阅读说明：本技术 一种提高固相微萃取进样方式色谱峰容量的方法 (Method for improving chromatographic peak capacity of solid phase microextraction sample injection mode ) 是由 向章敏 陈啸天 刘舒芹 方舒婷 肖雪 于 2020-12-16 设计创作，主要内容包括：本发明公开了一种提高固相微萃取进样方式色谱峰容量的方法,包括：将待测样品至于顶空瓶内,并用顶空瓶盖密封；将顶空瓶置于加热器上,加热平衡温度为室温至80℃,加热时间为10-60min；取固相微萃取纤维头,插入顶空瓶内平衡吸附5-60min；将固相微萃取纤维头取出,插入气相色谱进样口200-300℃解析1-5min；在进样口后端使用调制器将进样口气化后的组分在-10℃至-50℃冷却富集3-10min；将冷却后的组分转移至250-320℃调制器加热释放,解析时间10-60s；将加热释放后的组分使用毛细管色谱柱进行分离；将分离后组分使用高分辨质谱进行化合物鉴定。本发明可有效改善固相微萃取进样后组分不能瞬间气化,导致峰型变化及灵敏度减低的问题。(The invention discloses a method for improving the peak capacity of a solid phase microextraction sample injection mode chromatogram, which comprises the following steps: placing a sample to be detected in a headspace bottle, and sealing the headspace bottle cap; placing the headspace bottle on a heater, and heating to balance the temperature of room temperature to 80 ℃ for 10-60 min; taking a solid phase micro-extraction fiber head, inserting into a headspace bottle for balanced adsorption for 5-60 min; taking out the solid phase micro-extraction fiber head, inserting the fiber head into a gas chromatography sample inlet for analysis at 200-; cooling and enriching the gasified components at the back end of the sample inlet at-10 deg.C to-50 deg.C for 3-10 min; transferring the cooled components to a modulator at the temperature of 250-320 ℃ for heating and releasing, and analyzing for 10-60 s; separating the heat released components by using a capillary chromatographic column; the separated components were identified for compounds using high resolution mass spectrometry. The invention can effectively solve the problems that the components can not be instantly gasified after solid phase microextraction sample injection, which causes peak type change and sensitivity reduction.)

1. A method for improving the peak capacity of a solid phase microextraction sample injection mode chromatographic column is characterized by comprising the following steps: the method comprises the following steps:

placing a sample to be detected in a headspace bottle, and sealing the headspace bottle with a headspace bottle cap;

step two, placing the headspace bottle on a heater, and heating to balance the temperature between room temperature and 80 ℃ for 10-60 min;

thirdly, taking the solid phase micro-extraction fiber head, inserting the solid phase micro-extraction fiber head into a headspace bottle for balanced adsorption for 5-60 min;

step four, taking out the solid phase micro-extraction fiber head, inserting the fiber head into a gas chromatography sample inlet for analysis at the temperature of 200-;

fifthly, cooling and enriching the components gasified by the sample inlet at the temperature of between 10 ℃ below zero and 50 ℃ below zero for 3 to 10min by using a modulator at the rear end of the sample inlet;

sixthly, transferring the cooled components to a modulator at the temperature of 250-320 ℃ for heating and releasing, and analyzing for 10-60 s;

step seven, separating the components after heating release by using a capillary chromatographic column;

and step eight, identifying the separated components by using a high-resolution mass spectrum.

2. The method for improving the peak capacity of the solid phase microextraction sample injection mode chromatogram according to claim 1, which is characterized in that: the modulator is a solid state thermal modulator.

3. The method for improving the peak capacity of the solid phase microextraction sample injection mode chromatogram according to claim 1, which is characterized in that: the high-resolution mass spectrum is a quadrupole flight time high-resolution mass spectrum.

Technical Field

The invention relates to the technical field of sample pretreatment and chromatographic analysis, in particular to a method for improving chromatographic peak capacity in a solid phase microextraction sample injection mode.

Background

The modulator technology was originally a mobile "sweet" modulator (Ledford, et al 2001), evolved to a two-stage four-jet thermal modulator (benns, et al 2001) and a ring modulator (Ledford, et al 2002), and then to an "LMCS" radial modulator (Phillips, et al 2004). However, since all the above preparations use liquid nitrogen as refrigerant, large consumption and extra equipment are necessarily generated, although after 2004, the compression refrigeration technology using circulating liquid refrigerant is used for replacing liquid nitrogen consumption and is used for full two-dimensional chromatographic preparation (Libardoni, et al 2005&2006&2010), in fact, the product still needs to use nitrogen as hot gas injection, the consumption is large, and the minimum refrigeration temperature is-90 ℃, which is far higher than that of liquid nitrogen, and only compounds above C7 can be supplemented.

In recent years, a modulator technology without liquid nitrogen consumption, namely a solid-state thermal modulator (Guan X S, et al.2016) of a semiconductor refrigeration technology, is developed by domestic research and development teams, and the modulator technology can be used as a full two-dimensional modulator and also can be used as a gas phase cooling mode, but is not reported to be used for gas phase online cooling and enrichment.

Solid Phase Microextraction (SPME) is a green and rapid sample pretreatment technology integrating extraction, purification, separation and enrichment, and is one of six innovations in the Analytical Chemistry field of the 20 th century and the 90 th century evaluated by the American society for Chemistry, journal of Analytical Chemistry, in 2001. In recent years, more and more researches are carried out on the analysis fields of food, environment, medicines and the like (Koutidou, et al,2017& Song X B, et al.2020) by adopting solid-phase microextraction and gas chromatography, and the characteristics of the method are mainly benefited by the fact that simple, quick and in-situ pretreatment of food flavor substances can be realized.

However, in this analysis method, since the solid-phase microextraction surface coating material usually adsorbs the target substance by bonding, and is often incompletely gasified instantaneously during analysis at the sample inlet, leading to problems of broadened chromatographic peak profile, severe peak tailing, low sensitivity of low-content substances, and the like, a method is urgently needed to overcome this drawback.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for improving the chromatographic peak capacity of a solid phase microextraction sample injection mode, which is characterized in that after a component is gasified, low-temperature enrichment is carried out, then the component is transferred to high temperature for instant release, then a capillary chromatographic column is adopted for chromatographic separation, and simultaneously a high-resolution mass spectrum is combined for carrying out qualitative analysis on the separated substance, so that the problems of peak type change and sensitivity reduction caused by the fact that the component cannot be gasified instantly after the solid phase microextraction sample injection can be effectively solved.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for improving the peak capacity of a solid phase microextraction sample injection mode chromatographic method comprises the following steps:

placing a sample to be detected in a headspace bottle, and sealing the headspace bottle with a headspace bottle cap;

step two, placing the headspace bottle on a heater, and heating to balance the temperature between room temperature and 80 ℃ for 10-60 min;

thirdly, taking the solid phase micro-extraction fiber head, inserting the solid phase micro-extraction fiber head into a headspace bottle for balanced adsorption for 5-60 min;

step four, taking out the solid phase micro-extraction fiber head, inserting the fiber head into a gas chromatography sample inlet for analysis at the temperature of 200-;

fifthly, cooling and enriching the components gasified by the sample inlet at the temperature of between 10 ℃ below zero and 50 ℃ below zero for 3 to 10min by using a modulator at the rear end of the sample inlet;

sixthly, transferring the cooled components to a modulator at the temperature of 250-320 ℃ for heating and releasing, and analyzing for 10-60 s;

step seven, separating the components after heating release by using a capillary chromatographic column;

and step eight, identifying the separated components by using a high-resolution mass spectrum.

Further, the modulator is a solid state thermal modulator.

Further, the high-resolution mass spectrum is a quadrupole time-of-flight high-resolution mass spectrum.

Compared with the prior art, the invention has the beneficial effects that:

1) according to the method, after being analyzed at the sample inlet through the solid-phase microextraction fiber head, sample components do not directly enter the gas chromatographic column for separation, but are enriched and focused at a low temperature through the solid-phase thermal modulator, and then are instantaneously released to the chromatographic column at a high temperature for separation, so that all the components can instantaneously enter the gas chromatographic column for separation and analysis, and the chromatographic peak type is improved, so that the chromatographic peak capacity is improved, and higher-flux substance information can be obtained.

2) The method can be realized on a gas chromatography-mass spectrometer equipped with a solid thermal modulator, has the advantages of simple and convenient operation, and better solves the problems of broadening of a chromatographic peak shape, serious peak tailing, low sensitivity, insufficient peak capacity and the like caused by incomplete instantaneous gasification during solid phase microextraction sample injection.

3) Compared with the conventional method, the method can improve the chromatographic peak capacity by about 1.5 to 4 times under the same condition, and is better applied to the high-flux substance detection in the fields of natural products, foods, medicines, life sciences, environment and the like.

Drawings

FIG. 1 is a flow chart of the method of the present invention.

FIG. 2 is a comparison of the method of the present invention and the conventional method for analyzing the volatile components of white spirit.

FIG. 3 is a comparative graph showing the volatile components of tea soup after the method of the present invention and the conventional method are used.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Because solid phase micro extraction sampling is different from liquid and headspace sampling, the solid phase micro extraction sampling can be instantly gasified, and the analysis of the adsorbed target substance needs a certain time, so that the chromatographic peak shape is widened and the peak is trailing, thereby influencing the detection.

Therefore, the invention adopts a solid phase microextraction sample injection mode, and is designed in a certain temperature condition, volatile components are volatilized towards the upper space after a solid or liquid sample in a headspace bottle is heated, vapor pressure is generated, when a gas-liquid-solid three phase reaches thermodynamic dynamic equilibrium, a target substance is adsorbed on a fiber head material through a bonding effect, the substance adsorption concentration reaches equilibrium within a certain time, then a solid phase microextraction fiber head adsorbing the target substance is inserted into a gas chromatography sample injection port at the temperature of 200-:

placing a sample to be detected in a headspace bottle, and sealing the headspace bottle with a headspace bottle cap;

step two, placing the headspace bottle on a heater, and heating to balance the temperature between room temperature and 80 ℃ for 10-60 min;

thirdly, taking the solid phase micro-extraction fiber head, inserting the solid phase micro-extraction fiber head into a headspace bottle for balanced adsorption for 5-60 min;

step four, taking out the solid phase micro-extraction fiber head, inserting the fiber head into a gas chromatography sample inlet for analysis at the temperature of 200-;

fifthly, cooling and enriching the components gasified by the sample inlet at the temperature of between 10 ℃ below zero and 50 ℃ below zero for 3 to 10min by using a modulator at the rear end of the sample inlet;

sixthly, transferring the cooled components to a modulator at the temperature of 250-320 ℃ for heating and releasing, and analyzing for 10-60 s;

step seven, separating the components after heating release by using a capillary chromatographic column;

and step eight, identifying the separated components by using a high-resolution mass spectrum.

The modulators used in the fifth step and the sixth step are solid-state thermal modulators, and a semiconductor refrigeration technology is adopted, so that liquid nitrogen consumption is not needed.

And the high-resolution mass spectrum used in the step eight is a quadrupole flight time high-resolution mass spectrum and is used for qualitative analysis.

According to the method, after being analyzed at the sample inlet through the solid-phase microextraction fiber head, sample components are not directly separated after entering the gas chromatographic column, but are enriched and focused at a low temperature through the solid-phase thermal modulator, and then are instantly released to the chromatographic column at a high temperature for separation, so that all the components can instantly enter the gas chromatographic column for separation and analysis, the chromatographic peak type is improved, and the chromatographic peak capacity is improved.

The present invention will be described in further detail with reference to specific examples.

Example 1:

the application of the method in the detection and analysis of the volatile components of the white spirit is further explained by taking the detection of the volatile components of the white spirit as an example. According to the method for improving the chromatographic peak capacity of the solid-phase microextraction sample injection mode, the components are cooled and enriched and then subjected to chromatographic separation, and meanwhile, the method is compared with the method without the method.

Directly taking a white spirit sample from 5 mu L to 20mL in a headspace bottle, covering a sealing cover, balancing headspace at 50 ℃ for 20 min, then carrying out headspace adsorption by using a solid phase microextraction fiber head (DVB/CAR/PDMS) for 30min, then carrying out resolution at 250 ℃ for 5min by using a sample inlet, cooling and enriching, and then carrying out gas chromatography high-resolution mass spectrometry. Cooling conditions: cooling temperature: -50 ℃; cooling time: 5 min; resolving temperature: at 260 ℃. Gas chromatography conditions: a chromatographic column: HP-5MS (30 m.times.0.25 mm, 0.25 μm); a sample inlet: at 250 ℃, not shunting, and analyzing for 5min at a sample inlet; the carrier gas is helium, and the flow rate is constant and is 1.0 mL/min; the column temperature was decreased from an initial temperature of 200 deg.C (held for 5min), at a rate of 50 deg.C/min to 50 deg.C (held for 2min), and then increased at a rate of 5 deg.C/min to 240 deg.C (held for 2 min). Mass detector conditions: transmission line temperature 280 ℃, ion source temperature 200 ℃, quadrupole temperature 150 ℃, ionization mode: EI 70eV, mass range: 30-400m/z, qualitative method: the results of the spectral library matching method (NIST17) and the accurate mass calculation method are shown in FIG. 2.

As can be seen from FIG. 2, a small amount of white spirit is directly added into the headspace bottle for in-situ sampling analysis, and no other pretreatment method is adopted, and then the method of the present invention is adopted for comparative analysis with the conventional method. The result shows that under the same analysis condition, compared with the conventional method, 248 volatile components of the white spirit can be detected by the method (SPME-Online CT), while only 140 components are detected by the conventional method (SPME), the peak capacity is increased by 1.8 times, wherein 28 esters are increased, 18 alcohols are increased, 9 aldehydes are increased, 8 ketones are increased, 9 ethers are increased, 20 nitrogen heterocycles are increased, and 16 other types are increased. In addition, as seen from the peak comparison in fig. 1, the method of the present invention can significantly improve the peak intensity and the peak type, can make the peak height higher, the peak width smaller, reduce the tailing peak, and significantly improve the detection sensitivity, and is the key to obtain more substance chromatographic peaks.

Example 2:

taking the volatile component detection of the tea soup as an example, the application of the method in the volatile component detection and analysis of the tea soup is further explained. Similarly, the volatile chemical components of the tea soup are directly enriched in the experiment by adopting a headspace solid-phase microextraction method, and then the components are subjected to chromatographic separation after being cooled and enriched according to the method for improving the chromatographic peak capacity of the solid-phase microextraction sample injection mode, and are subjected to comparative analysis with the conventional method.

Pulverizing folium Camelliae sinensis, sieving with 60 mesh sieve, weighing 200mg folium Camelliae sinensis sample into 20mL headspace bottle, adding 100 deg.C boiled water 5mL, immediately covering with sealing cover, standing for 5min, and loading for analysis. The headspace was equilibrated at 60 ℃ for 30min, followed by headspace adsorption with a solid phase microextraction fiber head (DVB/CAR/PDMS) for 30min, followed by desorption at 260 ℃ for 4min at the inlet. Cooling conditions: cooling to-45 deg.c; cooling for 5 min; the desorption temperature was 250 ℃. Gas chromatography conditions: column DB-WAX (30 m.times.0.25 mm, 0.25 μm); the sample inlet is 250 ℃, the flow is not divided, and the sample inlet analysis time is 4 min; the carrier gas is helium, and the flow rate is constant and is 1.0 mL/min; the column temperature was decreased from an initial temperature of 200 deg.C (held for 4min), at a rate of 50 deg.C/min to 50 deg.C (held for 2min), and then increased at a rate of 5 deg.C/min to 240 deg.C (held for 3 min). Mass detector conditions: transmission line temperature 280 ℃, ion source temperature 200 ℃, quadrupole temperature 150 ℃, ionization mode: EI 70eV, mass range: 30-400m/z, qualitative method: the results of the spectral library matching method (NIST17) and the accurate mass calculation method are shown in FIG. 3.

As can be seen from FIG. 3, 217 components were detected by the method of the present invention (SPME-one CT), and also under the same analysis conditions, only 66 components were detected by the conventional method (SPME), which increased the peak capacity by nearly 3.3 times, and the classification analysis was performed according to the volatile fragrance precursor sources, in which 32 types of Maillard products were increased, 18 types of phenylalanine degradation products were increased, 14 types of lutein degradation products, 23 types of carotenoid degradation products, 21 types of sabanoid degradation products were increased, 18 types of chlorophyll degradation products were increased, and 25 types of other products were increased. In addition, from the chromatographic peak comparison in fig. 3, the on-line cooling technology can obviously improve the peak intensity and the peak type, and particularly, the tailing of the peak with stronger volatility (the substance with the retention time close to the front) is obviously reduced, the half-peak width is narrowed, and the detection sensitivity is obviously improved, so that the peak capacity and the component information of the tea soup volatile substance can be obviously improved.

The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention accordingly, and not to limit the protection scope of the present invention accordingly. All equivalent changes or modifications made in accordance with the spirit of the present disclosure are intended to be covered by the scope of the present disclosure.