Non-targeted screening analysis method for chlorinated and brominated organic pollutants in sludge

Abstract

The invention discloses a non-targeted screening analysis method for chlorinated and brominated organic pollutants in sludge. The method comprises the following steps: extracting target compounds with different polarities in a sludge sample by respectively adopting different extraction agents for the sludge sample, purifying and concentrating the sample by respectively adopting different solid-phase extraction columns, performing instrumental analysis based on liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS) and gas chromatography-tandem high-resolution mass spectrometry (GC-HRMS), and determining the content of the target compounds in the sludge sample. The information of chlorinated and brominated organic matters in the sample can be completely obtained as much as possible. The obtained instrument data is subjected to data format conversion, XCMS Online website mass spectrum data extraction and RStudio language processing by using MS Convert software, so that chloro and bromo organic matters in the sludge sample can be quickly and accurately screened. The technical problem that a halogenated organic pollutant analysis method in the prior art does not have the capability of performing non-targeted screening on multiple halogenated organic pollutants at the same time is solved.

Description

Technical Field

[0001] The present application relates to the technical field of environmental pollutant analysis, and in particular to a non-targeted screening and analysis method for chlorinated and brominated organic pollutants in sludge. Background Art

[0002] This section is intended to provide a background or context to what is stated in the claims or specification, and no admission is made that what is described herein is prior art by inclusion in this section.

[0003] Halogenated Organic Pollutants (HOPs) are a large class of organic compounds containing halogen atoms, which are widely distributed in the natural environment. Most HOPs have the characteristics of persistent organic pollutants (POPs). A study found that more than 60% of 610 POPs were halogenated organic pollutants after component analysis. Among them, halogenated aliphatic hydrocarbons, polychlorinated biphenyls, chlorinated phenols, organochlorine pesticides and other halogenated organic substances are listed as "priority controlled pollutants" by some countries. With the increasing number of halogenated organic substances detected in the environment, their environmental chemical behavior, control methods and ecotoxicological effects have attracted much attention, and the establishment of accurate and efficient analysis methods for halogenated organic substances is the basis for the above research. Therefore, the screening and identification of unknown halogenated organic substances in various complex environmental media are particularly important in environmental analysis. The current quantitative analysis methods for halogenated organic compounds based on liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), although highly sensitive, are only targeted at a certain type or class of halogenated organic compounds and do not have non-targeted screening capabilities. Therefore, there is an urgent need to establish a rapid and simple systematic screening method for multiple halogenated organic compounds.

[0004] To address the above-mentioned problems, no effective solution has been proposed yet. Summary of the invention

[0005] The embodiments of the present application provide a non-targeted screening and analysis method for chlorinated and brominated organic pollutants in sludge to solve the technical problem that the analysis method for halogenated organic pollutants in the related art does not have the ability to perform non-targeted screening of multiple halogenated organic pollutants at the same time.

[0006] According to one aspect of an embodiment of the present application, a non-targeted screening and analysis method for chlorinated and brominated organic pollutants in sludge is provided, comprising: cold-drying the collected sludge sample in a freeze dryer, grinding and passing through a 200-mesh sieve, taking 2 g of sample, of which 1 g is used as sample 1 and 1 g is used as sample 2, and they are placed in different 15 mL centrifuge tubes respectively; methanol is added as an extractant in the centrifuge tube of sample 1, and a mixed solution of n-hexane and dichloromethane (v:v=1:1) is added as an extractant in the centrifuge tube of sample 2; the extraction method is ultrasonic extraction, and the extracts obtained after the extraction of sample 1 and sample 2 are centrifuged in a centrifuge, and the supernatant in the centrifuge tube is collected; the supernatant of sample 1 and the supernatant of sample 2 obtained after repeated ultrasonic extraction and centrifugation are collected, and the supernatant of sample 1 and the supernatant of sample 2 are blown to near dryness with nitrogen using a nitrogen blower, and 5 mL Sample 1 was re-dissolved with a 5% methanol-water solution, and sample 2 was re-dissolved with a 1 mL mixed solution of n-hexane and dichloromethane (v:v=1:1); the sample 1 solution obtained after the re-dissolution was loaded onto an activated solid phase extraction column, and then rinsed with a 2% acetic acid-5% methanol-water solution (v:v:v=2:5:93), and then eluted with methanol to collect the eluate of sample 1; the sample 2 solution obtained after the re-dissolution was loaded onto an activated solid phase purification column, and then eluted with a mixed solution of n-hexane and dichloromethane (v:v=1:1), and the eluate of sample 2 was collected; the elution liquid nitrogen of sample 1 and sample 2 was purged using a nitrogen blower After blowing until nearly dry, sample 1 was re-dissolved in methanol and transferred to a 2mL injection vial, and the volume was adjusted to 1mL with methanol; sample 2 was re-dissolved in n-hexane and transferred to a 2mL injection vial, and the volume was adjusted to 1mL with n-hexane; sample 1 was tested on the machine using liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS) to obtain the instrumental analysis data of sample 1, and sample 2 was tested on the machine using gas chromatography tandem high-resolution mass spectrometry (GC-HRMS) to obtain the instrumental analysis data of sample 2; the instrumental analysis data of sample 1 and sample 2 were converted to mzXML data format using MSConvert software, and uploaded to the XCMS Online website for mass spectrometry data extraction and processing; the results obtained after extraction and processing on the XCMS Online website were screened by running the RStudio language program to obtain the mass-to-charge ratios of the candidate chlorinated and brominated organic parent ions. The XCMS Online website can extract the mass-to-charge ratio (m / z), corrected retention time (RT) of a single chromatographic peak in a total ion chromatogram (Total Ion Chromatogram) in a specific format (the data format in this application is mzXML), and calculate the response intensity (intensity) of the chromatographic peak.

[0007] Optionally, sample 1 is used to detect polar substances, and sample 2 is used to detect medium-polar or non-polar substances.

[0008] Optionally, when ultrasonic extraction is performed on Sample 1 and Sample 2: the volume of the extractant of Sample 1 and Sample 2 is 5 mL; and the extraction time of the ultrasonic extraction is 15 min.

[0009] Optionally, the centrifugal speed of the centrifuge is 8500 r / min, the centrifugal time is 10 min, and the temperature is 4°C.

[0010] Optionally, when using a solid phase extraction column to purify and concentrate sample 1, the solid phase extraction column is a Cleanert PEP solid phase extraction column; the solutions used for activation are 5 mL of methanol and 5 mL of water respectively; the solution used for elution is 5 mL of 2% acetic acid-5% methanol-water solution (v:v:v=2:5:93); and the solution used for elution is 5 mL of methanol.

[0011] Optionally, when using a solid phase extraction column to purify sample 2, the solid phase extraction column is Supelclean ENVI-Carb TM Solid phase extraction column; the solutions used for activation are 5 mL of dichloromethane and 5 mL of normal hexane; the solution used for elution is a mixed solution of 5 mL of normal hexane and dichloromethane (v:v=1:1).

[0012] In the present application embodiment, by taking different extractants to the sludge sample respectively, the target compound of different polarities in the sample can be extracted, and different solid phase extraction columns are used to purify and concentrate the sample, and instrumental analysis is performed based on liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS) and gas chromatography tandem high-resolution mass spectrometry (GC-HRMS), and the information of chlorinated and brominated organic matter in the sample can be obtained as completely as possible. The obtained instrumental data is converted to data format using MS Convert software, and XCMS Online website mass spectrometry information is extracted and RStudio language is processed, so that the chlorinated and brominated organic matter in the sludge sample can be screened quickly and accurately. The present application solves the technical problem that the halogenated organic pollutant analysis method in the related art does not have the ability to perform non-targeted screening on multiple halogenated organic pollutants at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0014] Figure 1 is a flow chart of a non-targeted screening and analysis method for chlorinated and brominated organic pollutants in sludge according to an embodiment of the present application;

[0015] Figure 2is a schematic diagram of an optional analytical method for detecting polar substances in sludge according to an embodiment of the present application;

[0016] Figure 3 It is a schematic diagram of an optional analytical method for detecting medium polar or non-polar substances in sludge according to an embodiment of the present application;

[0017] Figure 4 is a schematic diagram of chlorinated organic compounds screened during the data analysis process according to an embodiment of the present application;

[0018] Figure 5 It is a schematic diagram of brominated organic compounds screened out during the data analysis process according to the embodiments of the present application. DETAILED DESCRIPTION

[0019] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0020] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0021] According to one aspect of an embodiment of the present application, an example of a non-targeted screening analysis method for chlorinated and brominated organic pollutants in sludge is provided. Figure 1 is a flow chart of a non-targeted screening and analysis method for chlorinated and brominated organic pollutants in sludge according to an embodiment of the present application, such as Figure 1 As shown, the method may include the following steps:

[0022] Step S101, the collected sludge sample is freeze-dried by a freeze dryer, ground and sieved through a 200-mesh sieve, and 2 g of sample is taken, of which 1 g is used as sample 1 and 1 g is used as sample 2, and they are placed in different 15 mL centrifuge tubes (here 15 mL represents volume or capacity).

[0023] The above sample 1 is used to detect polar substances, and the sample 2 is used to detect medium polar or non-polar substances.

[0024] Step S102, adding methanol to the centrifuge tube of sample 1 and adding a mixed solution of n-hexane and dichloromethane (v:v=1:1) to the centrifuge tube of sample 2 as an extractant (v represents volume, v:v represents the volume ratio of n-hexane and dichloromethane, similar expressions below are similar to this and will not be repeated here).

[0025] Step S103, the extraction method is ultrasonic extraction, and the extracts obtained after the extraction of sample 1 and sample 2 are respectively centrifuged and the supernatant in the centrifuge tube is collected.

[0026] When ultrasonic extraction was performed on Sample 1 and Sample 2: the volume of the extractant in the centrifuge tubes of Sample 1 and Sample 2 was 5 mL, and the extraction time of ultrasonic extraction was 15 min.

[0027] Step S104, collect the supernatant of sample 1 and sample 2 obtained by repeating ultrasonic extraction and centrifugation for 3 times, use a nitrogen blower to blow the supernatant of sample 1 and sample 2 to nearly dryness, use 5mL 5% methanol-water solution to re-dissolve sample 1, and use 1mL mixed solution of n-hexane and dichloromethane (v:v=1:1) to re-dissolve sample 2. Nearly dry here means that the liquid content is lower than a specific value (such as 1%, 2%, which can be determined according to industry regulations or specific needs).

[0028] After the extract obtained after the sample extraction is centrifuged, the supernatant in the centrifuge tube is extracted: the centrifugal speed of the centrifuge is 8500r / min, the centrifugal time is 10min, and the temperature is 4°C.

[0029] Step S105, after loading the solution of sample 1 obtained after re-dissolution onto the activated solid phase extraction column, rinse with a 2% acetic acid-5% methanol-water solution (v:v:v=2:5:93), then elute with methanol, and collect the eluate of sample 1; after loading the solution of sample 2 obtained after re-dissolution onto the activated solid phase extraction column, elute with a mixed solution of n-hexane and dichloromethane (v:v=1:1), and collect the eluate of sample 2.

[0030] When using a solid phase extraction column to purify and concentrate sample 1: the solid phase extraction column is a Cleanert PEP solid phase extraction column; the solutions used for activation are 5 mL of methanol and 5 mL of water; the solution used for elution is 5 mL of a 2% acetic acid-5% methanol-water solution (v:v:v=2:5:93); the solution used for elution is 5 mL of methanol.

[0031] When using a solid phase extraction column to purify and concentrate sample 2: the solid phase extraction column is Supelclean ENVI-Carb TM Solid phase extraction column; the solutions used for activation are 5 mL of dichloromethane and 5 mL of normal hexane; the solution used for elution is a mixed solution of 5 mL of normal hexane and dichloromethane (v:v=1:1).

[0032] Step S106, use a nitrogen blower to blow the eluent of sample 1 and the eluent of sample 2 with nitrogen until they are almost dry, re-dissolve sample 1 with methanol and transfer it to a 2mL injection vial, and make the volume up to 1mL with methanol, re-dissolve sample 2 with n-hexane and transfer it to a 2mL injection vial, and make the volume up to 1mL with n-hexane.

[0033] Step S107, use LC-HRMS (i.e., liquid chromatography tandem high-resolution mass spectrometry) to detect the solution of sample 1 obtained after pretreatment to obtain instrumental analysis data of sample 1, and use GC-HRMS (i.e., gas chromatography tandem high-resolution mass spectrometry) to detect the solution of sample 2 obtained after pretreatment to obtain instrumental analysis data of sample 2.

[0034] Step S108, after converting the instrumental analysis data of sample 1 and the instrumental analysis data of sample 2 into the mzXML data format using the MSConvert software, upload them to the XCMS Online website for mass spectrometry data extraction and processing. The XCMSOnline website can extract the mass-to-charge ratio (m / z), corrected retention time (RT) of a single chromatographic peak in the total ion chromatogram (Total Ion Chromatogram) of data in a specific format, and calculate the response intensity (intensity) of the chromatographic peak.

[0035] Step S109, the results obtained after the extraction and processing on the XCMS Online website are screened by running an RStudio language program to obtain the mass-to-charge ratios of candidate chlorinated and brominated organic precursor ions.

[0036] In the present application embodiment, by taking different extractants for the sludge sample, the target compounds of different polarities in the sample can be extracted, and different solid phase extraction columns are used to purify and concentrate the sample, and instrumental analysis is performed based on liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS) and gas chromatography tandem high-resolution mass spectrometry (GC-HRMS), and the information of chlorinated and brominated organic matter in the sample can be obtained as completely as possible. The obtained instrumental data is converted into data format using MS Convert software, and the mass spectrum information of the XCMS Online website is extracted and processed by RStudio language, so that the chlorinated and brominated organic matter in the sludge sample can be screened quickly and accurately. This application solves the technical problem that the halogenated organic pollutant analysis method in the related art does not have the ability to perform non-targeted screening on multiple halogenated organic pollutants at the same time.

[0037] As an optional embodiment, the present application provides a pretreatment method for analyzing chlorinated and brominated organic matter in sludge samples, which is mainly based on the characteristics of the polarity difference of chlorinated and brominated organic matter in the sample, by taking different extractants for the sludge samples to extract target compounds of different polarities in the sample, and using different solid phase extraction columns to purify and concentrate the sample, and performing instrumental analysis based on liquid chromatography tandem high resolution mass spectrometry (LC-HRMS) and gas chromatography tandem high resolution mass spectrometry (GC-HRMS). The specific operation steps are:

[0038] (1) Figure 2 As shown, for sample 1 (detection of polar chlorinated and brominated organic matter in sludge): accurately weigh 1g of the sludge sample after cold drying, grinding and sieving, add 5mL of methanol to a 15mL centrifuge tube, and extract by ultrasonic. After the obtained extract is centrifuged at 8500r / min and 4℃ for 10min, extract the supernatant, repeat the extraction 3 times, and combine the supernatants; use a nitrogen blower to blow the sample supernatant to nearly dryness and then redissolve it in 5mL5% methanol-water solution. Use Cleanert PEP solid phase extraction column to purify and concentrate the sludge sample. Use 5mL of methanol and water to activate the solid phase extraction column respectively, and after loading the sample, use 5mL of 2% acetic acid-5% methanol-water (v:v:v=2:5:93) solution to elute, and finally use 5mL of methanol for elution. Collect the eluate, blow it to nearly dryness with nitrogen, redissolve it with methanol, transfer it to a 2mL injection vial, and make up to 1mL with methanol, and analyze it by liquid chromatography-tandem high-resolution mass spectrometry.

[0039] (2) Figure 3As shown, for sample 2 (detection of medium polar or non-polar chlorinated and brominated organic matter in sludge): accurately weigh 1 g of the sludge sample after freeze-drying, grinding and sieving, add 5 mL of a mixed solution of n-hexane and dichloromethane (v:v=1:1) into a 15 mL centrifuge tube, perform ultrasonic extraction, centrifuge the obtained extract at 8500 r / min, 4°C for 10 min, extract the supernatant, repeat the extraction 3 times, and combine the supernatants; use a nitrogen blower to blow the sample supernatant to nearly dryness with nitrogen, then redissolve it in 1 mL of a mixed solution of n-hexane and dichloromethane (v:v=1:1); use Supelclean ENVI-Carb TM The solid phase extraction column was used to purify and concentrate the sample. 5 mL of n-hexane and dichloromethane were used to activate the solid phase extraction column, and 5 mL of a mixed solution of n-hexane and dichloromethane (v:v=1:1) was used for elution after loading. The eluate was collected, blown to near dryness with nitrogen, and then re-dissolved with n-hexane and transferred to a 2 mL injection vial, and the volume was adjusted to 1 mL with n-hexane, and the instrumental analysis was performed by gas chromatography-tandem high-resolution mass spectrometry.

[0040] This application provides a non-targeted screening and analysis method for chlorinated and brominated organic matter in sludge samples, which mainly uses MS Convert software to convert the obtained instrument data into data format, extracts mass spectrometry information from the XCMS Online website, and processes it in RStudio language, and finally uses Xcalibur software to calculate and match the elemental composition of chlorinated and brominated organic matter to obtain the molecular formula. The specific operation steps are:

[0041] (1) The original instrument data obtained was converted into mzXML format using MS Convert software.

[0042] (2) Upload the data converted into mzXML format to the XCMS Online website for data preprocessing.

[0043] (3) Download the results of process (2) and change the file format to ".csv" format.

[0044] (4) Rename the "into" column header in the data file table obtained in (3) to "int", delete the other columns in the file table, and add a sequence column before the first column of the modified data table.

[0045] (5) The data table obtained in (4) is run on the RStudio language program to obtain the charge ratios (m / z) of alternative chlorinated and brominated organic substances.

[0046] (6) The elemental composition of chlorinated and brominated compounds was matched by m / z calculation using Xcalibur software to obtain the molecular formulas of chlorinated and brominated organic compounds.

[0047] The relevant process is implemented through computer software, and the specific implementation method is as follows (the character "#" represents the interpretation of a section or a line of code):

[0048]

[0049]

[0050] like Figure 4 and Figure 5 As shown, this application provides an illustration of the chlorinated and brominated organic compounds screened during the data analysis process.

[0051] Compared with the existing technology, the advantages and positive effects of this method are: by using different extractants for sludge samples, target compounds of different polarities in the samples can be extracted, and different solid phase extraction columns are used to purify and concentrate the samples, and instrumental analysis based on liquid chromatography tandem high-resolution mass spectrometry and gas chromatography tandem high-resolution mass spectrometry can obtain the information of chlorinated and brominated organic matter in the samples as completely as possible. By converting the obtained instrument data into data format using MS Convert software, extracting mass spectrometry information on the XCMS Online website, and processing with RStudio language, chlorinated and brominated organic matter in sludge samples can be screened quickly and accurately.

[0052] It should be noted that, for the aforementioned method embodiments, for the sake of simplicity, they are all expressed as a series of action combinations, but those skilled in the art should be aware that the present application is not limited by the described order of actions, because according to the present application, certain steps can be performed in other orders or simultaneously. Secondly, those skilled in the art should also be aware that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the present application.

Claims

1. A non-targeted screening and analysis method for chlorinated and brominated organic pollutants in sludge, characterized in that: include: The collected sludge samples were freeze-dried in a freeze dryer, ground and passed through a 200-mesh sieve, and 2 g of samples were taken, of which 1 g was used as sample 1 and 1 g was used as sample 2, and placed in different 15 mL centrifuge tubes respectively; Methanol was added to the centrifuge tube of sample 1, and a mixed solution of n-hexane and dichloromethane (v:v=1:1) was added to the centrifuge tube of sample 2 as an extractant; The extraction method is ultrasonic extraction. The extracts obtained after the extraction of sample 1 and sample 2 are centrifuged in a centrifuge and the supernatant in the centrifuge tube is collected; The supernatant of sample 1 and the supernatant of sample 2 obtained by repeated ultrasonic extraction and centrifugation for 3 times were collected, and the supernatant of sample 1 and the supernatant of sample 2 were nitrogen blown to nearly dryness using a nitrogen blower, and sample 1 was re-dissolved with 5 mL of 5% methanol-water solution, and sample 2 was re-dissolved with 1 mL of a mixed solution of n-hexane and dichloromethane (v:v=1:1); The solution of sample 1 obtained after redissolution was loaded onto the activated solid phase extraction column, and then rinsed with a 2% acetic acid-5% methanol-water solution (v:v:v=2:5:93), and then eluted with methanol to collect the eluate of sample 1; the solution of sample 2 obtained after redissolution was loaded onto the activated solid phase extraction column, and then eluted with a mixed solution of n-hexane and dichloromethane (v:v=1:1), and the eluate of sample 2 was collected; Use a nitrogen blower to blow the eluent of sample 1 and sample 2 to nearly dryness with nitrogen, re-dissolve sample 1 with methanol and transfer it to a 2 mL injection vial, and dilute it to 1 mL with methanol; re-dissolve sample 2 with n-hexane and transfer it to a 2 mL injection vial, and dilute it to 1 mL with n-hexane; The solution of sample 1 obtained after pretreatment was tested by liquid chromatography tandem high-resolution mass spectrometry (LC-HRMS) to obtain instrumental analysis data of sample 1, and the solution of sample 2 obtained after pretreatment was tested by gas chromatography tandem high-resolution mass spectrometry (GC-HRMS) to obtain instrumental analysis data of sample 2; The instrumental analysis data of sample 1 and sample 2 were converted into mzXML data format using MSConvert software, and then uploaded to the XCMS Online website for mass spectrometry data extraction. The XCMS Online website can extract the mass-to-charge ratio (m / z), corrected retention time (RT) of a single chromatographic peak in the total ion chromatogram (Total Ion Chromatogram) of data in a specific format, and calculate the response intensity (intensity) of the chromatographic peak; The results obtained after extraction and processing from the XCMS Online website were screened by running the RStudio language program to obtain the mass-to-charge ratios of candidate chlorinated and brominated organic precursor ions.

2. The method according to claim 1, characterized in that Sample 1 is used to detect polar substances, and sample 2 is used to detect medium-polar or non-polar substances.

3. The method according to claim 1, characterized in that When ultrasonic extraction was performed on samples 1 and 2: The volume of the extractant in the centrifuge tubes of Sample 1 and Sample 2 was 5 mL.

4. The method according to claim 1, characterized in that: When ultrasonic extraction was performed on samples 1 and 2: The extraction time of ultrasonic extraction was 15 min.

5. The method according to claim 1, characterized in that When the extract obtained after sample extraction is centrifuged and the supernatant in the centrifuge tube is extracted: The speed of the centrifuge is 8500 r / min, the centrifugation time is 10 min, and the temperature is 4°C.

6. The method according to claim 1, characterized in that When using a solid phase extraction column to purify and concentrate sample 1: The solid phase extraction column is a Cleanert PEP solid phase extraction column; The solutions used for activation were 5 mL of methanol and 5 mL of water; The solution used for elution was 5 mL of 2% acetic acid-5% methanol-water solution (v:v:v=2:5:93); The elution solution was 5 mL of methanol.

7. The method according to claim 1, characterized in that When using a solid phase extraction column to purify and concentrate sample 2: The solid phase extraction column is Supelclean ENVI-Carb TM Solid phase extraction columns; The solutions used for activation were 5 mL of dichloromethane and 5 mL of n-hexane; The solution used for elution was 5 mL of a mixed solution of n-hexane and dichloromethane (v:v=1:1).

Images