Method for separating multiple polycyclic aromatic hydrocarbons by serial mode chromatographic columns

Abstract

The invention belongs to the technical field of analytical chemistry, and discloses a method for separating multiple polycyclic aromatic hydrocarbons by using a series mode chromatographic column. The separation method comprises the following steps: preparing a polycyclic aromatic hydrocarbon mixed standard solution; connecting the first chromatographic column and the second chromatographic column in series to obtain a series mode chromatographic column; and separating the polycyclic aromatic hydrocarbon mixed standard solution by adopting a serial mode chromatographic column. According to the invention, the two chromatographic columns are combined in series, so that the column efficiency of each chromatographic column is brought into full play, and more sufficient and efficient separation of 16 polycyclic aromatic hydrocarbons is realized. According to the method, the defect that the separation effect is poor when a single liquid chromatographic column is used is overcome, and the target analyte is more sufficiently separated through the method that the two chromatographic columns are connected in series.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column. Background Art

[0002] As mutagenic and carcinogenic compounds, polycyclic aromatic hydrocarbons (PAHs) pose a serious threat to ecosystems and public health. Due to their semi-volatile, non-degradable and long-distance transport properties, PAHs emitted are ubiquitous in the global atmosphere, water and soil environment under natural process conditions such as atmospheric transmission and water cycle. PAHs are eventually transferred from the atmosphere and soil to water under atmospheric deposition and rainfall erosion, making receiving waters the main source of PAHs.

[0003] In the prior art, the methods commonly used to separate and detect PAHs include: gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS) and high performance liquid chromatography (HPLC), among which GC-MS is a common method for separating and detecting PAHs. However, considering that GC-MS instruments are relatively expensive and HPLC has been popularized in grassroots laboratories, high performance liquid chromatography is mainly considered. Among the 16 PAHs, since the molecular structures and molecular weights of fluorene and acenaphthylene, chrysene and benzo[a]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene are very similar, the peak times are too close during liquid chromatography analysis, resulting in large-area overlap of peak shapes. According to the existing standard (HJ-478-2009), the separation effect of a single L-C18 liquid chromatography column on the above six PAHs is not significant enough. Therefore, the superiority of the method can be judged by improving the separation effect of the above six PAHs.

[0004] Therefore, it is of great significance to develop a method for separating multiple PAHs using a tandem mode chromatographic column with good separation, high sensitivity, and strong resistance to matrix interference. Summary of the invention

[0005] In view of this, the present invention provides a method for separating multiple PAHs using a tandem mode chromatographic column, the purpose of which is to solve the problems of poor separation effect, low sensitivity and susceptibility to interference of liquid chromatography for PAHs in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides a method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column, comprising the following steps:

[0008] 1) Prepare a mixed standard solution of polycyclic aromatic hydrocarbons;

[0009] 2) connecting the first chromatographic column and the second chromatographic column in series to obtain a tandem mode chromatographic column;

[0010] 3) The PAHs mixed standard solution was separated using a tandem mode chromatographic column.

[0011] Preferably, in step 1), the polycyclic aromatic hydrocarbons include naphthalene, acenaphthene, fluorene, acenaphthylene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene.

[0012] Preferably, in step 1), the concentrations of naphthalene, acenaphthene, fluorene, acenaphthylene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene in the mixed standard solution of polycyclic aromatic hydrocarbons are independently 8 to 12 mg / L;

[0013] The solvent of the PAHs mixed standard solution is acetonitrile.

[0014] Preferably, in step 2), the first chromatographic column and the second chromatographic column independently include an L-C18 column, an S-C18 column, a Ph column or a PFP column.

[0015] Preferably, when the first chromatographic column is an L-C18 column, the second chromatographic column is an S-C18 column, a Ph column or a PFP column;

[0016] When the second chromatographic column is an L-C18 column, the first chromatographic column is an S-C18 column, a Ph column or a PFP column.

[0017] Preferably, in step 3), the mobile phase in the tandem mode chromatographic column is: firstly maintained with an acetonitrile aqueous solution for a period of time, and then the acetonitrile content is increased until the acetonitrile content in the mobile phase reaches 100% until a peak appears.

[0018] Preferably, in the acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 60-70:30-40;

[0019] The period of time is 25 to 30 minutes;

[0020] The rate of increase of acetonitrile content was 2-3% acetonitrile / min.

[0021] Preferably, the flow rate of the mobile phase is 1 to 1.5 mL / min.

[0022] Preferably, in step 3), the injection volume of the PAH mixed standard solution is 15 to 25 μL;

[0023] The column temperature of the tandem mode column is 25-35°C.

[0024] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention makes full use of the column efficiency of each chromatographic column by connecting two chromatographic columns in series, and achieves more complete and efficient separation of 16 polycyclic aromatic hydrocarbons. The method of the present invention makes up for the disadvantage of poor separation effect of using a single liquid chromatography column, and achieves more complete separation of target analytes by connecting two chromatographic columns in series.

[0026] The method of the invention has the advantages of good separation degree and good resistance to matrix interference, and can simultaneously realize the separation and quantitative detection of 16 kinds of polycyclic aromatic hydrocarbons.

[0027] The present invention provides a new separation and detection idea. When the column efficiency of a single chromatographic column is insufficient to achieve complete separation of the target analyte, it is possible to consider using two or more chromatographic columns in series to enhance the separation column efficiency and achieve complete separation of the target analyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 The chromatograms of the separation of polycyclic aromatic hydrocarbons in Example 1, Comparative Example 1 and Comparative Example 2, wherein 1 is naphthalene, 2 is acenaphthene, 3 is fluorene, 4 is acenaphthene, 5 is phenanthrene, 6 is anthracene, 7 is fluoranthene, 8 is pyrene, 9 is chrysene, 10 is benzo[a]anthracene, 11 is benzo[b]fluoranthene, 12 is benzo[k]fluoranthene, 13 is benzo[a]pyrene, 14 is dibenzo[a,h]anthracene, 15 is benzo[g,h,i]perylene, and 16 is indeno[1,2,3-cd]pyrene;

[0030] Figure 2 The chromatograms of the separation of polycyclic aromatic hydrocarbons in Comparative Examples 1 to 4 are as follows;

[0031] Figure 3 The chromatograms for separating polycyclic aromatic hydrocarbons in Examples 1 to 3;

[0032] Figure 4 The chromatograms for separating polycyclic aromatic hydrocarbons in Examples 4 to 6;

[0033] Figure 5 The chromatograms for separating polycyclic aromatic hydrocarbons in Example 1, Example 4 and Comparative Example 2 are shown. DETAILED DESCRIPTION

[0034] The present invention provides a method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column, comprising the following steps:

[0035] 1) Prepare a mixed standard solution of polycyclic aromatic hydrocarbons;

[0036] 2) connecting the first chromatographic column and the second chromatographic column in series to obtain a tandem mode chromatographic column;

[0037] 3) The PAHs mixed standard solution was separated using a tandem mode chromatographic column.

[0038] In the present invention, in the step 1), the polycyclic aromatic hydrocarbons include naphthalene, acenaphthene, fluorene, acenaphthylene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene.

[0039] In the present invention, in the step 1), the concentrations of naphthalene, acenaphthene, fluorene, acenaphthylene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene in the mixed standard solution of polycyclic aromatic hydrocarbons are independently preferably 8 to 12 mg / L, more preferably 9 to 11 mg / L, and more preferably 10 mg / L;

[0040] The solvent of the polycyclic aromatic hydrocarbons mixed standard solution is preferably acetonitrile, and the acetonitrile is preferably a chromatographic acetonitrile solution.

[0041] In the present invention, in the step 2), the first chromatographic column and the second chromatographic column independently include an L-C18 column, an S-C18 column, a Ph column or a PFP column.

[0042] In the present invention, when the first chromatographic column is an L-C18 column, the second chromatographic column is preferably an S-C18 column, a Ph column or a PFP column;

[0043] When the second chromatographic column is an L-C18 column, the first chromatographic column is preferably an S-C18 column, a Ph column or a PFP column.

[0044] In the present invention, in the L-C18 column, the filler is preferably silica gel particles, the particle size of the filler is preferably 4 to 6 μm, more preferably 4.5 to 5.5 μm, more preferably 5 to 5.2 μm, and the specification is preferably a VenusilXBP C18 (A) chromatographic column with a length of 250 mm and an inner diameter of 4.6 mm;

[0045] In the S-C18 column, the filler is preferably organic hybrid silica gel, the particle size of the filler is preferably 4 to 6 μm, more preferably 4.5 to 5.5 μm, more preferably 5 to 5.2 μm, and the specification is preferably a YMC-Triart C18 column with a length of 50 mm and an inner diameter of 4.6 mm;

[0046] In the Ph column, the filler is preferably a phenyl-bonded organic hybrid silica gel, the particle size of the filler is preferably 4 to 6 μm, more preferably 4.5 to 5.5 μm, more preferably 5 to 5.2 μm, and the specification is preferably a Kromasil100-5-Phenyl column with a length of 50 mm and an inner diameter of 4.6 mm;

[0047] In the PFP column, the filler is preferably an organic hybrid silica gel bonded with pentafluorophenyl, the particle size of the filler is preferably 4-6 μm, more preferably 4.5-5.5 μm, more preferably 5-5.2 μm, and the specification is preferably a YMC-Triart PFP column with a length of 50 mm and an inner diameter of 4.6 mm.

[0048] In the present invention, in the step 2), the first chromatographic column and the second chromatographic column are preferably connected by a joint dedicated to liquid chromatography columns.

[0049] In the present invention, in step 3), the mobile phase in the tandem mode chromatographic column is preferably: firstly maintaining the acetonitrile aqueous solution for a period of time, and then increasing the acetonitrile content until the acetonitrile content in the mobile phase reaches 100% until a peak appears.

[0050] In the present invention, in the acetonitrile aqueous solution, the volume ratio of acetonitrile to water is preferably 60-70:30-40, more preferably 62-68:32-38, and more preferably 65-66:34-35;

[0051] The period of time is preferably 25 to 30 minutes, more preferably 26 to 29 minutes, and more preferably 27 to 28 minutes;

[0052] The rate of increase of the acetonitrile content is preferably 2 to 3% acetonitrile / min, more preferably 2.2 to 2.8% acetonitrile / min, and more preferably 2.5 to 2.6% acetonitrile / min.

[0053] In the present invention, the flow rate of the mobile phase is preferably 1 to 1.5 mL / min, more preferably 1.1 to 1.4 mL / min, and even more preferably 1.2 to 1.3 mL / min.

[0054] In the present invention, in step 3), the injection volume of the PAH mixed standard solution is preferably 15 to 25 μL, more preferably 17 to 23 μL, and more preferably 19 to 20 μL;

[0055] The column temperature of the tandem mode column is preferably 25 to 35°C, more preferably 27 to 32°C, and even more preferably 28 to 30°C.

[0056] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0057] In the embodiments of the present invention and the comparative examples:

[0058] The filler of the L-C18 column is 5 μm silica gel particles, and the specification is a VenusilXBP C18 (A) chromatographic column with a length of 250 mm and an inner diameter of 4.6 mm;

[0059] The filler of the S-C18 column is 5 μm organic hybrid silica gel, and the specification is a YMC-Triart C18 column with a length of 50 mm and an inner diameter of 4.6 mm;

[0060] The filler of the Ph column is a 5 μm phenyl-bonded organic hybrid silica gel, with a specification of a Kromasil 100-5-Phenyl column of 50 mm in length and 4.6 mm in inner diameter;

[0061] The filler of the PFP column is 5 μm pentafluorophenyl-bonded organic hybrid silica gel, and the specification is a YMC-Triart PFP column with a length of 50 mm and an inner diameter of 4.6 mm.

[0062] Example 1

[0063] Prepare the PAHs mixed standard solution: 10 mg / L naphthalene solution, 10 mg / L acenaphthene solution, 10 mg / L fluorene solution, 10 mg / L acenaphthene solution, 10 mg / L phenanthrene solution, 10 mg / L anthracene solution, 10 mg / L fluoranthene solution, 10 mg / L pyrene solution, 10 mg / L chrysene solution, 10 mg / L benz[a]anthracene solution, 10 mg / L A 10 mg / L benzo[b]fluoranthene solution, a 10 mg / L benzo[k]fluoranthene solution, a 10 mg / L benzo[a]pyrene solution, a 10 mg / L dibenzo[a,h]anthracene solution, a 10 mg / L benzo[g,h,i]perylene solution and a 10 mg / L indeno[1,2,3-cd]pyrene solution were mixed in equal amounts to obtain a polycyclic aromatic hydrocarbon mixed standard solution, wherein the solvents in the above solutions are all chromatographic acetonitrile solutions;

[0064] The S-C18 column (in front) and the L-C18 column (in the back) are connected through a special connector for liquid chromatography columns to obtain a tandem mode chromatography column;

[0065] Take 1mL of the PAH mixed standard solution and enter it into a liquid chromatograph using a tandem mode chromatographic column for separation. The liquid chromatographic conditions are as follows: mobile phase: first maintain acetonitrile water solution with a volume ratio of 65:35 for 27 minutes, then increase the acetonitrile content in the mobile phase at a rate of 2.5% acetonitrile / min until the acetonitrile content reaches 100%, and maintain until the peak is completely eluted. The flow rate of the mobile phase is 1.2mL / min; the liquid input volume is 20μL; the column temperature is 30°C. This embodiment is recorded as "S-C18+L-C18".

[0066] Example 2

[0067] The tandem mode chromatographic columns in Example 1 are replaced by "connecting the Ph column (in front) and the L-C18 column (in the back) via a special connector for liquid chromatography columns", and the other steps are the same as Example 1. This example is recorded as "Ph+L-C18".

[0068] Example 3

[0069] The tandem mode chromatographic columns in Example 1 are replaced by "connecting the PFP column (in front) and the L-C18 column (in the back) via a special connector for liquid chromatography columns", and the other steps are the same as Example 1. This example is recorded as "PFP+L-C18".

[0070] Example 4

[0071] The tandem mode chromatographic columns in Example 1 are replaced by "connecting the L-C18 column (in front) and the S-C18 column (in the back) through a special connector for liquid chromatography columns", and the other steps are the same as Example 1. This example is recorded as "L-C18+S-C18".

[0072] Example 5

[0073] The tandem mode chromatographic columns in Example 1 are replaced by "connecting the L-C18 column (in front) and the Ph column (in the back) via a special connector for liquid chromatography columns", and the other steps are the same as Example 1. This example is recorded as "L-C18+Ph".

[0074] Example 6

[0075] The tandem mode chromatographic columns in Example 1 are replaced by "connecting the L-C18 column (in front) and the PFP column (in the back) via a special connector for liquid chromatography columns", and the other steps are the same as Example 1. This example is recorded as "L-C18+PFP".

[0076] Comparative Example 1

[0077] The tandem mode chromatographic column in Example 1 was replaced with a single S-C18 column, and the other steps were the same as in Example 1. This comparative example is recorded as "S-C18".

[0078] Comparative Example 2

[0079] The tandem mode chromatographic columns in Example 1 were replaced with a single L-C18 column, and the other steps were the same as in Example 1. This comparative example is recorded as "L-C18".

[0080] Comparative Example 3

[0081] The tandem mode chromatographic columns in Example 1 were replaced with a single Ph column, and the other steps were the same as in Example 1. This comparative example is denoted as "Ph".

[0082] Comparative Example 4

[0083] The tandem mode chromatographic columns in Example 1 were replaced with a single PFP column, and the other steps were the same as in Example 1. This comparative example is recorded as "PFP".

[0084] The liquid chromatograms and separations of Examples 1 to 6 and Comparative Examples 1 to 4 were tested respectively, and the results are shown in Tables 1 to 2 and Figures 1 to 5 shown.

[0085] Table 1 Separation of adjacent component peaks of polycyclic aromatic hydrocarbons in Example 1 and Comparative Example 2

[0086]

[0087]

[0088] The chromatograms of separating polycyclic aromatic hydrocarbons in Example 1, Comparative Example 1 and Comparative Example 2 are as follows: Figure 1 As shown. Combined Figure 1 As can be seen from Table 1, compared with the single L-C18 column in Comparative Example 2 and the series connection technology of S-C18 and L-C18 columns in Example 1, the separation effect of Example 1 on polycyclic aromatic hydrocarbons is much better than that of Comparative Example 1; the separation effect of polycyclic aromatic hydrocarbons using the column series connection technology in Example 1 is significantly improved compared with the separation effect of using a single L-C18 column to separate polycyclic aromatic hydrocarbons in Comparative Example 2, the peak elution time of each component substance is overall moved forward, and the overall peak elution time is shortened. For Comparative Example 2, the separation degree of the more difficult components of polycyclic aromatic hydrocarbons such as fluorene and acenaphthylene, chrysene and benzo[a]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene separated by the column series connection technology in Example 1 is also significantly improved.

[0089] Table 2 Separation degree of difficult-to-separate components in polycyclic aromatic hydrocarbons in Examples 1 to 6 and Comparative Example 2

[0090]

[0091]

[0092] The chromatograms of the separation of polycyclic aromatic hydrocarbons in Comparative Examples 1 to 4 are as follows: Figure 2 As shown, the chromatograms of Examples 1 to 3 for separating polycyclic aromatic hydrocarbons are as follows Figure 3 As shown, the chromatograms of Examples 4 to 6 for separating polycyclic aromatic hydrocarbons are as follows Figure 4 As shown, the chromatograms of separating polycyclic aromatic hydrocarbons in Example 1, Example 4 and Comparative Example 2 are as shown in Figure 5 As shown. Combined Figures 2 to 5 As well as Table 2, the following conclusions can be drawn:

[0093] Depend on Figure 2 It can be seen that compared with a single L-C18 chromatographic column, a single S-C18 chromatographic column, a single Ph column and a single PFP chromatographic column cannot separate 16 kinds of PAHs; from the content shown in Table 2 about Comparative Example 2, it can be seen that a single L-C18 column is used to separate the components that are difficult to separate in PAHs, and the separation degree of fluorene and acenaphthylene is 0.881, the separation degree of chrysene and benzo[a]anthracene is 0.760, and the separation degree of benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene is 1.094.

[0094] Depend on Figure 3 to Figure 5 It can be seen that the six series connection modes shown in Examples 1 to 6 have significant separation effects on polycyclic aromatic hydrocarbons. Combined with Table 2, it can be seen that for the components of polycyclic aromatic hydrocarbons that are difficult to separate, such as fluorene and acenaphthylene, chrysene and benzo[a]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene, the series connection modes shown in Examples 1 to 6 still have excellent separation effects.

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column, characterized in that: The steps include: 1) Prepare a mixed standard solution of polycyclic aromatic hydrocarbons; 2) connecting the first chromatographic column and the second chromatographic column in series to obtain a tandem mode chromatographic column; 3) The PAHs mixed standard solution was separated using a tandem mode chromatographic column.

2. The method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column according to claim 1, characterized in that: In the step 1), the polycyclic aromatic hydrocarbons include naphthalene, acenaphthene, fluorene, acenaphthylene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene.

3. The method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column according to claim 2, characterized in that: In the step 1), the concentrations of naphthalene, acenaphthene, fluorene, acenaphthylene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene and indeno[1,2,3-cd]pyrene in the mixed standard solution of polycyclic aromatic hydrocarbons are independently 8 to 12 mg / L; The solvent of the PAHs mixed standard solution is acetonitrile.

4. The method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column according to claim 1, characterized in that: In the step 2), the first chromatographic column and the second chromatographic column independently include L-C18 column, S-C18 column, Ph column or PFP column.

5. The method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column according to claim 4, characterized in that: When the first chromatographic column is an L-C18 column, the second chromatographic column is an S-C18 column, a Ph column or a PFP column; When the second chromatographic column is an L-C18 column, the first chromatographic column is an S-C18 column, a Ph column or a PFP column.

6. The method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column according to claim 5, characterized in that: In the step 3), the mobile phase in the tandem mode chromatographic column is: firstly maintained with an acetonitrile aqueous solution for a period of time, and then the acetonitrile content is increased until the acetonitrile content in the mobile phase reaches 100% until a peak appears.

7. The method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column according to claim 6, characterized in that: In the acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 60-70:30-40; The period of time is 25 to 30 minutes; The rate of increase of acetonitrile content was 2-3% acetonitrile / min.

8. The method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column according to claim 7, characterized in that: The flow rate of the mobile phase is 1-1.5 mL / min.

9. The method for separating multiple polycyclic aromatic hydrocarbons using a tandem mode chromatographic column according to claim 8, characterized in that: In the step 3), the injection volume of the PAH mixed standard solution is 15-25 μL; The column temperature of the tandem mode column is 25-35°C.

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