Extraction method of dibutyl ester_Kain Industrial Additive

Background and overview^[1][2]

Dibutyl ester, also known as Dibutylphthalate (DBP), is an excellent plasticizer. Due to its relatively low price and good processability, it is widely used at home and abroad. Dibutyl ester has good solubility for a variety of resins and has the characteristics of light color, good electrical properties, low volatility, low odor and low temperature resistance. Dibutyl ester is an ideal plasticizer for nitrocellulose, cellulose acetate, polyvinyl chloride, etc. It has good softening effect and excellent stability, adhesion, flex resistance and waterproofness. In addition, dibutyl ester is also used in the manufacture of chemical products such as paints, adhesives, artificial fibers, printing inks, safety glass, dyes, pesticides, perfume solvents and fabric lubricants.

Dibutyl ester can cause functional changes in the central nervous system and peripheral nervous system, and then further cause changes in their tissues; it has hepatotropism, can cause mild sensitization, has a moderate accumulation effect and mild It has a stimulating effect and is a suspected endocrine disruptor. At present, many countries such as the United States and the European Union have explicitly banned or restricted the use of dibutyl ester. my country also encourages the use of other chemicals to replace the toxic and harmful dibutyl ester in daily chemical products.

Detection^[1]

CN201310185247.0 provides a dibutyl ester detection kit and a preparation and use method of a dibutyl ester detection kit. When detecting dibutyl ester, the kit can continuously detect multiple samples at one time and is convenient. Fast, sensitive and low cost. The detection kit mainly includes:

1) ELISA plate coated with dibutyl ester-specific antigen: The concentration of dibutyl ester-specific antigen in each microwell of the ELISA plate is 1-5 μg/mL;

2) Dibutyl ester monoclonal antibody working solution: The concentration of the dibutyl ester monoclonal antibody working solution is 1‑5μg/mL;

The preparation method of the above dibutyl ester detection kit mainly includes the following steps:

1) Preparation of dibutyl ester-specific antigen

Mix phthalic acid and 2-ethylhexanol in cyclohexane, add concentrated sulfuric acid as a catalyst, and perform a reflux reaction to obtain 2-(2-ethylhexyl) benzoic acid; add the above 2- (2-Ethylhexyl) benzoic acid and 2-ethyl-6 tert-butoxyamide propanol are mixed in cyclohexane, concentrated sulfuric acid is added, and the reaction is refluxed to obtain dibutyl ester which is aminated with tert-butoxyamide. Product; then add the above intermediate product to trifluoroacetic acid, perform a reflux reaction, remove the protection of the tert-butoxyacyl group, expose the amine group, and prepare the hapten of aminodibutyl ester that can be coupled to the protein; in the N-hydroxyl group Under the catalysis of succinamide and carbodiimide, and then coupled with a carrier protein, dibutyl ester-specific antigen is prepared; the carrier protein is bovine serum albumin, ovalbumin, rabbit serum albumin or thyroid globulin protein;

2) Coated enzyme plate

The above-mentioned dibutyl ester-specific antigen is coated in a microplate, and the concentration of the dibutyl-specific antigen in each microwell of the microplate is 1-5 μg/mL;

3) Animal immunity

Use the dibutyl ester-specific antigen synthesized in step 1) as the immunogen to immunize mice, take the serum of the immunized mice, detect the anti-dibutyl ester titer and inhibitory potency of the serum, and select the potency and inhibitory potency. The highest immune mice;

4) Cell fusion and screening

Take the spleen cells of the immunized mice obtained in step 3) and perform cell fusion with myeloma cells SP2/0; use the limiting dilution method to screen hybridoma cells to obtain completely homogeneous monoclonal antibodies and stable monoclonal hybridoma cells. strain, and mice that had been sensitized by intraperitoneal injection of incomplete adjuvant were taken, hybridoma cell suspension was injected into the abdominal cavity of the mice, ascites was collected, and the ascites was purified by the octanoic acid-ammonium sulfate precipitation method to obtain the purified Butyl ester monoclonal antibody; prepare a dibutyl ester monoclonal antibody working solution with a concentration of 1-5 μg/mL.

The method of using the above dibutyl ester detection kit mainly includes the following steps:

1) Sample pretreatment

Accurately weigh the paint sample. After the organic solvent is dissolved, dilute it with water to a sample weight:solution volume ratio of 1:106. For example: a 1g sample is finally diluted to 1000L, a 0.1g sample is finally diluted to 100L, constant volume, and ultrasonic extraction 10‑30min, get the sample detection solution;

2) Detection

Use the above kit for detection, add standard or sample detection solution to the enzyme plate coated with dibutyl ester specific antigen, then add dibutyl ester monoclonal antibody working solution, incubate and wash the plate , add enzyme-labeled secondary antibody working solution to amplify the enzyme activity, wash the plate again, add chromogenic solution and stop solution, and measure the OD value with a microplate reader;

3) Result analysis

With the inhibition rate 1% as the ordinate and the logarithm of dibutyl ester concentration lg [dibutyl ester (μg/L)] as the abscissa, draw a dibutyl ester competitive inhibition curve; substitute the inhibition rate of the sample into the standard Curve regression equation, read the concentration corresponding to the sample from the standard curve, multiply it by the corresponding dilution factor to get the actual content of dibutyl ester in the sample.

Wastewater treatment^[2]

CN201410225022.8 discloses a method for treating organic wastewater containing high concentration of dibutyl ester. The method for treating organic wastewater containing high concentration of dibutyl ester according to the present invention first adjusts the pH to alkalinity and stirs it thoroughly to separate the oil phase into an upper layer rich in dibutyl ester and a lower layer containing dibutyl ester and other organic matter. Aqueous solution, separate the upper oil phase and return it�Dibutyl ester, the lower aqueous phase solution is oxidized using hydrogen peroxide-wet oxidation method. The COD removal rate of the treated lower aqueous phase solution is 65~80%, and the mass content of dibutyl ester is reduced to 0.01~0.55 %, can achieve harmless treatment. The method for treating organic wastewater containing high concentration dibutyl ester of the present invention fully realizes resource recovery and reduces the energy consumption required for treating wastewater.

Extraction method^[3]

CN201710367720.5 provides a method for extracting dibutyl ester from marine coccolithophore, aiming to use thin layer chromatography, silica gel column chromatography, gel column chromatography, preparative thin layer PTLC, etc. Methods The dibutyl ester of the ethyl acetate phase extract of marine coccolithophore was separated and purified to facilitate the subsequent large-scale production of dibutyl ester.

The method of extracting dibutyl ester specifically includes the following steps:

Step 1. Prepare the ethyl acetate phase extract of marine coccolithophore: first extract with ethanol solution to obtain ethanol extract, then perform extraction treatment with ethyl acetate as the extractant, combine the extracts, and concentrate under reduced pressure to obtain Marine coccolithophore ethyl acetate phase extract;

Grow the marine coccolithophore to a stable phase, harvest, and freeze-dry to obtain 19.3740g of marine coccolithophore algae powder. Extract with 75% ethanol to obtain 5.1247g of ethanol extract. Add an equal volume of ethyl acetate and water, repeatedly extracted, collected the ethyl acetate phase, combined, and concentrated to dryness under reduced pressure to obtain 4.2357g of the ethyl acetate phase extract of marine coccolithophore;

Step 2: Preparation of primary crude dibutyl ester by reversed-phase silica gel column chromatography: chromatograph the obtained ethyl acetate phase extract of marine coccolithophore on a reversed-phase silica gel column, collect, detect, combine, and reduce pressure Concentrate to dryness to obtain crude dibutyl ester;

After dissolving 4.2357g of marine coccolithophore ethyl acetate phase extract with an appropriate amount of analytically pure methanol, perform reverse-phase silica gel (180g) column chromatography; sequentially use water, a mass fraction of 25% methanol solution, and a mass fraction of It is a 50% methanol solution, with a mass fraction of 75% methanol solution, methanol elution, each gradient elution is 1.5L, the flow rate is controlled to 30mL/min, collect 1 tube for every 200mL, spin evaporate, and preliminarily analyze the effluent with TLC , combine the palmitic acid high-concentration flow segments, and concentrate to dryness under reduced pressure to obtain a crude dibutyl ester product;

Step 3: Preparation of the secondary crude dibutyl ester product by chromatography on SephadexLH-20 Sephadex gel column: Chromatograph the primary crude dibutyl ester product on SephadexLH-20 Sephadex gel column, collect, detect, Combine and concentrate to dryness under reduced pressure to obtain the secondary crude product of dibutyl ester;

After dissolving the crude ethyl acetate phase extract of Marine Coccolithophore with an appropriate amount of methanol, put it on SephadexLH-20, a Sephadex LH-20 column, and use methanol as the eluent for elution. The flow rate is controlled at 12-13S/drop. , use an automatic collector to collect each fraction, and collect one tube every 30 minutes. Preliminarily analyze the effluent with TLC, combine the fractions from 80 to 90 tubes, and concentrate to dryness under reduced pressure to obtain the secondary crude product of dibutyl ester;

Step 4: Preparation of the primary product of dibutyl ester by reversed-phase silica gel column chromatography: The obtained secondary crude product of dibutyl ester is subjected to reversed-phase silica gel column chromatography, collected, detected, combined, and concentrated under reduced pressure to dryness. Obtain a fine product of dibutyl ester;

The secondary crude dibutyl ester was eluted with a methanol water gradient with a mass fraction of 30%, 40%, 50%, 60%, and 70%, and finally was eluted with methanol for reversed-phase silica gel column chromatography. Each gradient elutes 300 mL, and the flow rate is controlled to 5 mL/min. Rotary evaporate, and the effluent is initially analyzed by TLC. The high-concentration dibutyl ester flow segments are combined and concentrated to dryness under reduced pressure to obtain the primary fine product of dibutyl ester;

Step 5. Preparation of the secondary fine product of dibutyl ester by normal phase silica gel column chromatography: perform chromatography on the obtained primary fine product of dibutyl ester using a normal phase silica gel column, collect, detect, combine, and concentrate to dryness under reduced pressure. Obtain the secondary fine product of dibutyl ester;

Put the primary product of dibutyl ester through normal phase silica gel column chromatography, mix the sample with silica gel powder, saturate the silica gel with petroleum ether and load it into the column. After loading the sample on the column, use petroleum ether and acetone system to elute. Petroleum ether and acetone The volume ratio is 200:1. Collect the fractions and concentrate to dryness under reduced pressure to obtain the secondary fine product of dibutyl ester;

Step 6. Preparation of pure dibutyl ester by PTLC: Perform PTLC on the obtained secondary fine product of dibutyl ester, spotting, unfolding, scraping, sample recovery, and storing at -20°C to obtain pure dibutyl ester;

Put the secondary fine product of dibutyl ester through PTLC to finally obtain dibutyl ester: use a high-efficiency thin layer chromatography plate to develop in a developing agent with a volume ratio of: petroleum ether: ethyl acetate = 80:1. After color development, scrape the middle part with a razor blade, combine, dissolve with methanol, centrifuge at high speed, collect the supernatant, concentrate to dryness under reduced pressure, and finally obtain dibutyl ester.

Main reference materials

[1][China invention, China invention authorization] CN201310185247.0 dibutyl phthalate detection kit and its preparation and use methods

[2]CN201410225022.8 A method for treating organic wastewater containing high concentration of dibutyl phthalate

[3][Chinese invention] CN201710367720.5 A method for extracting dibutyl phthalate from marine coccolithophore