JPS61128165A - Liquid chromatograph apparatus - Google Patents

Abstract

PURPOSE:To accurately perform the simultaneous analysis of a plurality of components having different max. absorption wavelengths, by providing a control mechanism capable of performing the wavelength change-over setting of a detected optimum UV wavelength to the required component from a separation column on the holding time thereof by an UV detector. CONSTITUTION:A liquid chromatograph apparatus 1 is constituted of a flow passage formed of a moving phase storage tank 2, a high pressure liquid feed pump 3, a specimen injection part 4, a separation column 5 equipped with a thermostatic tank 51 and the flow cell set in an UV detection part. The UV detection part is constituted of an optical system equipped with a deuterium lamp 12, a slit 13, a diffraction lattice 10, a concave mirror 14, a slit 15 and a photomultiplier tube. The diffraction lattice 16 is adjusted in its angle by a stepping motor 11. The change-over of a wavelength suitable for each component is performed by said optical system, a control part 18, an injection detection sensor 41 and a key input part 9 to perform accurate analysis.

Description

[Detailed description of the invention] (b) Industrial application fields The present invention relates to a liquid chromatograph device.

More specifically, the present invention relates to a liquid chromatography device that has an ultraviolet absorption (Uv) detector as a detector and is particularly useful for separating and analyzing samples containing a plurality of components with significantly different sensitivities to a single UV wavelength.

(b) Prior Art Liquid chromatography devices using UV detectors as detectors, particularly flat liquid chromatography devices, have been widely used in the past.

However, in such liquid chromatography devices, the detection wavelength is set to a single wavelength, so there is a significant difference in peak size between components that have significantly different sensitivities to that wavelength, that is, molar extinction coefficients. It will be done. (ζRecently, drinks containing various vitamin components and stimulant components, such as nutritional tonics and soft drinks, are on the market, but the representative components contained in these are vitamins C9, B1, B3, B6, and Café). When trying to analyze using in-it liquid chromatography, the maximum absorption wavelength of each component is different, so there are significant differences in the size of the peaks obtained, making it impossible to perform accurate quantitative analysis for each component. was there.

P→Purpose of the Invention The present invention was made to solve the above-mentioned conventional problems, and provides a liquid chromatograph capable of performing accurate separation theorem 1 analysis even when the maximum absorption wavelengths of each test component are significantly different. It is intended to provide a graphical representation of glt.

B) Structure of the Invention According to the present invention, a mobile phase storage tank, a liquid feeding pump,
It is equipped with a sample injection section, a separation column, and a UV detection section in sequence, and detects the optimal υV wavelength for detection of a predetermined fraction component from the separation column based on its retention time.
A liquid chromatograph device is provided, which is characterized by being provided with a wavelength switching control mechanism that can be appropriately switched and set in a detection section.

This invention enables accurate detection of each component of interest by setting the measurement UV wavelength in the UV detection section to the optimum detection wavelength (usually the maximum absorption wavelength) for each fraction. It is constructed in such a way that it is possible to conduct a constant foot analysis.

The apparatus of the present invention is suitable for separating and analyzing water-soluble organic substances in samples containing various mixed components, and is particularly suitable for analyzing samples containing multiple vitamin components, stimulants, etc., such as the aforementioned drink. .

As for the analysis conditions for such samples, it is suitable to set the reversed phase mode, and as a mobile phase, an acidic phosphate JII solution with a pH of around 2.1 is mixed with a small amount of polar organic solution i, and a small amount of ion pairs (e.g. , anionic surfactants such as sodium octadecyl sulfonate), and a so-called ODS column in which silicone polymer is chemically bonded to a silica carrier as the stationary phase. Furthermore, as the polar organic solvent to be added in a small amount, one having a polarity higher than that of acetone is suitable, and acetonitrile is preferable.

(E) Examples The present invention will be explained in detail with reference to examples below, but the invention is not limited by these examples.

Fi+ shown in FIG. 1 is a configuration explanatory diagram showing a high performance liquid chromatograph g1 which is an embodiment of the apparatus of the present invention. In the figure, high-speed limb chromatography equipment fffl+
Basically, mobile phase 1fi (2+, high pressure liquid pump (
3), sample injection M5 (<1, separation column (5) and UV
A flow path is formed from a flow cell ( ) set in the detection section, and is connected to a drain H. On the other hand, the UV detection section includes a light source consisting of a deuterium lamp, a diffraction grating side,
It consists of an optical system equipped with a light receiver (6) consisting of a slit, an α-shape, a concave a-shape, and a photomultiplier tube. The diffraction grating u (M is configured to adjust the setting angle It by a stepping motor (1), and the g control part (
8), the injection detection sensor and the key manual section (9) constitute the wavelength switching control mechanism of the present invention.

As shown in FIG. 2, the control section a (8) consists of a microcomputer system WIJm using a microprocessor (81i). (82) is a ROM section, which includes a system program section (821), a wavelength switching program section (822 ) and an arithmetic program section (82B), (8a) is an RA
MP, (84) is a pass line. The system program section (821) has a timer that is linked to the microprocessor (81) based on the output (sample injection signal) of the injection detection sensor (υ), which is a photocoupler attached to the sample injection section (4). The RAM section (8
8) is the basis of each component intended for analysis. Retention time a+b
, c+d...and the maximum absorption wavelengths α, β171δ... of each of these components are recorded! These data are based on 8! of each ingredient. It is input from the key human power section (9) before analysis based on sML. The calculation program section (82ft) calculates the time immediately before the rise of the fraction of each component corresponding to the reference retention times a, bc, d, etc. of each component input to the RAM t?f (8a). (Corrected holding time) A, B, C, D... f: g
A program is set to output and store it again in RAMP (813), and normally the standard holding time alb+c1d.
The times A, B, C, D, etc. before the rising edge M are calculated by subtracting the time of about half m width from each of them.

On the other hand, the wavelength switching program @ (822) includes the above F
rfr/iJ data A, B, C, D...
A program is set for comparing the time data and time data of the timer, and a program for outputting a motor drive signal (data) to the D/A converter (85) based on the comparison result.

The motor drive signal is based on the maximum absorption wavelengths α, β, γ, δ...
It is output to the D/A converter (85) as data with a weight proportional to..., and correspondingly the data is output to the D/A converter (85).
6) The stepping motor (11) is driven for a predetermined time via t'', and the angle of the diffraction grating (]0) is detected as vR;
As a result, predetermined wavelengths α, β+7rδ, . . . are appropriately switched and set as measurement wavelengths. The main routine of the wavelength switching program n (822) is shown in FIG. 8, linked to a timer.

In the liquid chromatograph apparatus fl) having such a configuration,
A sample such as a drink is injected from an injection port (4) and introduced into a separation column (5) set at about 35 to 45°C by a constant temperature bath (51). Next, each separated fraction is sent to the flow cell (7), where it is UV detected and recorded in the integrator (61). At this time, U'l/wavelength is analyzed by the wavelength switching side aSS described above. Since the maximum absorption wavelength is controlled for each intended fraction component, accurate quantitative analysis of each component can be performed.
Commercially available drinks containing + B3 + B6 and caffeine as main components were analyzed. The data of (retention time)/(III large absorption wavelength) input in advance are respectively vitamin B1: (10,9 minutes)/(254 nm+, vitamin B3: (8,05 minutes)/(290 nm), vitamin B6
:(4,98 minutes)/(270nm), Caffeine:
(7,8 minutes)/(265 nm). Moreover, the analysis conditions are as follows.

Mobile phase: 60mM phosphate buffer (pH 2,1; 80mM
Mixture of M monosodium phosphate + 80 mM phosphoric acid (90 parts by volume), acetonitrile (10 parts by volume), and sodium octadecylsulfone (1 mM) Stationary phase: C18 ODS column Flow rate: 1.5 d/min Separation column Temperature: 40°C This analysis result was calculated using a fixed wavelength (254n) without wavelength switching.
Figures 4(a) and 4(b) show the results of a comparative example using a conventional device for UV detection in (m). In the figure, (a) shows the above-mentioned example, and (b) shows a comparative example. As shown in the figure, fζ is almost undetectable by the conventional fixed wavelength detection type device and is constant. A clear peak was detected for the difficult vitamin B6, and it can be seen that the sensitivity is increased even for caffeine. Therefore, an accurate and ideal determination of each component can be made.

Note that the wavelength switching control mechanism of the present invention is not limited to the above-mentioned embodiments, and may be, for example, one using a rotary switching type interference filter or one using a quartz prism.

Furthermore, the wavelength does not necessarily have to be set to the maximum absorption wavelength in the ultraviolet region, but may be set to an appropriate maximum absorption wavelength, that is, a UV wavelength suitable for detection.

However, at least as shown in the above configuration,
A sample introduction detection sensor, a timer driven by a signal from the sample introduction detection sensor, a data storage section that stores the retention time and maximum absorption wavelength of each predetermined component, and a correction of about half width to the retention time of the data storage section. An arithmetic unit that subtracts the value and stores this corrected holding time in the data storage unit again, and a calculation unit that compares the corrected holding time in the data storage unit and the timer time, and mechanically calculates the value (based on ζ) It is suitable to configure a wavelength switching control mechanism from a UV wavelength in the UV detector: a--a length setting section for appropriately switching and setting.

(to) Effect of the invention The device of this invention [! According to lζ, it is possible to eliminate peak irregularities and errors caused by the difference in sensitivity of separated components due to the fixed wavelength of conventional liquid chromatography equipment, and it is possible to accurately quantify multiple components having different maximum absorption wavelengths simultaneously. :
You can do 4 things.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing the configuration of an embodiment of the liquid chromatograph apparatus of the present invention, FIG. 2 is an explanatory diagram showing the basic configuration of the control section in FIG. FIG. 4 is a flowchart showing the main routine of the wavelength switching program section that can be performed in FIG. +11...High performance liquid chromatography device, (2)...
・Mobile phase storage tank, (3)...High pressure liquid pump, (4
)...Sample injection section, (6)...Separation column, (
6)...Receiver, 17+...Flow cell, (8)...
...Control @ Department, ('J)...Key Human Resources Department, Shout-
...Diffraction grating, (11)...Stepping motor, +121...Light source, IJ3, 05)
...Slit, (141...concave m, on...drain, circle...injection detection sensor, (61)...integrator. Published by:

Claims (1)

[Claims] 1. A mobile phase storage tank, a liquid sending pump, a sample injection section, a separation column, and a UV detection section are sequentially provided, and the optimum UV wavelength for detection of a predetermined fraction component from the separation column is determined based on the retention time of the UV wavelength. 1. A liquid chromatograph apparatus characterized by being provided with a wavelength switching control mechanism that can be appropriately switched and set in a detection section.