CN111239266B - Thin-inner-diameter capillary liquid chromatographic column filling method and homogenizing tank - Google Patents
Thin-inner-diameter capillary liquid chromatographic column filling method and homogenizing tank Download PDFInfo
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- CN111239266B CN111239266B CN201811441661.2A CN201811441661A CN111239266B CN 111239266 B CN111239266 B CN 111239266B CN 201811441661 A CN201811441661 A CN 201811441661A CN 111239266 B CN111239266 B CN 111239266B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Abstract
The invention discloses a filling method and a homogenate tank of a capillary liquid chromatographic column with a small inner diameter. The conical discharge end of the proposed homogenate tank is made of high polymer materials, so that filler particles only collide with the high polymer materials in a friction mode in the filling process of the filler, the filling flow rate is low, and the impact force between the filler particles and the diameter-variable channel in the filling process can be greatly reduced by combining the two factors. The capillary liquid chromatographic column has high column efficiency, and the stuffing has low breaking rate and thus high inertness. Can be used for high-efficiency separation of medicine, protein and hormone components.
Description
Technical Field
The invention relates to the field of high performance liquid chromatography, in particular to a method for filling a capillary liquid chromatography column with a small inner diameter and a homogenate tank used in the method, which are particularly suitable for filling the capillary liquid chromatography column with the small inner diameter of 25-250 mu m.
Background
The chromatographic column is the core part of the high performance liquid chromatography analysis. Capillary liquid chromatography columns are an important branch of chromatography columns with internal diameters between tens of microns and hundreds of microns. In recent years, capillary chromatographic columns have been rapidly developed in biochemical and biological analyses because of their small column volume (0.01 to 30 μ L), small volume of mobile phase consumed (0.1 to 10L/year), and small required sample volume.
In recent years, capillary liquid chromatography has played an increasingly important role in analyzing micro or trace biological samples and the like. However, the packing is partially broken in the filling process, and the abundant silicon hydroxyl on the surface of the packing can generate adsorption to alkaline drugs, protein biomolecules, hormones and the like, so that the integrity of the packing is more and more emphasized in the filling process of the capillary liquid chromatography column, namely the breaking rate of the packing in the filling process needs to be reduced. At present, the mainstream filling methods include an uplink method and a downlink method. The adoption of the upward filling method requires the addition of a magnetic stirring device, and the crushing rate of the filler can be increased due to the friction and collision between the filler and the magnetons in the filling process. And the downlink method does not need a magnetic stirring device, so that the damage caused by friction and collision between the filler and the magneton is avoided. However, when the downward filling method is adopted, in order to avoid coagulation of the filler under the action of gravity, a rapid filling mode is mostly adopted, the downward speed of the filler in the filling process is very high, and the filler collides with the reducing part of the discharge channel of the homogenizing tank, so that the filler is crushed, and the inertia of the chromatographic column is reduced, the peak is trailing, the column efficiency is reduced, and the front pressure of the column is increased.
Disclosure of Invention
Aiming at the technical problems, the invention provides a filling method of a capillary liquid chromatographic column with a small inner diameter and an improved scheme of a filling column homogenate tank. The conical discharge end of the proposed homogenate tank is made of high polymer materials, so that filler particles only collide with the high polymer materials in a friction mode in the filling process of the filler, the impact force with the variable-diameter channel in the filling process of the filler is relieved, and the breakage rate of the filler in the column filling process is reduced.
The technical scheme of the invention is as follows:
a method for packing a capillary liquid chromatography column with a small inner diameter comprises the following steps:
a. connecting and fixing a capillary tube with a sieve plate and a small inner diameter to the discharge end of the homogenizing tank;
b. mixing a filler with a high-density homogenizing solvent to prepare a homogenizing liquid, wherein the density of the homogenizing solvent is close to that of the filler, and ultrasonically mixing uniformly for later use;
c. adding a proper amount of the homogenate into a homogenate tank, and filling the vacant area with a solvent to ensure that no air bubbles exist in the homogenate tank;
d. the feed end of the homogenization tank is connected with a high-pressure liquid phase pump, and the pump is filled with a given solvent in advance. Starting a high-pressure liquid phase pump, pressing a mobile phase into a homogenate tank, and filling at a low flow rate by adopting a pressure control mode;
e. the initial filling pressure is 0.3-0.5 MPa, the pressure is increased at the speed of 1.5-2 MPa/min along with the filling until 30-40 MPa, and the high-pressure liquid phase pump is closed after the constant pressure is kept for 10-30 min;
f. and after the pressure of the high-pressure liquid phase pump is reduced to zero, the filled capillary liquid phase chromatographic column with the small inner diameter is unloaded.
The capillary chromatographic column sieve plate in the step a is an integral column sieve plate and is prepared from a polymer containing a filler bonding phase or a carrier, and the length of the integral column sieve plate is 2-3 mm; the inner diameter of the capillary is 25-250 μm, and the outer diameter is 365 μm.
The filler in the step b can be silica gel matrix, and the homogenizing solvent can be carbon tetrachloride/methanol mixed liquor or tetrabromoethane/methanol mixed liquor, wherein the volume fraction of carbon tetrachloride or tetrabromoethane is 90-95%; the concentration range of the filler is 25-30 mg/mL; the ultrasonic time range is 1-2 min.
And d, the mobile phase in the step d is methanol or ethanol, and the flow rate of the mobile phase is 15-20 mu L/min.
The utility model provides a homogenate jar for filling method comprises jar body (1), high-pressure tube connecting piece (2), PEEK sleeve pipe (3) and conical ejection of compact piece (4), its characterized in that: the conical discharging piece (4) of the homogenate tank is conical and is made of high polymer materials.
The tank body (1) of the homogenate tank is of a closed hollow barrel-shaped structure, the interior of the tank body is a cylindrical cavity, a conical discharging piece (4) is placed in the cavity, and the conical lower end of the conical discharging piece (4) is an outlet end;
the upper end of the tank body (1) is connected with a high-pressure pipe connecting piece (2), and the lower port of the high-pressure pipe connecting piece (2) extends into the tank body (1);
the lower end of the tank body (1) is connected with a high-pressure pipe connecting piece (2), the lower port of the high-pressure pipe connecting piece (2) extends into the tank body (1), one end of a PEEK sleeve (3) extends into the high-pressure pipe connecting piece (2), and a capillary to be filled extends into the tank body (1) through the PEEK sleeve (3) through the high-pressure pipe connecting piece (2) and is connected with the lower port of the conical discharging piece (4);
the homogenate tank body (1) and the conical discharging piece (4) are of discrete structures, the tank body (1) is made of stainless steel, the outer diameter of the upper port of the conical discharging piece (4) is matched with the inner diameter of the tank body (1), and the conical discharging piece (4) is directly embedded into the tank body, so that the conical outer surface of the conical discharging piece (4) and the inner surface of the tank body form a closed space.
The high polymer material is nylon or Polyaryletherketone (PAEK).
Compared with the prior art, the method for filling the capillary high performance liquid chromatography column with the small inner diameter and the homogenate tank have the following advantages:
1. in the traditional downward capillary column filling method, because the filling speed is high, the filler is in frictional impact with a metal reducing port in a homogenizing tank, so that the filler is crushed, and the inertia of a chromatographic column is reduced, the peak is trailing, the column efficiency is reduced, and the front pressure of the column is increased. The invention provides a scheme for filling a capillary column at a low speed, which adopts a homogenizing solvent with the density close to that of the filler, and controls the filling pressure to slow down the coagulation speed of the filler, so that the arrangement of a filling layer is more ordered, and the column efficiency is improved. In addition, the adoption of a homogenate tank made of high polymer materials avoids the crushing of the filler,
2. the tank body of the homogenate tank is made of stainless steel, can resist high pressure and is suitable for filling capillary columns with small inner diameters. In order to avoid the damage caused by the fact that the filler impacts the stainless steel reducing port in the filling process, the discharge port of the homogenizing tank is embedded into the discharge port of the conical channel made of the high polymer material, so that the impact on the filler in the filling process is relieved, and the breaking rate of the filler can be effectively reduced. Therefore, the integral inertia of the chromatographic column is obviously improved, and when the chromatographic column is used for analyzing alkaline drugs, proteins and hormone components, the tailing factor of a chromatographic peak can be reduced, thereby being beneficial to the chromatographic separation of the proteins.
Drawings
FIG. 1 is a schematic structural view of an exemplary embodiment of a homogenate tank of the present invention; in the figure: 1-tank body, 2-high pressure tube connecting piece, 3-PEEK sleeve, 4-conical discharge piece and 5-capillary liquid chromatographic column.
FIG. 2 is a chromatogram of IgG protein separation using a capillary liquid chromatography column having an inner diameter of 50 μm in application example 1.
Detailed Description
In order that the invention may be better understood, it is illustrated by the following examples. The specific embodiments of the present invention are set forth herein for the purpose of illustration only and are not intended to be limiting of the invention.
Example 1:
a homogenate tank: the device comprises a tank body, a high-pressure pipe connecting piece, a PEEK sleeve and a conical discharging piece, wherein the tank body is made of stainless steel, a cylindrical cavity is formed in the tank body, the inner diameter of the tank body is 2.1mm, and the outer diameter of the tank body is 3.17 mm; conical ejection of compact spare has been placed in the cavity, and conical ejection of compact spare toper lower extreme is the exit end, and conical ejection of compact spare is made by the nylon materials, and the homogenate tank jar body is discrete structure with conical ejection of compact spare. The top diameter of conical ejection of compact piece is 2.1mm, and the bottom internal diameter is 0.4mm, and direct embedding jar is internal, its bottom and connecting piece in close contact with. The jar body upper end is connected with high-pressure tube connection spare (high-pressure tube connection spare in the embodiment comprises 1/16 inches stainless steel cutting ferrule and 1/16 inches stainless steel nut), this high-pressure tube connection spare lower port stretches into jar internal, jar body lower extreme is connected with high-pressure tube connection spare and the PEEK sleeve pipe that the internal diameter is 0.4mm, PEEK sleeve pipe one end stretches into in the high-pressure tube connection spare, high-pressure pipe spare stretches into jar internal, as shown in figure 1, the capillary that waits to fill stretches into jar internal portion through PEEK sleeve pipe and high-pressure connection spare and links to each other with the lower port of the conical ejection of compact piece. A quartz capillary tube having an outer diameter of 365 μm was used as a column tube, and the quartz capillary tube was inserted into a PEEK sleeve having a length of 2cm and an inner diameter of 0.4mm in advance, and then connected to the lower end of a conical discharge member of a homogenizer tank.
The method for filling the capillary liquid chromatographic column with the small inner diameter by adopting the homogenate tank comprises the following steps:
a. connecting and fixing a quartz capillary tube with a 3mm long C4 monolithic column sieve plate with a PEEK sleeve and a high-pressure connecting piece with a lower port of a conical discharging piece of a homogenizing tank, wherein the inner diameter of the quartz capillary tube is 50 micrometers, the outer diameter of the quartz capillary tube is 365 micrometers, and the length of the quartz capillary tube is 20 cm;
b. c4 bonded silica gel chromatographic packing with the particle size of 5 mu m is adopted for filling, the packing is mixed with a carbon tetrachloride/methanol homogenizing solvent with the volume fraction of 95 percent to prepare homogenate, and the concentration of the packing is 28.5 mg/mL. Ultrasonically mixing for 1 min;
c. adding 200 μ L of the homogenate into a homogenate tank, and filling the vacant area with a solvent to ensure that no air bubbles exist in the homogenate tank;
d. connecting the upper port of the high-pressure pipe connecting piece of the homogenizing tank to a high-pressure liquid phase pump, filling methanol in the pump in advance, starting the high-pressure liquid phase pump, and pressing the methanol into the homogenizing tank;
e. the initial filling pressure is 0.3MPa, the pressure is increased at the speed of 1.5MPa/min along with the filling, the final pressure is 30MPa, and the high-pressure liquid phase pump is closed after the constant pressure is kept for 30 min;
f. and after the pressure of the high-pressure liquid phase pump is reduced to the initial zero, the capillary liquid phase chromatographic column with the small inner diameter is unloaded after the filling is finished.
Application example 1:
the capillary liquid chromatography column filled in example 1 was connected to a high performance liquid chromatography system, and the column efficiency was evaluated by a laser-induced fluorescence detector. The liquid chromatography conditions were: mobile phase a acetonitrile: mobile phase B volume fraction 0.1% aqueous trifluoroacetic acid solution ═ 8: 2; flow rate of mobile phase: 200 nL/min; sample preparation: 5 μ L of 3X 10-10M Dylight 488-labeled IgG single label. The Sepu3010 chromatography workstation (university of Zhejiang) was used for signal acquisition. The experimental results are shown in fig. 2, and the column efficiency of the capillary chromatographic column is 61,500 and the tailing factor of the chromatographic peak is 1.17. The capillary chromatographic column filled by the filling method can be used for high-efficiency separation of protein.
Example 2:
the homogenization pot described in example 1 was used to fill a capillary liquid chromatography column of fine internal diameter, the procedure was as follows:
a. connecting and fixing a quartz capillary tube with a 3mm long silica gel monolithic column sieve plate with a lower port of a conical discharging part of a homogenizing tank through a PEEK sleeve and a high-pressure connecting piece, wherein the inner diameter of the quartz capillary tube is 250 micrometers, the outer diameter of the quartz capillary tube is 365 micrometers, and the length of the quartz capillary tube is 25 cm;
b. c18 bonded silica gel chromatographic packing with the particle size of 5 mu m is adopted for filling, the packing is mixed with a carbon tetrachloride/methanol homogenizing solvent with the volume fraction of 90 percent to prepare homogenate, and the concentration of the packing is 30 mg/mL. Ultrasonically mixing for 1 min;
c. adding 200 μ L of the homogenate into a homogenate tank, and filling the vacant area with a solvent to ensure that no air bubbles exist in the homogenate tank;
d. connecting the upper port of the high-pressure pipe connecting piece of the homogenizing tank to a high-pressure liquid phase pump, filling methanol in the pump in advance, starting the high-pressure liquid phase pump, and pressing the methanol into the homogenizing tank;
e. the initial filling pressure is 0.5MPa, the pressure is increased at the speed of 1.5-2 MPa/min along with the filling, the final pressure is 27MPa, and the high-pressure liquid phase pump is closed after the constant pressure is kept for 10 min;
f. and after the pressure of the high-pressure liquid phase pump is reduced to zero, the filled capillary liquid phase chromatographic column with the small inner diameter is unloaded.
Example 3:
the slurry tank of embodiment 1 wherein the discharge end is made of Polyaryletherketone (PAEK) having a conical inner hole. The homogenate tank is adopted to fill a capillary liquid chromatographic column with a small inner diameter, and the steps are as follows:
a. connecting and fixing a quartz capillary tube with a 3mm long silica gel monolithic column sieve plate with a lower port of a conical discharging part of a homogenizing tank through a PEEK sleeve and a high-pressure connecting piece, wherein the inner diameter of the quartz capillary tube is 75 micrometers, the outer diameter of the quartz capillary tube is 365 micrometers, and the length of the quartz capillary tube is 25 cm;
b. the filler is filled by C18 bonded silica gel chromatographic filler with the particle size of 5 mu m, and the filler is mixed with a tetrabromoethane/methanol homogenizing solvent with the volume fraction of 95 percent to prepare a homogenate liquid, wherein the concentration of the filler is 25 mg/mL. Ultrasonically mixing for 1 min;
c. adding 230 μ L of the homogenate into a homogenate tank, and filling the vacant area with a solvent to ensure that no air bubbles exist in the homogenate tank;
d. connecting the upper port of the high-pressure pipe connecting piece of the homogenizing tank to a high-pressure liquid phase pump, filling methanol in the pump in advance, starting the high-pressure liquid phase pump, and pressing the methanol into the homogenizing tank;
e. the initial filling pressure is 0.3MPa, the pressure is increased at the speed of 1.5-2 MPa/min along with the filling, the final pressure is 35MPa, and the high-pressure liquid phase pump is closed after the constant pressure is kept for 30 min;
f. and after the pressure of the high-pressure liquid phase pump is reduced to zero, the filled capillary liquid phase chromatographic column with the small inner diameter is unloaded.
Claims (5)
1. A method for filling a capillary liquid chromatographic column with a small inner diameter is characterized by comprising the following steps: the method comprises the following steps:
a. connecting and fixing a capillary tube with a sieve plate and a small inner diameter with a lower port of a conical discharge part (4) of a homogenizing tank through a PEEK sleeve (3); the inner diameter of the capillary is 25-250 mu m, and the outer diameter is 365 mu m;
b. mixing a filler with a high-density homogenizing solvent to prepare a homogenizing liquid, wherein the density of the homogenizing solvent is close to that of the filler, and ultrasonically mixing uniformly for later use; the filler in the step b is a silica gel matrix, and the homogenizing solvent is a carbon tetrachloride/methanol mixed solution or a tetrabromoethane/methanol mixed solution, wherein the volume fraction of carbon tetrachloride or tetrabromoethane is 90-95%;
c. adding a proper amount of the homogenate liquid into the homogenate tank and the conical discharging piece (4), and filling the vacant area with a solvent to ensure that no air bubble exists in the homogenate tank;
d. connecting the upper port of the high-pressure pipe connecting piece (2) of the homogenizing tank with a high-pressure liquid phase pump, filling a given solvent in the pump in advance, starting the high-pressure liquid phase pump, pressing the mobile phase into the homogenizing tank, and filling at a low flow rate by adopting a pressure control mode; the mobile phase in the step d is methanol or ethanol, and the flow rate of the mobile phase is 15-20 mu L/min;
e. the initial filling pressure is 0.3-0.5 MPa, the pressure is increased at the speed of 1.5-2 MPa/min along with the filling until 30-40 MPa, and the high-pressure liquid phase pump is closed after the constant pressure is kept for 10-30 min;
f. and after the pressure of the high-pressure liquid phase pump is reduced to zero, the filled capillary liquid phase chromatographic column with the small inner diameter is unloaded.
2. The method of claim 1, wherein: the capillary chromatographic column sieve plate in the step a is an integral column sieve plate and is prepared from a polymer containing a filler bonding phase or a carrier, and the length of the integral column sieve plate is 2-3 mm.
3. The method of claim 1, wherein: the concentration range of the filler is 25-30 mg/mL; the ultrasonic time range is 1-2 min.
4. A homogenate tank for the filling method according to claim 1, consisting of a tank body (1), a high-pressure pipe connector (2), a PEEK bushing (3) and a conical discharge piece (4), characterized in that: the conical discharging piece (4) of the homogenate tank is conical and is made of high polymer materials;
the tank body (1) of the homogenate tank is of a closed hollow barrel-shaped structure, the interior of the tank body is a cylindrical cavity, a conical discharging piece (4) is placed in the cavity, and the conical lower end of the conical discharging piece (4) is an outlet end;
the upper end of the tank body (1) is connected with a high-pressure pipe connecting piece (2), and the lower port of the high-pressure pipe connecting piece (2) extends into the tank body (1);
the lower end of the tank body (1) is connected with a high-pressure pipe connecting piece (2), the lower port of the high-pressure pipe connecting piece (2) extends into the tank body (1), one end of a PEEK sleeve (3) extends into the high-pressure pipe connecting piece (2), and a capillary to be filled extends into the tank body (1) through the PEEK sleeve (3) via the high-pressure pipe connecting piece (2) and is connected with the lower port of the conical discharging piece (4);
the homogenate tank body (1) and the conical discharging piece (4) are of discrete structures, the tank body (1) is made of stainless steel, the outer diameter of the upper port of the conical discharging piece (4) is matched with the inner diameter of the tank body (1), and the conical discharging piece (4) is directly embedded into the tank body, so that the conical outer surface of the conical discharging piece (4) and the inner surface of the tank body form a closed space.
5. The homogenate tank of claim 4, wherein: the high polymer material is nylon or polyaryletherketone.
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