents. However, because the solvent containing chloride has certain corrosivity to it, it should be noted that such solvent cannot be left in the column and connecting pipe for a long time to avoid corrosion

b, column packing:

the separation function of liquid chromatography column is between packing and mobile phase, and the classification of column is based on the type of packing

normal phase column: silica gel is often used as column filler. According to the appearance, it can be divided into amorphous and spherical types, and its particle diameter is within the range of 310 M. The other kind of normal phase filler is the silica gel surface bonded with CN, -nh2 and other functional groups, which is the so-called bonded phase silica gel

reversed phase column: it is mainly a non-polar filler with Octadecyl functional groups (ODS) bonded on the surface of silica gel. There are also amorphous and spherical types

other commonly used inverse fillers include bonded C8, C4, C2, phenyl, etc., with particle sizes between 310 M

2, installation of chromatographic column:

a. unpack the column packing box and confirm the type, size, ex factory date and solvent stored in the column

b unscrew the sealing plugs at both ends of the column, taking the plastic bag as an example, and put them back into the packaging box for standby

c. According to the flow direction of the mobile phase marked on the column pipe, connect the inlet end of the color outlook future spectrum column with the outlet of the sampling valve through the connecting pipe (if conditions permit, it is recommended to use a protective column in front of the column); The outlet of the column is connected with the detector. The connecting pipe has an outer diameter of 1.57mm and an inner diameter of 0 3mm stainless steel pipe. Both ends of the connecting pipe are provided with hollow screws and pressure rings for sealing. Try to reduce the dead volume outside the column when connecting. After the connecting pipe passes through the hollow screw and pressing ring, try to insert it to the bottom as hard as possible, then tighten the hollow screw clockwise until it can't be screwed, and then continue to tighten it clockwise with a wrench for 1//2 turns. Remember not to use too much force. If there is liquid leakage after the chromatographic column is pressurized through the mobile phase, please continue to screw it clockwise for 1/4 turn with a wrench until there is no liquid leakage

II. Use of liquid chromatographic column:

before using the chromatographic column, it is best to test the performance of the column and save the results as a reference for evaluating the performance changes of the column in the future. However, it should be noted that the performance of the column may be different due to the differences of the samples used, mobile phase, column temperature and other conditions; In addition, the column performance test is carried out according to the conditions in the chromatographic column factory report (the conditions used in the factory test are the best conditions). Only in this way can the measured results be comparable

1. Sample pretreatment:

a. it is best to use mobile phase to dissolve the sample

b. use a pretreated column to remove impurities with strong polarity in the sample or irreversibly adsorbed with the column filler

c, filter with 0.45 m filter membrane to remove particulate impurities

2. Preparation of mobile phase:

liquid chromatography is that the sample components are filled in the column and reliably grounded. It is very important to protect personal safety and computer safety. The mass exchange between the material and the mobile phase is required to achieve the purpose of separation. Therefore, the mobile phase is required to have the following characteristics:

a. the mobile phase has a certain solubility relative to the sample 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 has a certain inertia and does not produce chemical reaction with the sample (except for 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, reduce the column pressure drop and prolong the service life of the liquid 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, resulting in the failure of the experiment

f. Degassing must be carried out after the mobile phase is prepared. Removing the trace gas dissolved in the mobile phase is not only conducive to detection, but also can prevent the trace oxygen in the mobile phase from interacting with the sample

3. Selection of mobile phase flow rate:

because the column effect is a function of the linear flow rate of the mobile phase in the column, different column effects can be obtained by using different flow rates. For a specific chromatographic column, to pursue the best column efficiency, it is best to use the best flow rate. For the chromatographic column with an inner diameter of 4.6mm, the flow rate is generally 1ml/min. for the column with an inner diameter of 4.0mm, the flow rate of 0.8ml/min is the best

when the optimal flow rate is selected, the analysis time may be prolonged. The method of changing the washing intensity of the mobile phase can be used to shorten the analysis time (for example, when using the reversed-phase column, the content of methanol or acetonitrile can be appropriately increased)

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