CN105273038A - Preparation method for multistage separation extraction of earthworm active proteins and method - Google Patents
Preparation method for multistage separation extraction of earthworm active proteins and method Download PDFInfo
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- CN105273038A CN105273038A CN201510095719.2A CN201510095719A CN105273038A CN 105273038 A CN105273038 A CN 105273038A CN 201510095719 A CN201510095719 A CN 201510095719A CN 105273038 A CN105273038 A CN 105273038A
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Abstract
The invention discloses a preparation method for multistage separation extraction of earthworm active proteins and a method. The preparation device comprises a constant temperature foam separating column, an air pump, an air distributer, a rotor flowmeter and a foam collection device. The preparation method comprises the following steps: firstly, collection and detection of an earthworm protein homogenate material liquid is carried out; secondly, the earthworm protein homogenate material liquid is subjected to pretreatment; thirdly, first stage foam separation is carried out; fourthly, second-stage foam separation is carried out. A two-stage foam separation technology is established at a high temperature based on influences on earthworm active protein foam separating effects, and the two-stage foam separation technology can raise the enrichment ratio and can raise the recovery rate.
Description
Technical field
The present invention relates to earthworm protein separation and Extraction field, specifically a kind of stage trapping extracts earthworm active protein preparation facilities and method thereof.
Background technology
The multiple activated protein with economic worth is had in earthworm.The extracting method of current activated protein mainly contains biochemical process, salting-out process, membrane separation process and ion-exchange-resin process etc.But, due to these methods exist investment and productive expense high, operating procedure complexity etc. defect, add enterprise cost, reduce enterprise profit.Therefore need that search operation is simple, expense is low, free of contamination novel process.
Foamet developed the more important isolation technique than faster in recent years.It is according to surface adsorption principle, making to have in solution on surface-active solute or granular absorption to liquid-vapo(u)r interface and with bubble by bubbling is that carrier is separated with liquid phase main body, as long as therefore have surface-active material or can carry out foam separation with the material of surfactant generation complexing.Foam separating technology is also more and more subject to the attention of investigator because it has the advantages such as equipment is simple, less investment, energy consumption are low and pollution-free.20 beginnings of the century, foam separating technology is applied to metallurgical industry, is applied to environment-protecting industrial afterwards, the application of recent researches in bioseparation process in proteins extraction.The extraction of application foamet is applicable to very much because earthworm active protein has good solubility, emulsifying property, whipability, retentiveness and viscoelasticity.
Why the concentration ratio improving protein in the foam separation aqueous solution is difficult to, and is because current research operates under being only confined to normal temperature.During normal temperature, when Proteins In Aqueous Solutions concentration exceedes its micelle-forming concentration, foam layer discharge opeing difficulty in foam separation process, the liquid holdup of foam column outlet is large, thus makes protein-enriched ratio be difficult to improve.
Summary of the invention
The present invention provides a kind of stage trapping to extract earthworm active protein preparation facilities and method thereof in order to solve the problem.
The present invention for addressing this problem taked technical scheme is:
A kind of stage trapping extracts earthworm active protein preparation facilities, comprise constant temperature foam separation post, air pump, gas distributor, spinner-type flowmeter, foam collection device, it is characterized in that: in constant temperature foam separation post, foam separation post is that synthetic glass is made, foam separation column bottom has a thief hole, opening for feed is had in the middle part of foam separation post, foam separation post top set temperature meter also connects foam collection device, foam separation post outside is wound around by emulsion tube, on emulsion tube, lower end arranges circulating water outlet respectively, recirculated water entrance, pass into recirculated water, use as control temperature, air pump passes into the gas distributor of foam separation column bottom after connecting spinner-type flowmeter, valve successively.
A kind of stage trapping extracts earthworm active protein preparation method, comprises the following steps:
The collection of I earthworm protein matter homogenate feed liquid detects: collecting earthworm protein matter homogenate material liquid volume is 2500ml, detects protein concn and should be 30.79g/L and pH value 3.9;
II earthworm protein matter homogenate feed liquid pre-treatment: use pH value regulator solution to regulate the pH value to 3.9 of the earthworm protein matter homogenate feed liquid described in the first step, as the charging of first step foam separation technique in Step II I;
III first step foam separation: superficial gas velocity be 40-60ml/min, temperature operates under being the condition of 50-60 DEG C, Froth is carried out to foam layer, ventilation is stopped when foam no longer overflows from constant temperature foam separation column top, collect foam and carry out froth breaking in foam collection device, froth breaking liquid is as earthworm active protein extracting solution, and the residual night work in knockout tower is the charging of step IV second stage foam separation;
IV second stage foam separation: charging dress liquid amasss as 2500mL, in feed liquid, earthworm protein matter homogenate feed liquid albumen initial mass concentration is 4.61g/L, initial pH value is 3.9, superficial gas velocity degree is 40-60ml/min, temperature is carry out under the condition of 20-30 DEG C, Froth is carried out to foam layer, when foam can not overflow from constant temperature foam separation column top, again superficial gas velocity is raised to 100-120ml/min, ventilation is stopped when foam can not overflow from constant temperature foam separation post, the foam collected carries out froth breaking in foam collection device, froth breaking liquid joins in the charging of first step foam separation.
The advantage that the present invention has and positively effect are:
Stage trapping of the present invention extracts earthworm active protein preparation facilities and method thereof, at comparatively high temps (50-60 DEG C), on basis to earthworm active protein foam separation influential effect, establish two-stage foam separation technique: first step foam separation makes the concentration ratio of earthworm protein high as far as possible, earthworm protein concentration in froth breaking liquid exceedes solubleness and separates out, and can be used as the raw materials for production of earthworm active protein; Second stage foam separation makes the rate of recovery of earthworm protein high as far as possible.Such two-stage foam separation technique can improve concentration ratio, can increase the rate of recovery again, and the earthworm active protein total yield of two-stage foam separation technique is 83.46%.
Accompanying drawing explanation
Fig. 1 is that stage trapping of the present invention extracts earthworm active protein preparation facilities structural representation.
Embodiment
Referring to drawings and Examples, the present invention will be described in detail.
As shown in Figure 1, a kind of stage trapping extracts earthworm active protein preparation facilities, comprise constant temperature foam separation post 1, air pump 2, gas distributor 3, spinner-type flowmeter 4, foam collection device 5, and foam separation post 6 is made for synthetic glass in constant temperature foam separation post 1, at the bottom of foam separation post, 6 have a thief hole 7, opening for feed 8 is had in the middle part of foam separation post 6, foam separation post 6 top set temperature meter 9 also connects foam collection device 5, foam separation post 6 outside is wound around by emulsion tube 10, on emulsion tube 10, lower end arranges circulating water outlet 11 respectively, recirculated water entrance 12, pass into recirculated water, use as control temperature, air pump 2 passes into the gas distributor 3 bottom foam separation post 6 after connecting spinner-type flowmeter 4, valve 13 successively.
Foam separation post high 6 is 610mm, and diameter is 80mm.
Described recirculated water is provided by ultra thermostat.
A kind of stage trapping extracts earthworm active protein preparation method, comprises the following steps:
The collection of I earthworm protein matter homogenate feed liquid detects: collecting earthworm protein matter homogenate material liquid volume is 2500ml, detects protein concn and should be 30.79g/L and pH value 3.9;
II earthworm protein matter homogenate feed liquid pre-treatment: use pH value regulator solution to regulate the pH value to 3.9 of the earthworm protein matter homogenate feed liquid described in the first step, as the charging of first step foam separation technique in Step II I;
III first step foam separation: superficial gas velocity be 40-60ml/min, temperature operates under being the condition of 50-60 DEG C, Froth is carried out to foam layer, ventilation is stopped when foam no longer overflows from constant temperature foam separation post 1 top, collect foam and carry out froth breaking in foam collection device 5, froth breaking liquid is as earthworm active protein extracting solution, the concentration ratio of earthworm active protein is 5.19, in raffinate, earthworm protein mass concentration is 4.61g/L, and the residual night work in constant temperature foam separation post 1 is the charging of step IV second stage foam separation;
IV second stage foam separation: charging dress liquid amasss as 2500mL, in feed liquid, earthworm protein matter homogenate feed liquid protein mass starting point concentration is 4.61g/L, initial pH value is 3.9, superficial gas velocity degree is 40-60ml/min, temperature is carry out under the condition of 20-30 DEG C, Froth is carried out to foam layer, when foam can not overflow from constant temperature foam separation post 1 top, again superficial gas velocity is raised to 100-120ml/min, ventilation is stopped when foam can not overflow from constant temperature foam separation post 1, the foam collected carries out froth breaking in foam collection device 5, froth breaking liquid joins in the charging of first step foam separation.The earthworm protein mass concentration of raffinate is 1.45g/L.
The earthworm active protein total yield of two-stage foam separation technique is 83.46%.
Stirring means froth breaking is adopted in described Step II I and step IV.
Described pH value regulator solution adopts sodium hydroxide.
Carry out foam separation to the earthworm protein matter aqueous solution, the factor such as initial pH value superficial gas velocity and temperature of investigating is on the impact of separating effect.Initial pH value hydrochloric acid in specific embodiment and sodium hydroxide regulate, by temperature in the recirculated water control tower of ultra thermostat, actual temperature in thermometer measure tower, spinner-type flowmeter regulates superficial gas velocity, when foam can not overflow from foam separating tower, stop ventilation, the foam in foam collection device adopts stirring means froth breaking.Measure protein concentration in froth breaking liquid, raffinate volume and raffinate.
Specific embodiment one:
What this specific embodiment adopted prepares material: large No. two, level ground earthworm, Baiming Science & Technology Co., Ltd.; Coomassie brilliant blue G250 biological reagent.
The concrete model of apparatus for preparation adopted: spinner-type flowmeter 3WB, Changzhou dicyclo; Tool sieve plate foaming tower 610mm × 80mm, Beijing Xin Weier customizes; Air pump FB-280, Shanghai Jaguar; Spectrophotometer L6S, upper Nereid section; Thermostat water bath HH-3, Jiangsu high honour.
Preparation process:
Fresh earthworm 100g clear water is cleaned, adds distilled water 250ml and make thick homogenate, the centrifugal 20min of 4000r/min, collect supernatant liquor; Precipitation adding distil water 250ml continues homogenate, the centrifugal 20min of 4000r/min, collects supernatant liquor; Till being repeatedly extracted into supernatant liquor clarification (totally 10 times).Supernatant liquor several times before merging, adjust ph to 3.9, by warming-in-water to 55 DEG C.Volume is settled to 2500ml to load in one-level foaming tower, measures Proteins In Aqueous Solutions concentration (following determination of protein concentration method is identical) in tower by Coomassie brilliant blue G250 method.Regulate gas speed to 50ml/min, carry out foaming and be separated earthworm active protein, until foam no longer overflows foaming tower.Measure volume and the concentration of isolated foam enrichment liquid.Measure residual liquid sum concentration in foaming tower simultaneously.
Load residual solution 2500ml at secondary foaming tower, regulate gas speed to 100ml/min, carry out foaming and be separated earthworm active protein, until foam no longer overflows foaming tower.Measure volume and the concentration of isolated foam enrichment liquid.Measure residual liquid sum concentration in foaming tower simultaneously.
Preparation result:
Former homogenate protein concn 30.79mg/ml, the protein concn 159.87mg/ml that one-level foaming tower is separated, residual solution protein concn 4.61mg/ml, concentration ratio is 5.19; The protein concn 19.61mg/ml that secondary foaming tower is separated, residual solution protein concn 1.45mg/ml.The overall rate of recovery is 83.46%.
Specific embodiment two:
What this specific embodiment adopted prepares material: large No. two, level ground earthworm, Baiming Science & Technology Co., Ltd.; Coomassie brilliant blue G250 biological reagent.
The concrete model of apparatus for preparation adopted: spinner-type flowmeter 3WB, Changzhou dicyclo; Tool sieve plate foaming tower 610mm × 80mm, Beijing Xin Weier customizes; Air pump FB-280, Shanghai Jaguar; Spectrophotometer L6S, upper Nereid section; Thermostat water bath HH-3, Jiangsu high honour.
Preparation process:
Fresh earthworm 100g clear water is cleaned, adds distilled water 250ml and make thick homogenate, the centrifugal 20min of 4000r/min, collect supernatant liquor; Precipitation adding distil water 250ml continues homogenate, the centrifugal 20min of 4000r/min, collects supernatant liquor; Till being repeatedly extracted into supernatant liquor clarification (totally 10 times).Supernatant liquor several times before merging, adjust ph to 3.9, by warming-in-water to 50 DEG C.Volume is settled to 2500ml to load in one-level foaming tower, measures Proteins In Aqueous Solutions concentration (following determination of protein concentration method is identical) in tower by Coomassie brilliant blue G250 method.Regulate gas speed to 50ml/min, carry out foaming and be separated earthworm active protein, until foam no longer overflows foaming tower.Measure volume and the concentration of isolated foam enrichment liquid.Measure residual liquid sum concentration in foaming tower simultaneously.
Load residual solution 2500ml at secondary foaming tower, regulate gas speed to 100ml/min, carry out foaming and be separated earthworm active protein, until foam no longer overflows foaming tower.Measure volume and the concentration of isolated foam enrichment liquid.Measure residual liquid sum concentration in foaming tower simultaneously.
Preparation result:
Former homogenate protein concn 30.79mg/ml, the protein concn 159.87mg/ml that one-level foaming tower is separated, residual solution protein concn 4.61mg/ml, concentration ratio is 5.19; The protein concn 19.61mg/ml that secondary foaming tower is separated, residual solution protein concn 1.45mg/ml.The overall rate of recovery is 83.46%.
Specific embodiment three
What this specific embodiment adopted prepares material: large No. two, level ground earthworm, Baiming Science & Technology Co., Ltd.; Coomassie brilliant blue G250 biological reagent.
The concrete model of apparatus for preparation adopted: spinner-type flowmeter 3WB, Changzhou dicyclo; Tool sieve plate foaming tower 610mm × 80mm, Beijing Xin Weier customizes; Air pump FB-280, Shanghai Jaguar; Spectrophotometer L6S, upper Nereid section; Thermostat water bath HH-3, Jiangsu high honour.
Preparation process:
Fresh earthworm 100g clear water is cleaned, adds distilled water 250ml and make thick homogenate, the centrifugal 20min of 4000r/min, collect supernatant liquor; Precipitation adding distil water 250ml continues homogenate, the centrifugal 20min of 4000r/min, collects supernatant liquor; Till being repeatedly extracted into supernatant liquor clarification (totally 10 times).Raffinate in supernatant liquor and example 1 center pillar several times before merging, adjust ph to 3.9, by warming-in-water to 60 DEG C.Volume is settled to 2500ml to load in one-level foaming tower, measures Proteins In Aqueous Solutions concentration (following determination of protein concentration method is identical) in tower by Coomassie brilliant blue G250 method.Regulate gas speed to 50ml/min, carry out foaming and be separated earthworm active protein, until foam no longer overflows foaming tower.Measure volume and the concentration of isolated foam enrichment liquid.Measure residual liquid sum concentration in foaming tower simultaneously.
Load residual solution 2500ml at secondary foaming tower, regulate gas speed to 100ml/min, carry out foaming and be separated earthworm active protein, until foam no longer overflows foaming tower.Measure volume and the concentration of isolated foam enrichment liquid.Measure residual liquid sum concentration in foaming tower simultaneously.
Preparation result:
Former homogenate protein concn 35.88mg/ml, the protein concn 186.33mg/ml that one-level foaming tower is separated, residual solution protein concn 5.37mg/ml, concentration ratio is 5.20; The protein concn 22.86mg/ml that secondary foaming tower is separated, residual solution protein concn 1.69mg/ml.The overall rate of recovery is 84.32%.
Extract earthworm active protein compared to conventional biochemical process and salting-out process, often extract and do not need repeatedly repetitive operation or progressive operation comprehensively, introduce a large amount of chemical reagent, contaminating protein is polluted water resources also; Membrane filter method and ion exchange resin introduce the pollution decreased environment, but the cost of filter membrane, and the cost of resin, whole plant treatment scale defines this kind for the treatment of process and is more suitable for laboratory and the production application of nonbusiness's particularly middle-size and small-size science-and-technology enterprise.Foamet only needs to inject clean air to foam column bottom just can isolate the multiple active protein being dissolved in water, energy-conservation, pollution-free, low cost, high-level efficiency.
Stage trapping of the present invention extracts earthworm active protein preparation facilities and method thereof, at comparatively high temps (50-60 DEG C), on basis to earthworm active protein foam separation influential effect, establish two-stage foam separation technique: first step foam separation makes the concentration ratio of earthworm protein high as far as possible, earthworm protein concentration in froth breaking liquid exceedes solubleness and separates out, and can be used as the raw materials for production of earthworm active protein; Second stage foam separation makes the rate of recovery of earthworm protein high as far as possible.Such two-stage foam separation technique can improve concentration ratio, can increase the rate of recovery again.
Claims (6)
1. a stage trapping extracts earthworm active protein preparation facilities, comprise constant temperature foam separation post (1), air pump (2), gas distributor (3), spinner-type flowmeter (4), foam collection device (5), it is characterized in that: foam separation post (6) is made for synthetic glass in constant temperature foam separation post (1), (6) portion at the bottom of foam separation post has a thief hole (7), foam separation post (6) middle part has opening for feed (8), foam separation post (6) top set temperature meter (9) also connects foam collection device (5), foam separation post (6) outside is wound around by emulsion tube (10), on emulsion tube (10), lower end arranges circulating water outlet (11) respectively, recirculated water entrance (12), pass into recirculated water, use as control temperature, air pump (2) passes into the gas distributor (3) of foam separation post (6) bottom after connecting spinner-type flowmeter (4), valve (13) successively.
2. stage trapping according to claim 1 extracts earthworm active protein preparation facilities, it is characterized in that: foam separation post high (6) is 610mm, and diameter is 80mm.
3. stage trapping according to claim 1 extracts earthworm active protein preparation facilities, it is characterized in that: described recirculated water is provided by ultra thermostat.
4. stage trapping extracts an earthworm active protein preparation method, comprises the following steps:
The collection of I earthworm protein matter homogenate feed liquid detects: collecting earthworm protein matter homogenate material liquid volume is 2500ml, detects protein concn and should be 30.79g/L and pH value 3.9;
II earthworm protein matter homogenate feed liquid pre-treatment: use pH value regulator solution, regulates the pH value to 3.9 of the earthworm protein matter homogenate feed liquid described in the first step, as the charging of first step foam separation technique in Step II I;
III first step foam separation: superficial gas velocity be 40-60ml/min, temperature operates under being the condition of 50-60 DEG C, Froth is carried out to foam layer, ventilation is stopped when foam no longer overflows from constant temperature foam separation post (1) top, collect foam and carry out froth breaking in foam collection device (5), froth breaking liquid is as earthworm active protein extracting solution, and the residual night work in knockout tower is the charging of step IV second stage foam separation;
IV second stage foam separation: charging dress liquid amasss as 2500mL, in feed liquid, earthworm protein matter homogenate feed liquid protein mass starting point concentration is 4.61g/L, initial pH value is 3.9, superficial gas velocity degree is 40-60ml/min, temperature is carry out under the condition of 20-30 DEG C, Froth is carried out to foam layer, when foam can not overflow from constant temperature foam separation post (1) top, again superficial gas velocity is raised to 100-120ml/min, ventilation is stopped when foam can not overflow from constant temperature foam separation post (1), the foam collected carries out froth breaking in foam collection device (5), froth breaking liquid joins in the charging of first step foam separation.
5. stage trapping according to claim 4 extracts earthworm active protein preparation method, it is characterized in that: adopt stirring means froth breaking in described Step II I and step IV.
6. stage trapping according to claim 4 extracts earthworm active protein preparation method, it is characterized in that: described pH value regulator solution adopts hydrochloric acid or sodium hydroxide.
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| CN106107242A (en) * | 2016-07-29 | 2016-11-16 | 舟山市瑞丰生物技术有限公司 | A kind of nonreactive aquatic immune reinforcing agent |
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| CN1789220A (en) * | 2005-11-15 | 2006-06-21 | 江西汇仁药业有限公司 | Method for separating Chinese medicinal active substances |
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| CN106040722A (en) * | 2016-07-15 | 2016-10-26 | 天津市百鸣科技发展有限公司 | Method for purifying earthworms contaminated by heavy metal |
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Application publication date: 20160127 |