JPH11271291A - Pre-column derivation method and device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To broaden the sample selection by performing the introduction and reaction of a reaction reagent with a sample, the delivery of the sample, the trapping of a sample band, and the delivery of the trapped sample to an analyzing device in different processes. SOLUTION: When a solvent containing a derivation reagent is carried from a pump 1, and a sample is fed from an injector 2, they reaches a reactor 4 through a 6-way valve 3, where the reagent reacts with the sample. The 6-way valve 3 is switched to carry the solvent from a second pump 5 to the reactor 4 and carry a sample band to a first column 10, and the sample is trapped in the first column 10. When the 6-way valve 3 is then switched, the solvent from the pump 1 and the solvent from the second pump 5 are discharged from drains 6, 11 through the reactor 4 and the first column 10, respectively, and the sample band is trapped in the first column 10. When an analyzing eluant is inputted by a third pump 9, the sample trapped in the first column 10 is sent to a second column 12 and detected by a detector 13.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a precolumn derivatization method and apparatus.

[0002]

2. Description of the Related Art Derivatization of a sample is a method of increasing the sensitivity by adding another substance having high detection sensitivity when there is a substance to be analyzed and quantified, but detection is difficult due to low sensitivity. HPLC has particularly high utility value and is a very effective means for detection. The so-called post-column derivatization method, in which pre-column derivatization, in which a reaction is performed beforehand, and analysis is automatically performed online after the separation of a sample in a column, from reaction to detection, is a general technique. . In the latter method, the reaction is performed automatically, so that the result can be obtained without bothering the analyst. On the other hand, there have been some reports on automation in the precolumn method using OPA or the like, but automation using other reagents is rare.

[0003] In the post-column method, a sample solution can be directly injected into an HPLC without requiring a complicated derivatization operation as in the pre-column method. Since the reaction is managed on-line, it can be applied even when the derivatization reaction has not reached the end point or when there is a problem in the stability of the reaction product. Automatic analysis at night is also easy by using an autosampler. On the other hand, it is not possible to use a derivatization reagent that has strong fluorescence. It is also unsuitable for a reaction requiring a long time reaction or a reaction under strong acidity or strong alkalinity. The system and its control are complicated, and HPLC, which should be a general-purpose analytical instrument, becomes a dedicated device for only the target substance. Since the reaction reagent solution is mixed with the components separated by the HPLC column, the peak shape becomes broad. There is a disadvantage called.

[0004]

On the other hand, the pre-column method can convert any substance into a compound which can be detected by HPLC using one or more steps of organic chemical reaction. it can. The components are simple. The amount of reagent used is small. On the other hand, the derivatization operation becomes complicated. The sample components are diluted. Where the resulting derivative is unstable, application becomes difficult. Separation of excess reagents and reaction by-products from the target components must be considered. Has the disadvantage of: Therefore, the present inventors have previously proposed a pre-column derivatization method by a column switching method using a corresponding valve. (Japanese Unexamined Patent Publication No.
This method can stabilize the reaction by eliminating complicated operations by automating derivatization, and prevent peak broadening due to dilution by trapping the sample. Also, since the derivatization reagent itself does not directly enter the analytical column, it is a great feature that deterioration of the column can be prevented. However, in the above invention, since the reaction channel and the trap channel are common, it is difficult to control the reaction conditions and the trap conditions. In addition, the derivatization reagent remains in the piping connected to the six-way valve before and after the trap column, and there is a disadvantage that the obtained chromatogram becomes complicated.

[0005]

Means for Solving the Problems Accordingly, in the present invention,
By separately providing the reaction channel and the trap channel, the conditions are easily controlled, and the control of the trap conditions is also easy. From this, the range of choice of the derivatization reagent can be expanded. In addition, it is possible to eliminate adverse effects on the chromatogram, such as the derivatization reagent does not remain in the piping of the trap column. The method is characterized by comprising a sending step, a step of trapping the sent sample, and a step of sending the trapped sample to an analyzer.

[0006]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to an embodiment shown in FIG. Reference numeral 1 denotes a first pump, which is a six-way valve 3 serving as a first switching valve via an injector 2.
Is communicated with the entrance 31 of. Reference numeral 4 denotes a reactor, which communicates between the port 32 and the port 35 of the six-way valve 3. Reference numeral 5 denotes a second pump, which communicates with the opening 33 of the six-way valve 3.
Reference numeral 6 denotes a drain, which communicates with a port 36 of the six-way valve 3. Reference numeral 7 denotes a static mixer, which is an opening 34 of the 6-way valve 3 and an opening 84 of the 6-way valve 8 as a second switching valve.
Is in communication with Static mixer 7 is also in communication with pump 5.

Reference numeral 9 denotes a third pump, which is a port 8 of the six-way valve 8.
Connected to 1. 10 is a first column, a 6-way valve 8
Are communicated between the openings 83 and 86. Reference numeral 11 denotes a drain, which communicates with a port 85 of the six-way valve 8. The detector 82 is connected through a second column 12 to an opening 82 of the six-way valve 8.
Is in communication with The second column 12 and the detector 13 constitute an analyzer, and various analyzers can be used. It is convenient to use a UV detector as the detector 13, but another detector may be selected. Six-way valves 3 and 8 are mounted in the column oven.

The reactor 4 preferably has a heater and can be adjusted in temperature. It is convenient to use an autosampler to supply the sample. The first column 10 is a trap column, and the second column 12 is an analytical column. Any column can be used as long as it can hold a derivatized sample, and there is no particular difference between the two. The sample used amino acids such as glycine, methionine, valine, and phenylalanine and amikacin, but it can be used for most substances by using one or more steps of organic chemical reaction.

The method of use will be described. A solvent containing a derivatization reagent, for example, a DNFB solution is flowed from the pump 1 and a sample is fed from the injector. At that time, the solvent carries the sample and reaches the reactor 4 via the ports 31 and 32 of the six-way valve 3, where the derivatizing reagent and the sample react. The remainder larger than the total capacity of the reactor is discharged from the drain 6. In the meantime, a solvent for trap, for example, water flows from the second pump 5 and is discharged from the static mixer 7 to the drain 11 through the first columns 10 through the openings 84 and 83 of the six-way valve 8 for cleaning. .

Next, the 6-way valve 3 is switched, and the
Then, the solvent from the second pump 5 flows through the reactor 4, the sample band flows through the first column 10 via the static mixer 7, and is discharged from the drain 11. Therefore, the first precolumn
The sample is trapped in the column 10. At this time, the solvent containing the derivatizing reagent is discharged from the drain 6 through the openings 31 and 36 of the six-way valve 3.

Therefore, the six-way valve 3 is switched. At that time, the solvent containing the derivative reagent flowing from the first pump 1 fills this via the reactor 4 and drains it.
Is more exhausted. On the other hand, the solvent sent from the second pump 5 passes through the openings 33 and 34 of the six-way valve 3, passes through the static mixer 7, passes through the openings 84 and 83 of the six-way valve 8, passes through the first column 10, and is discharged from the drain 11. Is done. The sample band is trapped in the first column 10.

Next, the six-way valve 8 is switched, and the solvent from the second pump 5 is discharged from the drain 11. Also, the third
By the operation of the pump 9, the mobile phase for analysis, that is, the sample trapped in the first column 10 by the supply of the eluent for analysis, for example, phosphate buffer solution / acetonitrile, is sent to the second column 12 and the detector 13 is detected.

[0013]

Example 1 Example 1 (FIG. 2) Reagent: FDNB (2.4-dinitrofluorobenzene) Derivatization reaction conditions Reaction solution: 25 mg / 50 ml, MeOH time: 20 minutes (500 μl reactor, flow rate: 0.025 ml)
/ Min) Temperature: room temperature Analysis conditions Trap column: Inertsil ODS-3 10 * 4.0mmI.
D. 5 μm Eluent for trap: 2% MeOH in H ₂ O Analysis column: Inertsil ODS-3 150 * 4.6 mm I.D.5
eluent for analysis: 25mM phosphate (pH2.3) / ACN = 60/40 (W
/ W) Detection: UV360nm Temperature: 40 ° C Flow rate: 1.0ml / min

Example 2 (FIG. 3) Reagent: DABSCl (dimethylaminoazobenzenesulfonyl chloride) derivatization reaction conditions Reaction solvent: 25 mg / 50 ml, MeOH Reaction time: 10 minutes Reaction temperature: 40 ° C. Analysis conditions Trap column : Inertsil ODS-3 10 * 4.0mmI.
D.5 μm Eluent for trap: 2% MeOH in 50m boric acid Analysis column: Inertsil ODS-3 150 * 4.6mm ID.5
μm eluent for analysis: 25 mM phosphate (pH7.3) / ACN = 75/25 (W
/ W) Detection: UV460nm Temperature: 40 ℃ Flow Rate: 1.0ml / min

Example 3 (FIG. 4) Reagent: Dns-Cl (dansyl chloride) derivatization reaction conditions Reaction solvent: 25 mg / 50 ml, MeOH Reaction time: 10 minutes Reaction temperature: 40 ° C. Analysis conditions Trap column: Inertsil ODS-3 10 * 4.0mmI.
D. 5 μm Trap solvent: 2% MeOH in 50 mM boric acid Analysis column: Inertsil ODS-3 150 * 4.6 mm I.D.5
μm Analysis solvent: 25 mM phosphate (pH7.3) / ACN = 75/25 (W /
W) Detection: UV460nm Temperature: 40 ° C Flow rate: 1.0ml / min

Example 4 (FIG. 5) Reagent: NBD-Cl (4-chloro-7 nitrobenzofurazan) Derivatization reaction conditions Reaction solvent: 25 mg / 50 ml, MeOH Reaction time: 20 minutes Reaction temperature: 75 ° C. Analysis Conditions Trap column: Inertsil ODS-3 10 * 4.0mmI.
D. 5 μm Trap solvent: 2% MeOH in 50 mM boric acid Analysis column: Inertsil ODS-3 150 * 4.6 mm I.D.5
μm Analysis solvent: 25 mM phosphate (pH7.3) / ACN = 75/25 (W /
W) Detection: UV460nm Temperature: 40 ° C Flow rate: 1.0ml / min

Example 5 (FIG. 6) Reagent: FDNB (2,4-dinitrofluorobenzene) Sample: Amikacin Derivatization reaction conditions Reaction solvent: 0.2 g / 100 ml ACN // 28 mM K ₂ HPO ₄ = 50 // 50 Reaction time: 20 minutes (500μReactor, Flow rate: 0.025ml
/ Min) Reaction temperature: 80 ° C Analysis conditions Trap column: Inertsil ODS-3 10 * 4.0mmI.
D. 5 μm Eluent for trap: 2% ACN in H ₂ O Column for analysis: Inertsil ODS-3 150 * 4.6 mm I.D.5
μm Analysis solvent: 25 mM phosphate (pH 7.2) / ACN = 60/40 (W /
W) Detection: UV460 nm Temperature: 40 ° C Flow Rate: 1.0 ml / min Sample amikacin (1.3 mg / ml) Reproducibility V (%) 1.66% (510 μg / ml) Linearity Correlation coefficient: 0.9999977 (42 μg / ml → 10.5 mg / m)
l)

Example 6 (FIG. 7) Reagent: FDNB (2,4 dinitrofluorobenzene) derivatization reaction conditions Reaction solvent: 100 mg / 30 ml MeOH // 25 mM K ₂ HPO ₄ = 30 // 20 Reaction time: 11 minutes Reaction temperature: 75 ° C Analysis conditions Trap column: Inertsil ODS-3 10 * 4.0mmI.
D.5 μm Eluent for trap: 2% ACN in M boric acid Analysis column: Inertsil ODS-3 150 * 4.6 mm I.D.5
μm Analysis solvent: 25 mM phosphate (pH7.3) / ACN = 60/40 (W /
W) Detection: UV360nm Temperature: 40 ° C Sample: Urine with amikacin

[0019]

According to the present invention as described above, a step of introducing and reacting a reaction reagent and a sample, a step of sending out a sample by a different route from the reaction step, a step of trapping the sent out sample band, Since the method comprises a step of sending the sample to an analyzer, the steps from derivatization to separation and detection can be performed automatically by injecting the sample. or,
Samples can be made to HP by one-stage or multi-stage organic chemical reaction.
It became detectable by LC, and it became possible to correspond to most samples.

Since the reaction conditions and trap conditions are easy to control, and there is no residual derivatizing reagent in the reaction channel, the chromatography is stabilized, the range of choice of reagents is widened, and the amount of reagent used is small. As a result, the practical effect is large, such as efficient use.

[Brief description of the drawings]

FIG. 1 is a block diagram of a precolumn derivatization apparatus according to the present invention.

FIG. 2. Chromatogram of derivatization with FDNB

FIG. 3 Chromatogram of derivatization with dabsyl chloride

FIG. 4 Chromatogram when analysis was performed with dansyl chloride

FIG. 5: Chromatogram when analysis was performed with NBC-chloride

FIG. 6: Chromatogram of DBC-amikacin

FIG. 7 is a chromatogram showing the analysis state of amikacin added to urine.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 1st pump 2 Injector 3 6-way valve 4 Reactor 5 2nd pump 6 Drain 7 Static mixer 8 6-way valve 9 3rd pump 10 1st column 11 Drain 12 2nd column 13 Detector

Claims (3)

[Claims] A step of introducing and reacting a reaction reagent and a sample;
A precolumn derivatization method comprising: a step of sending out a sample by a different route from the reaction step; a step of trapping the sent out sample; and a step of sending the trapped sample to an analyzer. 2. A first switching valve is provided with a first pump, a system for feeding a reaction reagent and a sample to a reactor is formed, and a second pump provided in the first switching valve is connected to the second switching valve via the reactor. A system for communicating the sample held in the trap column by a third pump provided for the second switching valve to the detection device. Pre-column derivatization device. 3. The precolumn derivatization apparatus according to claim 2, wherein the first switching valve and the second switching valve are housed in an oven.