Comparison among three anion exchange chromatographic supports to capture erythropoietin f

合集下载

Q Sepharose Fast Flow 产品说明书

Q1126pis Rev 01/221Product InformationQ Sepharose ® Fast FlowQ1126Product DescriptionQ Sepharose ® Fast Flow is an ion exchangechromatography resin with a quaternary amine (Q) functional group [-CH 2-N +(CH 3)3] attached to Sepharose ® Fast Flow. The Q group serves as astrong anion exchanger, which is completely ionized over a broad pH range. The terms “s trong" and"weak" in ion exchange chromatography refer to the extent of ionization with pH, and not to the binding strength of the functional group to the target species. The parent Sepharose ® Fast Flow is a cross-linked derivative of Sepharose ®. The particle size range is 45-165 µm. The average bead diameter is ~90 µm. The counterion in the product is sulfate (SO 4-2). Recommended cation buffers to use with Q Sepharose ® Fast Flow include alkylamines,ammonium, ethylenediamine, imidazole, pyridine, or Tris. In terms of pH, it is suggested to operate within 0.5 pH unit of the buffer's pK a . With proteins, it is suggested to operate at least 1 pH unit above the pI of the protein, to facilitate binding. Oxidizing agents, and anionic detergents and buffers, should not be used with Q Sepharose ® Fast Flow. Likewise,extended exposure of Q1126 to pH < 4 should be avoided. Several publications 1,2 and dissertations 3-5 cite use of product Q1126 in their research.ReagentQ Sepharose ® Fast Flow is offered as a suspension in 20% ethanol.Approximate Exclusion Limit: average molecular mass of ~4 × 106 DaltonsIonic Capacity: 0.18-0.24 mmol Cl -/mL gel Binding Capacity: ~42 mg BSA per mL gel pH Stability: 2-12Working temperature: 4-40 °CPrecautions and DisclaimerFor R&D use only. Not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. General Resin Preparation Procedure1. Allow the ion exchange medium and ~10 columnvolumes (CV) of buffer to equilibrate to thetemperature chosen for the chromatographic run. 2. Mix the pre-swollen suspension with startingbuffer to form a moderately thick slurry, which consists of ~75% settled gel and 25% liquid. 3. Degas the gel under vacuum at the temperatureof column operation.4. Mount the column vertically on a suitable stand,out of the way of direct sunlight or drafts, which may cause temperature fluctuations.5. Pour a small amount of buffer into the emptycolumn. Allow the buffer to flow through spaces to eliminate air pockets.6. Pour the suspension of ion exchange mediumprepared in Step 3 into the column by allowing it to flow gently down the side of the tube, to avoid bubble formation.7. For consistent flow rates and reproducibleseparations, connect a pump to the column. 8. Fill the remainder of the column to the top withbuffer. Allow ~5 CV of buffer to drain through the bed at a flow rate at least 133% of the flow rate to be used in the procedure. The bed height should have settled to a constant height.9. Using a syringe or similar instrument, apply thesample dissolved in starting buffer to the column. For isocratic separations, the sample volumeshould range from 1-5% of the column volume. If the chromatographic run involves elution with a gradient, the applied sample mass is of much greater importance than the sample volume, and the sample should be applied in a low ionicstrength medium. Ion exchange is used both to concentrate and to fractionate the sample. 10. Elution:• If only unwanted substances in the sample areadsorbed, or if sample components aredifferentially retarded under isocratic conditions, the starting buffer can also be used as the eluent.The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.Merck and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Q1126pis Rev 01/22 JJJ,MAM,GCY2•Normally, however, separation and elution are achieved by selectively decreasing the affinity of the molecules for the charged groups on the resin by changing the pH and/or ionic strength of the eluent. This procedure is termed gradient elution. 11. Regeneration: •Either (a) washing the column with a high ionic strength salt solution, such as 1 M NaCl, or (b) changing the pH to the tolerable low and high pH extremes, is usually sufficient to remove reversibly bound material.• When needed, lipids and precipitated proteins canbe removed by washing with 1 CV of 1-2 M NaCl, followed by 1 CV of 0.1 M NaOH in 0.5 M NaCl. • Rinse with several CV of water. Thenre-equilibrate the resin with starting buffer.• If base such as NaOH was used, adjust the pH ofthe resin to neutral before storing or using.12. Storage: Q Sepharose ® Fast Flow may be storedat 2-8 °C in water with 20% ethanol added as an antibacterial agent.General NotesCation versus Anion Exchanger• If sample components are most stable below their pI values, a cation exchanger should be used. • If sample components are most stable above their pI values, an anion exchanger should be used. •If stability is good over a wide pH range on both sides of the pI, either or both types of ion exchanger may be used.Strong versus Weak Ion Exchanger•Most proteins have pI values within the range 5.5-7.5, and can thus be separated on both strong and weak ion exchangers.•When maximum resolution occurs at an extreme pH and the molecules of interest are stable at that pH, a strong ion exchanger should be used. Choice of Buffer, pH, and Ionic Strength• The highest ionic strength which permits binding should normally be used.•The required buffer concentration varies fromsubstance to substance. Usually, an ionic strength of at least 10 mM is required to ensure adequate buffering capacity.•As salts (such as buffers) help to stabilize proteins in solution, their concentration should be highenough to prevent denaturation and precipitation.References1. López, G. et al ., Eukaryot. Cell , 14(6), 564-577(2015).2. Bhargava, V. et al ., Dev. Cell., 52(1), 38-52.e10(2020).3. Fu , Yinan, “Structure and dynamics ofPseudomonas aeruginosa ICP”. University ofGlasgow, Ph.D. dissertation, p. 126 (April 2009). 4. Redmond, Miranda , “The Role of N-TerminalAcidic Inserts on the Dynamics of the Tau Protein ”. University of Vermont, Ph.D. dissertation, p. 22 (May 2017).5. Taylor-Whiteley, Teresa Rachel , “RecapitulatingParkinson’s disease pathology in athree-dimensional neural cell culture mode ”. Sheffield Hallam University, Ph.D. dissertation, p. 58 (September 2019).NoticeWe provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve ourcustomers of their own responsibility for checking the suitability of our products for the envisaged purpose. The information in this document is subject to change without notice and should not be construed as acommitment by the manufacturing or selling entity, or an affiliate. We assume no responsibility for any errors that may appear in this document.Technical AssistanceVisit the tech service page at /techservice .Standard WarrantyThe applicable warranty for the products listed in this publication may be found at /terms .Contact InformationFor the location of the office nearest you, go to /offices .。

离子交换分离原理及设备课件PPT

具
有
块
石
支
持
1-进料口 2-视镜 3-液位计
层
的
4-树脂层 5-卵石层 6-出液口
离
子
交
换
罐
淋洗水、解吸液及再生剂进口
被交换溶液进口
废液出口
反
吸
分布器
附
离
子
交
换
罐
分布器
淋洗水、解吸液及再生剂出口，反洗水进口
application
扩交换容量：表示方法有质量交换容量(mmol/g树脂)和体积交换容量(mmol/mL树脂)；
SSeOOnUUtRReCCrEE
填料设计的填料设计的
FineLINE FineLINE
柱柱
antib•odSymcolaulmlnms moolneocculolnealsawntibiloldytaatktaechleodntogceolrumton pass 解吸离d子oBw+自n 树th脂e内c部ol的u活m性n中th心a扩n散la到rg树e脂o表n面es；because
的 (生产力)
(峰越少越好)
CHROMAFLOW 自动装卸层析柱
流
程
筛
漩
板
涡
式
式
连
连
续
续
离
离
子
子
交
交
换
换
设
设
备
备
离子交换设备的计算
✓ 罐体积
罐体积Vt： Vt V y
罐高径比一般取Hi/D=2～3。
✓ 交换设备的放大
1.根据交换罐负荷相同的原则放大； 2.根据交换罐中溶液空塔流速相同的原则放大； 3.溶液通过床层的压力降

Waters Protein-Pak Hi Res Q Column 分离 Low Range ss

Size and Purity Assessment of Single-Guide RNAs by Anion-Exchange Chromatography (AEX)Hua Yang,Stephan M. Koza,Ying Qing YuWaters CorporationAbstractSingle-guide RNA (sgRNA) is a critical element in the CRISPR/Cas9 Technology for gene editing, the size of which usually ranges from 100 to 150 bases. In this application note, we show that the size of several sgRNAs could be estimated by comparison to a Low Range ssRNA Ladder (50–500 bases) using an optimized anion-exchange method developed on a Waters Protein-Pak Hi Res Q Column. In addition, the purity of the sgRNA samples can be assessed using the same anion exchange method, providing an informative and non-complex method for sgRNA product consistency.BenefitsWaters Protein-Pak Hi Res Q Column separation of a Low Range ssRNA Ladder with the size ranging from ■50 to 500 basesWaters Protein-Pak Hi Res Q Column separation of ssRNAs and their impurities■Size and purity estimation of ssRNAs having a size range of 100–150 mer under the same gradient conditions ■using the AEX method on Waters Protein-Pak Hi Res Q ColumnIntroductionThe discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) bacterial immunity systems and the rapid adaptation of RNA guided CRISPR/CRISPR Associated Protein 9 (Cas9) Technology to mammalian cells have had a significant impact in the field of gene editing.1–3 The Cas9 protein, a non-specific endonuclease, is directed to a specific DNA site by a guide RNA (gRNA), where it makes a double-strand break of the DNA of interest. The gRNA consists of two parts: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The crRNA is usually a 17–20 nucleotide sequence complementary to the target DNA, and the tracrRNA serves as a binding scaffold for the Cas9 nuclease. While crRNAs and tracrRNAs exist as two separate RNA molecules in nature, the single-guide RNA (sgRNA), which combines both the crRNA sequence and the tracrRNA sequence into a single RNA molecule, has become a commonly used format. The length of a sgRNA is in the range of 100–150 nucleotides. It is critical to characterize the sgRNA, as it is the core of the CRISPR/Cas9 technology.Anion-exchange chromatography (AEX) separates molecules based on their differences in negative surface charges. This analytical technique can be robust, reproducible, and quantitative. It is also easy to automate, requires small amounts of sample, and allows for the isolation of fractions for further analysis. AEX has been utilized in multiple areas related to gene therapy, including adeno-associated virus empty and full capsid separation, plasmid isoform separation, and dsDNA fragment separation.4–6 Since the sgRNAs are negatively charged due to the phosphate groups on the backbone, we investigated AEX for size and purity assessment of sgRNAs.In this application note, we show that using a Waters Protein-Pak Hi Res Q strong Anion-Exchange Column on an ACQUITY UPLC H-Class Bio System, a single-stranded RNA (ssRNA) ladder ranging from 50 to 500 bases can be separated and used for estimating the size of ssRNAs in the approximate range of 100–150 bases, including the sgRNAs for CRISPR/Cas9 System. Moreover, the purity of these ssRNAs can be estimated with the same gradient conditions.ExperimentalSample DescriptionHPRT (purified and crude) is a pre-designed CRISPR/Cas9 sgRNA (Hs.Cas9.HPRT1.1AA, 100 mer). GUAC is acustomized ssRNA (150 mer), which contains repeats of GUAC sequence. HPRT sgRNA and GUAC ssRNA were purchased from Integrated DNA Technologies (IDT). Rosa26 and Scrambled #2 are both pre-designedCRISPR/Cas9 sgRNAs purchased from Synthego (100 mer). Low Range ssRNA Ladder was purchased from New England Biolabs (N0364S).Method ConditionsLC ConditionsLC system:ACQUITY UPLC H-Class BioDetection:ACQUITY UPLC TUV Detector with 5 mm titaniumflow cellWavelength:260 nmVials:Polypropylene 12 x 32 mm Screw Neck Vial, withCap and Pre-slit PTFE/Silicone Septum, 300 µLVolume, 100/pk (P/N 186002639)Column(s):Protein-Pak Hi Res Q Column, 5 µm, 4.6 x 100 mm(P/N 186004931)Column temp.:60 °CSample temp.:10 °CInjection volume:1–10 µLFlow rate:0.4 mL/minMobile phase A:100 mM Tris-HClMobile phase B:100 mM Tris baseMobile phase C: 3 M Tetramethylammonium chloride (TMAC)Mobile phase D:WaterBuffer conc. to deliver:20 mMGradient Table (an AutoBlend Plus Method, Henderson-Hasselbalch derived).In the above gradient table, the buffer is 20 mM Tris pH 9.0. The initial salt concentration is set to 0 mM to ensure all the analytes are strongly bound onto the column. After 5 mins, the salt concentration is increased to 1400 mM where most of the impurities will elute, based on prior investigation. After 4 mins equilibration, the separation gradient starts. The salt concentration increases linearly from 1400 m to 2100 mM in 20 mins for the Low Range ssRNA Ladder separation, as well as individual ssRNAs. Then it is ramped up to 2400 mM to strip off any remaining bound molecules. Finally, an equilibration step to the initial condition takes place, preparing for the next injection.An equivalent gradient table for a generic quaternary LC system is shown above.Data ManagementChromatography software:Empower 3 (FR 4)Results and DiscussionSize AssessmentVarious mobile phase conditions were tested using a Low Range ssRNA Ladder for size assessment of the ssRNAs, including pH (7.4 and 9.0), column temperature (30 °C and 60 °C) and salt (NaCl and TMAC).The results from the optimal conditions are shown in Figure 1B. Using a pH 9.0 Tris buffer with 60 °C column temperature and a TMAC salt gradient, the Low Range ssRNA Ladder (50–500 bases) along with four pre-made sgRNAs (100 mer), and one customized ssRNA (150 mer) were separated on a Waters Protein-Pak Hi Res Q Column. The separation for the Low Range ssRNA Ladder on this strong anion exchange column was very similar to that on an agarose gel, as shown in Figure 1A. A calibration curve was constructed based on the retention time and the logarithm of the number of bases of each ssRNA in the ladder (Figure 1C, blue dots). Thelinear fit from the Low Range ssRNA Ladder indicates a strong correlation between the logarithm of the size andthe retention time (R2=0.993). Using this plot, the size of the ssRNAs was calculated from their individual retention time. The percent error is calculated using the formula {(calculated size – theoretical size)/theoretical size}. The percent error was less than 6% for all the RNAs tested (Figure 1d), as evidenced by the orange data points residing on or very closely to the trendline of the calibration curve. Notice that small percent error was obtained from four pre-made sgRNAs from two different manufacturers and a customized ssRNA with an artificial sequence. Although ssRNAs with shorter than 100 bases and larger than 150 bases were not tested, it is possible that this method can be used for the ssRNAs size assessment in the range of 50–500 bases.Figure 1A.Agarose gel separation of Low Range ssRNA Ladder (Reprinted from (2021) with permission from New England Biolabs); 1B. Anion-exchange separation of Low Range ssRNA Ladder and ssRNAs on a Waters Protein-Pak Hi Res Q Column; 1C. A plot of log(size) vs. retention time of Low Range ssRNA Ladder (blue dots) and individual ssRNAs (orange dots); 1D. Size estimation of individual ssRNAs based on retention time and calibration curve. Small percent error was obtained for all ssRNAs.It is noteworthy that a mobile phase condition with pH 7.4 Tris buffer, 60 °C column temperature and a TMAC salt gradient also resulted in good size estimation with percent error <5% for all pre-made sgRNAs (100 mer) and ~12% for the artificially made GUAC ssRNA (150 mer). Overall, 60 °C column temperature resulted in one singlepeak for each ssRNA which is needed to determine the retention time of the peak for size assessment. 30 °C column temperature resulted in more than one major peaks, which are presumably the isomers of the ssRNAs. Multiple peaks were also observed when using NaCl as the salt, regardless of the pH and column temperature.Purity AssessmentPurified and crude HPRT sgRNA was separated on the Protein-Pak Hi Res Q Column (Figure 2) using the same gradient conditions for size assessment. The relative purities of the crude and purified samples were measured as 37.4% and 88.0%, respectively, based on the peak areas indicated. The majority of the impurities eluted prior to 50 bases although lower abundance impurities appear to be present up to the size of the HPRT sgRNA.Figure 2. Crude and purified HPRT sgRNA for CRISPR/Cas 9 System were separated on a Waters Protein-Pak Hi Res Q Column using the same conditions as in Figure 1B (see Experimental for details).ConclusionAnion-exchange chromatography is robust, reproducible, easy to automate, yields quantitative information, andrequires a small amount of sample. We demonstrate here that the components of a Low Range ssRNA Ladder, ranging from 50 to 500 bases, can be separated on a Waters Protein-Pak Hi Res Q Column with a linear correlation between the log of base-number and observed retention time when TMAC is used as an elution salt. The size of ssRNAs ranging from 100 to 150 bases can be estimated by comparing the retention time of the ssRNAs with that of the Low Range ssRNA Ladder. In addition, the purity of a sgRNAs may also be observed from the same chromatographic separation. This method can potentially be applied to the analysis of sgRNAs which are the key element for CRISPR/Cas9 gene editing technology.ReferencesDunbar C E, High K A, J. Joung K, Kohn D B, Ozawa K, Sadelain M. Gene Therapy Comes of Age. Science 1.2018; 359: 175.2.Rath D, Amlinger L, Rath A, Lundgren M. The CRISPR-Cas Immune System: Biology, Mechanisms and Applications. Biochimie 2015; 117: 119–128.3.Patrick D. Hsu P D, Eric S. Lander E S, and Zhang F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell 2014; 157: 1262–1278.Yang H, Koza S and Chen W. Anion-Exchange Chromatography for Determining Empty and Full Capsid4.5.Yang H, Koza S and Chen W. Plasmid Isoform Separation and Quantification by Anion-Exchange6.Yang H, Koza S and Chen W. Separation and Size Assessment of dsDNA Fragments by Anion-ExchangeFeatured Products■■720007428, November 2021© 2021 Waters Corporation. All Rights Reserved.。

3种测定皂类产品中氯含量方法的比较研究

第7卷第8期食品安全质量检测学报 Vol. 7 No. 82016年8月Journal of Food Safety and Quality Aug. , 2016*通讯作者: 杨园园, 工程师, 主要研究方向为食品安全及产品质量检测。

E-mail: ylyf2001@*Corresponding author: YANG Yuan-Yuan, Engineer, Shanghai Institute of Quality Inspection and Technical Research, Shanghai 200233, China. E-mail: ylyf2001@3种测定皂类产品中氯含量方法的比较研究杨园园*, 程白羽, 吴秀萍(上海市质量监督检验技术研究院, 上海 200233)摘要: 目的采用反滴定法、电位滴定法以及离子色谱法分别对皂类产品中的氯含量进行分析和比较。

方法反滴定法采用硫氰酸铵和硝酸银标准溶液进行滴定。

电位滴定法采用银电极作为指示电极, 硝酸银作为标准溶液滴定。

离子色谱法采用IonPac AS 19阴离子交换色谱柱分离, 淋洗液为20 mmol/L KOH 溶液, 以电导检测器检测。

通过对市售6个香皂样品的检测结果分析, 进行3种方法的比较。

结果反滴定法、电位滴定法、离子色谱法的相对误差为 1.2%~2.0%、1.5%~2.2%和0.9%~1.7%; 相对标准偏差分别为0.009%~0.014%、0.013%~0.016%和0.008%~0.012%; 加标回收率分别为101.9%±4.6%、99.8%±8.3%和101.9%±3.0%。

结论从精密度、准确度和加标回收率来看, 离子色谱法检测效果最好, 值得推广应用。

关键词: 香皂; 氯含量; 反滴定法; 电位滴定法; 离子色谱法Comparison of 3 kinds of test methods for determination of chloride content in soap productsYANG Yuan-Yuan *, CHEN Bai-Yu, WU Xiu-Ping(Shanghai Institute of Quality Inspection and Technical Research , Shanghai 200233, China )ABSTRACT: Objective To compare 3 kinds of methods for determination of chloride content in soap products, including back titration, potentiometric titration and ion chromatography. Method The back titration method used ammonium thiocyanate and silver nitrate standard solution for titration. In potentiometric titration method, the silver electrode was used as indicator electrode, and silver nitrate was used as standard solution for titration. In ion chromatography method, IonPac AS 19 anion exchange column was used for separation with leacheate of 20 mmol/L KOH and detector of conductivity. Six soap samples were detected to compare the 3 different kinds of methods. Results The relative errors of back titration, potentiometric titration, ion chromatography were 1.2%~2.0%, 1.5%~2.2% and 0.9%~1.7%, respectively; the relative standard deviations of 3 test methods were 0.009%~0.014%, 0.013%~0.016% and 0.008%~0.012%, respectively; and the recoveries were 101.9%±4.6%, 99.8%±8.3% and 101.9%±3.0%, respectively. Conclusions Based on the precision, accuracy and recovery, the ion chromatography method is the best, which deserves to be popularized and applied.KEY WORDS: soap; chloride content; back titration; potentiometric titration; ion chromatography3404 食品安全质量检测学报第7卷1引言自从出现餐具洗涤剂、沐浴液等洗涤用品, 香皂在日化产品中的市场份额越来越小; 但随着市场日益细分[1], 目前又有了厨房清洁皂、果蔬液体皂等产品。

Unit 3 Comparison and Contrast

Task 1: Familiarizing Yourself with Comparison & Contrast
Reading 1 Electric Vehicles and Gas Vehicles
Questions: (P66) 3) What are the similarities and differences between the two subjects? Differences: c. Cost EV--$6 to $12 a week GV---$5 a gallon d. Convenience GV---many people and much energy are used to refuel EV---only need to plug and recharge it into an outlet
Task 1: Familiarizing Yourself with Comparison
& Contrast
Reading 1
Electric Vehicles and Gas Vehicles (P66)
New Words: 1) Electric vehicle=电动汽车 2) Gas vehicle=燃油汽车 3) Auto industry=汽车产业 4) Churn out=快速且大量地生产 5) Dodge Circuit EV=道奇电路电动车 (electric-powered) 6) Dodge Challenger=道奇挑战者 (gas-powered) 7) Muscle car=肌肉车 8) Bold look=狂野的外形 9) Standard feature=标准配置 10) lithium-ion battery=锂电池

Multiple comparisons

Multiple comparisons (1 of 3)If more than one comparison among means is conducted at a given PCER,the EER will be higher than the PCER. The following inequality can be used to control the EER:EER (c)(PCER)where c is the number of comparisons performed. For example, if six comparisons are performed at the .05 significance level(PCER = .05), then the EER is less than or equal to (6)(.05) = .30. If a researcher wishes to perform six comparisons and keep the EER at the .05 level, the .05/6 = .0083 significance level should be used for each comparison. That way, the EER would be less than or equal to (6)(.0083) = .05.In general, to keep the EER at or below .05, the PCER should be: PCER = .05/c where c is the number of comparisons. More generally, to insure that the EER is less than or equal to α, use PCER = α/c.Adjusting the PCER in this manner is called either the "Bonferroni adjustment" or "Dunn's procedure" after the statisticians who developed it.Since Scheffé's test can also be used to test multiple comparisons, it is important to use the Scheffé's test if it is more powerful than the Bonferroni adjustment. Scheffé's test will be more powerful if more than three comparisons are planned among three means or more than seven comparisons are planned among four means. In almost all realistic situations, the Bonferroni adjustment is more powerful. As in the case of pairwise comparisons,there is a tradeoff between controlling the EER and power. The Bonferroni adjustment provides control over the EER at a substantial cost in power. Some statisticians argue that it is not always necessary to control the EER. The "experiment," after all, is an arbitrary unit of analysis. Why is it necessary to control the error rate in one experiment but not in a whole series of experiments. For example, if a researcher conducted a series of five related experiments, few statisticians would recommend that the probability of a Type I error in any of the comparisons in any of the experiments be controlled. Nonetheless, the experiment is a convenient unit of analysis. The general consensus is that steps should be taken to control the EER.There is room for argument, however, about whether it should be strictly contolled, or whether it should be allowed to rise slightly.。

重金属方面的英文文献

Analytica Chimica Acta 515(2004)343–348A continuous approach for the determination of Cr(VI)in sediment and soil based on the coupling of microwave-assisted water extraction,preconcentration,derivatization and photometric detectionS.Morales-Muñoz,J.L.Luque-Garc´ıa ∗,M.D.Luque de CastroDepartment of Analytical Chemistry,University of Córdoba,Marie Curie Building,Campus of Rabanales,E-14071Córdoba,SpainReceived 19January 2004;received in revised form 19March 2004;accepted 19March 2004Available online 25May 2004AbstractA dynamic system for the continuous leaching of Cr(VI)from sediment and soil based on both microwave assistance and iterative changeof the ﬂow direction of the extractant through the sample cell has been developed.The microwave-assisted extractor has been coupled to a photometric detector through a ﬂow injection interface in order to develop a fully automated method.The Cr(VI)extracted was monitored after derivatization with 1,5-diphenylcarbazide.Two approaches are proposed which differ in the inclusion of a preconcentration minicolumn packed with a strong anion exchange resin.A 0.04M ammonium buffer solution was used as extractant and 0.2g of sample—river sediment spiked with 50and 5␮g g −1for the method without preconcentration (method A)and with preconcentration (method B)—was subjected to 8–14min of 300W microwave-assisted extraction.The within-laboratory reproducibility and repeatability were 2.6and 1.9for method A,and 4.0and 2.6for method B.The proposed methods have been compared with the reference EPA method 3060/7196.©2004Elsevier B.V .All rights reserved.Keywords:Microwave-assisted extraction;Hexavalent chromium;Sediment;Photometric detection1.IntroductionThe predominant use of chromium in industry (in pro-cesses such as plating,tanning,and paint and pigment production)unfortunately causes environmental concern.The toxicity of a metal depends on its oxidation state.In the case of chromium,Cr(III)is considered an essential microelement [1,2]while Cr(VI)is thought to be toxic [3]and carcinogenic.Thus,the identiﬁcation of Cr(VI)species in environmental samples is a problem of great concern be-cause of their toxicity to aquatic and terrestrial organisms,including humans.It is not sufﬁcient to give a total concen-tration of metal;instead,what is required to understand the potential toxicity of a sample is the concentration of Cr(VI)in the sample.An ideal extraction method would extract the metal ef-ﬁciently without converting metal ions from one oxidation state to another.Although much research has been focused∗Corresponding author:Tel.:+34-957-218615;fax:+34-957-218615.E-mail address:952lugaj@uco.es (J.L.Luque-Garc´ıa).on the extraction and detection of chromium species in liq-uid samples such as natural and waste waters [4–6],the ex-traction of Cr(VI)from soil samples requires an additionaleffort [7].The difﬁculty in determining Cr(VI)species in solid samples arises from the possible changes taking place in the chromium oxidation state.The environmental protection agency (EPA)recog-nises four methods for sample preparation of hexavalent chromium:7195,coprecipitation;7196,colorimetry with 1,5-diphenylcarbazide (DPC);7197,chelation/extraction;and 7198,differential pulse polarography.An ofﬁcial method,namely EPA method 3060,using alkaline diges-tion of Cr(VI)is also suggested for sample preparation of Cr(VI).A study was conducted by Gurknecht in 1983to evaluate the above four methods [8].The study concluded that 7195and 7197methods were vulnerable to effects of matrix composition.The 7196colorimetric method based on the coloured complex formed between DPC and Cr(VI)[9]is one of the most sensitive and selective for Cr(VI)determination.Several methods based on batch extraction have been proposed for the speciﬁc extraction of Cr(VI)from solid0003-2670/$–see front matter ©2004Elsevier B.V .All rights reserved.doi:10.1016/j.aca.2004.03.092344S.Morales-Muñoz et al./Analytica Chimica Acta515(2004)343–348samples[3,10–12].There is only one case in the literature in which a dynamic extraction system has been used[13]. Almost all these methods are based on the use of ultrasound energy to accelerate the extraction step[3,10,11,13].This energy has proved to be of great help in the extraction of Cr(VI)from solid samples without disturbing the species distribution.Microwave-assisted extraction is also an expeditious,in-expensive and efﬁcient extraction technique.Over the past few years,this technique has been used in a discontinu-ous mode for accelerating the sample preparation step and avoiding some potential problems—namely,analyte losses and contamination[14,15]—related to conventional meth-ods.Dynamic microwave-assisted sample treatment has also been used[16–19],allowing the automation of the prelimi-nary step of the analytical process.In the present research,a dynamic microwave-assisted ex-traction method has been developed and compared with the recommended EPA method3060[21].A commercial fo-cused microwave device(Soxwave-100)has been employed. The use of a dynamic extraction system[20]facilitates the automation of the whole analytical process.Thus,a fully automated approach in which aﬂow injection(FI)manifold is used as interface for the coupling of a microwave-assisted extractor with a photometric detector is proposed.The inclu-sion of a minicolumn packed with a strong anion-exchange (SAE)resin in the FI system allowed preconcentration of the Cr(VI)extracted before detection,thus allowing the analysis of samples with low levels of the target analyte.2.Experimental2.1.Instruments and apparatusMicrowave extraction was performed with a Soxwave-100focused microwave digestor(Prolabo,Fontenay-sous-Bois,France)with a maximum irradiation power of300W.A TX32device(Prolabo)was used for the control of the mi-crowave unit.A Gilson Minipuls-3low-pressure peristaltic pump(Gilson,Worthington,OH,USA)programmed for changing the rotation direction a preset intervals,three Rheo-dyne low-pressure selection valves(Rheodyne,Cotati,CA, USA),a laboratory-made chamber of Teﬂon(7cm×7.5mm i.d.)and Teﬂon tubing of0.8mm i.d.were used to build the leaching system.Two Gilson Minipuls-3low-pressure peristaltic pumps, a Rheodyne Model5041low-pressure injection valve,a laboratory-made minicolumn(3cm×2mm i.d.)packed with SAE resin,a laboratory-made debbubler and Teﬂon tubing of0.8mm i.d.were used to construct the two dynamic man-ifolds.One of the channel of PP2was used as PP1.A Waters model490liquid chromatographic spectropho-tometer equipped with a low-volumeﬂow-cell and Knauer x–t recorder was employed for the photometric monitoring of the Cr–DPC coloured complex at540nm.2.2.Reagents and standardsCr(VI)and Cr(III)working standard solutions were prepared from1000␮g ml−1stock standard solutions prepared by K2CrO4and Cr2O3(Merck,Darmasdt, Germany).The Cr(VI)complexing reagent solution of 1,5-diphenylcarbazide(DPC)(Panreac,Barcelona,Spain) was prepared by dissolving0.2g of DPC in40ml of ethanol, diluting to100ml with water and storing in a light-excluding bottle.Ammonia solution and ammonium sulfate(Panreac) were used for preparation of0.04and0.5M ammonium buffer(pH8)solution used as extractant and eluent,respec-tively.Ultrapure water from a Milli-Q system(Millipore, Canada,USA)was used throughout.All reagents were of analytical reagent grade.An SAE resin(Dowex1-X8-400; Sigma–Aldrich,Steinheim,Germany)was used for precon-centration of Cr(VI).The resin was supplied in the chloride form and was cleaned prior to use by slurring it with3M HCl,allowing it to stand for10min and then decanting off the acid.This procedure was repeated three times.After pouring off the last portion of the cleaning acid,the resin was slurried with1M HCl and dried prior to use.2.3.SamplesTwo hundred andﬁfty grams of river sediment as ma-trix,spiked with Cr(VI)to obtain aﬁnal concentration of 50␮g g−1,was used to carry out the optimisation study.A 250g portion of river sediment was spiked with25␮g g−1 of Cr(VI)and other250g portion with5␮g g−1of Cr(VI). These spiked levels were selected in order to obtain sedi-ments with environmentally representative concentrations. Six aliquots of50g were spiked with Cr(VI)to obtain aﬁnal concentration in the sediment of50␮g g−1in all them and with Cr(III)to aﬁnal concentration of:5,10, 15,20,25and30␮g g−1from theﬁrst to the last aliquot. The sediment thus prepared was aged for three months in order to simulate the matrix–analyte interaction in natural samples.Two types of natural contaminated soil(namely, clayey and slimy)were selected for validate the pro-posed method by comparison with the EPA method3060 [21].2.4.Procedures2.4.1.EPA method3060/71962.4.1.1.Leaching step.A50ml volume of extractant(a solution consisting of a mixture of0.5M NaOH and0.28M Na2CO3at pH11.5)and2.5g of sediment sample were poured into a beaker and heated to90–95◦C on a hot-plate during1h.The cooled extract(pH>12)wasﬁltered through a0.45␮m membraneﬁlter,and then,theﬁltrate was neutralised to pH7.5with concentrated HNO3and stored until the following step.S.Morales-Muñoz et al./Analytica Chimica Acta515(2004)343–348345 LEACHING STEP DETERMINATION STEPFig.1.Experimental set-up for development of the methods for Cr(VI)without(A)and with preconcentration step(B).LC,leacher-carrier;St,standard; PP,peristaltic pump;PPP,programmable peristaltic pump;SV,selection valve;W,waste;ME,microwave extractor;EC,extraction cell;R,refrigerant; MEC,microwave extraction controller;EX,extract;MC,mixing coil;RC,reaction coil;DB,debubbler;D,detector;E,eluent;IV,injection valve;SAEC, strong anion-exchange column.2.4.1.2.Determination step.A proper amount of the neu-tralisedﬁltrate and1ml of DPC solution(2mg ml−1)werepoured into a50ml calibratedﬂask and1%HCl was usedto adjust the solution to pH2,necessary for the formationof the red–violet complex,which was monitored photomet-rically at540nm.2.4.2.Proposed procedureTwo different methods were developed using the mani-folds in Fig.1,which differ in the inclusion of a preconcen-tration unit in Fig.1B in order to achieve lower determina-tion levels.2.4.2.1.Leaching step.An amount of0.2g sedimentwas placed into the sample chamber.The closed system(with a total volume of2ml)wasﬁlled with the extractant(0.04M ammonium buffer solution)impelled by peristalticpump PP1by maintaining selecting the leacher-carrier(LC)through valve SV1with the valve SV2in the closed-circuit position.The sample chamber was placed in themicrowave vessel,which contained∼=50ml of water,and irradiated at300W for a preset time depending on the sam-ple matrix.During microwave irradiation,the direction ofthe extractant(at1.2ml min−1)was changed each30s inan iterative manner,thus minimising both dilution of theextract and increased compactness of the sample in the ex-traction chamber,and avoiding overpressure in the systemas a result.In addition,a closer sample–extractant contactis achieved,thus accelerating the removal of the target an-alyte.After extraction,selection valve SV3was switched and the extract was driven either to the preconcentration system(at0.2ml min−1)or directly to the detection system (at0.25ml min−1).For introduction of the standards in the system,valve SV1selected the standard(St)channel with valve SV2in the closed-circuit position.In this way,the extraction sys-tem wasﬁlled with a given standard.An amount of0.2g of sediment without detectable level of the target analyte was placed into the extraction chamber in order to obtain a stan-dard volume equal to those of the extracts(when extraction is performed).Once the system wasﬁlled,selection valves SV1and SV2were switched.In this way,the leacher-carrier drives to the waste the standard volume between both valves (SV1and SV2).Then,selection valves SV2and SV3were switched to the closed-and open-circuit positions,respec-tively,in order to drive the standard either to the preconcen-tration or detection system.2.4.2.2.Preconcentration step.The extract from the closed system was driven(at0.2ml min−1)to a minicolumn packed with SAE resin where the analyte was retained.The minicolumn was located in the loop of an injection valve, thus allowing elution in the direction opposite to retention. Elution was carried out by passing through the minicolumn a0.5M ammonium buffer stream at0.25ml min−1.The elu-ate was driven to the spectrophotometer for determination after derivatization with DPC.2.4.2.3.Determination step.The extract or the eluate from the preconcentration step was merged with an1%HCl346S.Morales-Muñoz et al./Analytica Chimica Acta 515(2004)343–348stream and was driven to a mixing coil (1m ×0.8mm i.d.).Then,the resulting stream was merged with 2mg ml −1DPC solution and driven to a reaction coil (1.5m ×0.8mm i.d.)in order to form the coloured complex.Both the HCl and DPC streams were pumped at 0.25ml min −1.Photometric detection was performed at 540nm.A debubbler was con-nected in-line to remove occluded air in the solid,which could give rise to parasitic signals at the detector.3.Results and discussionThe order used for optimising the steps involved in the overall method was as follows:ﬁrst,the formation of the coloured complex was optimised for checking the other pre-vious steps;then,the variables affecting the leaching step and,ﬁnally,the preconcentration step,which is an optional step that provides a more sensitive alternative for the anal-ysis of soils with low levels of Cr(VI)(below 1.2␮g g −1).3.1.Optimisation of the coloured complex formation A multifactorial design methodology was used for the op-timisation of the main factors affecting the formation of the red–violet complex (namely,the ﬂow rates and the concen-tration of the HCl and DPC solutions and the ﬂow rate of the sample).The optimisation procedure and the results ob-tained were similar to those of Ref.[13].The ranges assessed and optimum values found are shown in Table 1.3.2.Optimisation of the continuous microwave-assisted extraction procedureThe variables optimised in the leaching step were the irradiation power,the irradiation time,the extractant ﬂow rate in the closed circuit during microwave irradiation and the time interval between successive changes of the ﬂow direction of the extractant.A 0.04M ammonium buffer solution was selected as extractant as it had proved to be efﬁcient.The volume of extractant (2ml)correspondedTable 1Ranges and optimum values for the variables affecting the different steps Step VariableTested range Optimum valueDetectionExtract ﬂow rate (ml min −1)0.25–1.750.25DPC ﬂow rate (ml min −1)0.25–1.750.25Acid ﬂow rate (ml min −1)0.25–1.750.25DPC concentration (mg ml −1)2–82Acid concentration (%,v/v)1–251LeachingIrradiation power (%)40–100100Irradiation time (min)2–16See textExtractant ﬂow rate (ml min −1)0.2–1.2 1.2PreconcentrationRetention ﬂow rate (ml min −1)0.1–10.2Elution ﬂow rate (ml min −1)–0.25Breakthrough volume (ml)–>5with the capacity of the closed circuit and was kept constant.A univariate approach was used for the optimisation of the delay time –time interval between successive changes of the ﬂow direction after the total volume of extractant had circulated once through the sample cell.The optimisation was developed under an irradiation power of 150W,a ﬂow rate of the extractant of 0.5ml min −1and 5min of irradi-ation time.After testing times of 10,20,30and 40s,the highest efﬁciency was achieved using a delay time of 20s for changing the ﬂow direction.Blockage of the sample cell was observed for longer times.The irradiation power,the irradiation time and the ex-tractant ﬂow rate were optimised by the experimental de-sign methodology,as they were presumably interrelated (Table 1).A full two-level factorial design involving an overall of 23=8experiments plus three centred points was built for a screening study of the behaviour of the main factors affecting the extraction process [22].The up-per and lower values given to each factor were selected from the available data and the experience gathered in the preliminary experiments.The conclusions of the study were that the extractant ﬂow rate was not an inﬂuential factor in the range under study.However,the results showed better recoveries with the highest value tested.Thus,the optimum ﬂow rate was 1.2ml min −1.The irradiation time and the irradiation power were the key factors with a positive effect on the extraction efﬁciency.Higher values should be tested;however,con-cerning the irradiation power,the upper value of the design was the maximum power provided by the extractor used (300W)which yielded the best value.3.3.Kinetics studyTo determine the optimum extraction time for total re-moval of Cr(VI)as a function of the sample matrix,a study of the extraction kinetics was performed for spiked river sediments and natural contaminated soils.The other extrac-tion variables were ﬁxed at their optimum values.As can beS.Morales-Muñoz et al./Analytica Chimica Acta 515(2004)343–34834702468101224681012141618Extraction time (min)C r V I e x t r a c t e dg )Fig.2.Kinetics study of the extraction of Cr(VI)from different matrices.seen in Fig.2,total removal of Cr(VI)was obtained after 10min for the river sediment spiked at 50␮g g −1and 8min for the sediment samples spiked at 25and 5␮g g −1,while 12and 14min were necessary for slimy and clayey natural contaminated soils,respectively.3.4.Optimisation of the preconcentration stepIn order to obtain a more sensitive method,a preconcen-tration minicolumn was included in the FI manifold.An SAE resin and 0.5M ammonium buffer were selected as sorbent material and eluent,respectively,based on the good results reported in the literature for the isolation of Cr(VI)from Cr(III)and other cations [3,12].The elution ﬂow rate was not optimised as preconcentration was per-formed in the same manifold as detection and an extract ﬂow rate of 0.25ml min −1was previously optimised for a good development of the derivatization reaction.The reten-tion ﬂow rate was optimised in the range 0.1–1ml min −1and the results were that the recovery increased when the ﬂow rate decreased from 1to 0.2ml min −1and levelled off for lower values,so a ﬂow rate of 0.2ml min −1was selected for further experiments (Table 1).Samples volumes between 1and 5ml,which contained 5␮g g −1of Cr(VI),were passed through the minicolumn.The signal remained constant up to 5ml,so the breakthrough volume should be higher than 5ml.Table 2Recoveries obtained for Cr(VI)in the presence of Cr(III)Sample Cr(VI)added (␮g g −1)Cr(III)added (␮g g −1)Cr(VI)recoveries (%)R.S.D.a (%)150093.1 2.6250592.8 3.03501094.1 3.545015101.80.355020107.80.265025110.40.175030112.60.6aRelative standard deviation (n =3).3.5.Determination of Cr(VI)in the presence of Cr(III)In order to evaluate the feasibility of the proposed ap-proach for the extraction of Cr(VI)in the presence of Cr(III)without disturbing the species distribution,several sediment samples containing the same amount of Cr(VI)(50␮g g −1)and variable amounts of Cr(III)(from 5to 30␮g g −1)were subjected to the extraction process.As can be seen in Table 2,the proposed approach does not disturb the species distribu-tion at Cr(VI)/Cr(III)ratios higher than 2:1.3.6.Features of the methodCalibration curves were obtained by using a linear plot of the peak area as a function of the standard con-centration of Cr(VI).The linear dynamic ranges for the two proposed methods were 1.5–7.5␮g ml −1—with cor-relation coefﬁcients (r 2)of 0.9894—for method A;and 0.5–6.5␮g ml −1—with r 2=0.9954—when the preconcen-tration step was included (method B).The detection limits were 0.12␮g ml −1and 1.2␮g g −1,for the extract and soil,respectively,in the case of method A,and 0.012␮g ml −1and 0.12␮g g −1for method B.The precision of the methods,expressed as within-laboratory reproducibility and repeatability,was studied in a single experimental set-up with duplicated for each method [23].The experiments were carried out using 0.2g348S.Morales-Muñoz et al./Analytica Chimica Acta515(2004)343–348Table3Comparison of the proposed methods with the reference EPA method3060Sample Method A a Method B a EPA method a Natural contaminated clayey soil7.32±0.167.15±0.257.40±5.71 Natural contaminated slimy soil 6.91±0.217.00±0.39 6.81±2.16a Amount of Cr(VI)extracted expressed as mean(␮g)±relative standard deviation(n=3).of spiked sediment containing50␮g g−1for the method without preconcentration and5␮g g−1for the method with preconcentration,under the optimum working conditions. The within-laboratory reproducibility and repeatability were2.6and1.9for method A,and4.0and2.6for method B.parison of the proposed methods with the EPA method3060The optimised proposed methods were validated by com-parison with the EPA method3060in terms of efﬁciency and precision.The recoveries,expressed as an average of three extractions,were similar for the three methods(Table3). However,the precision of the proposed methods was better than that obtained by the EPA method.4.ConclusionsThe automated approach based on the coupling of a microwave-assisted extractor with or without a precon-centration unit prior to a photometric detector allows the quantitative determination of Cr(VI)in spiked and natural sediment and soil samples.The dynamic microwave-assisted extraction based on the use of a closed extraction system with iterative change of theﬂow direction of the extractant during extraction overcomes the compactness of the sample that could cause overpressure of the system and favours a closer sample–extractant contact,thus achieving an efﬁcient extrac-tion of Cr(VI)without disturbing the original species distri-bution.The proposed approach also allows obtaining results similar to those provided by the reference procedure,but in a shorter time(the extraction was performed in10–14min versus1h for the reference method)and with better precision.AcknowledgementsSpain’s Comisión Interministerial de Ciencia y Tec-nolog´ıa(CICyT)is gratefully acknowledged forﬁnancial support(project BQU-2003-01333).References[1]A.Kortenkamp,M.Casadevall,S.P.Faux,A.Jenner,R.O.J.Shayer,N.Woodbridge,P.O’Brien,Arch.Biochem.Biophys.329(1996) 199.[2]S.L.Brauer,K.E.Wetterhahm,J.Am.Chem.Soc.113(1991)3001.[3]J.Wang,K.Ashley, E.R.Kennedy, C.Neumeister,Analyst122(1997)1307.[4]M.J.Marques,A.Salvador,A.Morales-Rubio,M.de la Guardia,Fresenius J.Anal.Chem.367(2000)601.[5]N.K.Hamadi,X.D.Chen,M.M.Farid,M.G.Q.Lu,Chem.Eng.84(2001)95.[6]Y.B.Martiúnez,N.A.F.Roig,F.J.López,F.Hernández,J.Chro-matogr.A926(2001)265.[7]M.J.Marques,A.Salvador,A.Morales-Rubio,M.de la Guardia,Fresenius J.Anal.Chem.362(1998)239.[8]W.Gurknecht,R.Handy,L.Michael,E.Pellizari,L.Sheldon,M.Umana,D.Whitaker,Evaluation of methods for determining hexava-lent chromium in extracts of industrial wastes,Contract No.68-03-3099,US Environmental Monitoring and Support Laboratory,1983.[9]US Environmental Protection Agency(EPA),Physical/ChemicalMethods,3rd ed.,Method7196,Test Methods for Evaluating Solid Waste,US Government Printing Ofﬁce,Washington,DC,1990. [10]J.Wang,K.Ashley,D.Marlow,E.C.England,G.Carlton,Anal.Chem.71(1999)1027.[11]K.Ndung’u,N.K.Djane,F.Malcus,L.Mathiasson,Analyst124(1999)1367.[12]G.P.Foy,G.E.Pacey,Talanta51(2000)339.[13]J.L.Luque-Garc´ıa,M.D.Luque de Castro,Analyst127(2002)1115.[14]H.M.kingston,L.B.Jassie,Introduction to Microwave Sample Prepa-ration,ACS Professional Reference Book,American Chemical So-ciety,Washington,DC,1998.[15]F.E.Smith,E.A.Arsnault,Talanta43(1996)1207.[16]E.S.Beary,P.J.Paulsen,L.B.Jassie,J.D.Fassett,Anal.Chem.69(1997)758.[17]T.Duelcks,LaborPraxis20(1996)68.[18]D.Lofty,Lab-Equip-Dig30(1992)13.[19]M.C.Herrera,M.D.Luque de Castro,J.Anal.Atom.Spectrom.17(2002)1530.[20]A.Rios,M.D.Luque de Castro,M.Valcárcel,M.Grasserbauer,Int.J.Environ.Anal.Chem.66(1997)285.[21]US Environmental Protection Agency(EPA),Test Methods for Eval-uating Solid Waste,Method3060A,Alkaline Digestion for Hexava-lent Chromium,US Government Printing Ofﬁce,Washington,DC, 1996.[22]Statgraphics Plus for Windows,Version2.2,Rockville,MD,USA,1992.[23]D.L.Massart,B.G.M.Vanderginste,L.M.C.Buydens,S.De Jong,P.J.Lewi,J.Smeyers-Verbeke,Handbook of Chemometrics and Quali-metrics,Part A,Elsevier,Amsterdam,1997.。

A COMPARISON BETWEEN RECURRENT NEURAL NETWORK ARCHITECTURES FOR DIGITAL EQUALIZATION

ABSTRACT
sical RNN architectures for a digital equalization task. Section 2 describes the network architectures used. Section 3 describes the parameters of the simulations and presents the results. Finally, the conclusions are given in Section 4. We have chosen three classical rst-order recurrent neural architectures: a simple, fully recurrent NN used by Kechriotis et al. 6], a partially recurrent neural network used by Robinson and Fallside 7] for speech recognition purposes, and an augmented recurrent architecture having a layer mapping states to outputs, used by Elman 4] to study temporal sequences. All the architectures are single-input, single-output (SISO) and have N hidden state units. The input, the state of the i-th hidden unit, the network output, and the desired output at time t are denoted by u t], x t], y t], and d t] respectively. The state vector will be denoted by x t]. The equations describing these architectures are:

uNGAL联合尿NAG对危重症患者急性肾损伤病情及预后的评估价值

临床研究uNGAL联合尿NAG对危重症患者急性肾损伤病情及预后的评估价值张艳，靳衡，曹超，寿松涛△摘要：目的探讨尿中性粒细胞明胶酶相关脂质运载蛋白（uNGAL）联合尿N-乙酰-β-D-氨基葡萄糖苷酶（NAG）对危重症患者急性肾损伤（AKI）病情及预后的评估价值。

方法选取危重症合并AKI的患者101例（AKI组）和同期收治的非AKI患者27例（对照组）。

比较2组患者入院后24h内血肌酐、血钾、血白蛋白和阴离子间隙（AG）、uNGAL、尿NAG、尿蛋白、尿微量白蛋白的差异。

AKI组患者根据肾功能损伤严重程度分为Stage1组（40例），Stage2组（36例）和Stage3组（25例），另根据患者出院时存活情况，分为存活组（63例）和死亡组（38例）。

比较不同严重程度及不同生存状态间上述指标的差异。

应用Logistic回归分析危重症患者AKI预后的独立影响因素，受试者工作特征（ROC）曲线评价uNGAL联合尿NAG对危重症患者AKI预后的评估价值。

结果（1）与非AKI组相比，AKI组uNGAL、尿NAG、尿蛋白、尿微量白蛋白、血肌酐水平升高，血清白蛋白水平降低（P＜0.01）。

亚组分析结果显示，随着病情的加重，AKI组uNGAL、尿NAG和血肌酐水平均出现明显升高（P＜0.05）；同时死亡组uNGAL、尿NAG、尿微量白蛋白、尿蛋白及血肌酐高于存活组，血清白蛋白低于存活组（P＜0.05）。

Logistic回归分析显示，uNGAL和尿NAG 升高是影响患者预后的独立危险因素，ROC曲线显示uNGAL联合尿NAG预测预后的曲线下面积（0.886）优于uNGAL （0.850）、尿NAG（0.784）。

结论uNGAL联合尿NAG检测有助于对危重症患者急性肾损伤的危险分层和预后评估。

关键词：危重病；急性肾损伤；死亡；尿中性粒细胞明胶酶相关脂质运载蛋白；尿N-乙酰-β-D-氨基葡萄糖苷酶中图分类号：R692.5文献标志码：A DOI：10.11958/20202746The value of uNGAL combined with urinary NAG in the evaluating the severity and prognosis ofcritically ill patients with acute kidney injuryZHANG Yan,JIN Heng,CAO Chao,SHOU Song-tao△Department of Emergency Medicine,Tianjin Medical University General Hospital,Tianjin300052,China△Corresponding Author E-mail:***************.cnAbstract:Objective To assess the role of urinary neutrophil gelatinase-associated lipocalin(uNGAL)and urinary N-acetyl-beta-D glucosaminidase(NAG)in risk stratification and prognosis for critically ill patients with acute kidney injury (AKI).Methods A total of101critically ill patients with AKI were included in the AKI group,and another27critically ill patients without AKI during the same period were included as the control group.Clinical and experimental indicators, including serum creatinine,serum potassium,serum albumin,anion gap,uNGAL,urinary NAG,urinary protein and urinary microalbuminuria were compared between the two groups.Based on the severity of kidney injury,patients were divided into Stage1group(n=40),Stage2group(n=36)and Stage3group(n=25).Besides,regarding the survival status of discharge，patients were divided into survival group(n=63)and death group(n=38).The indicators we collected were compared with different severity and living state.Logistic analysis was used to find out the independent risk factors affecting the prognosis of critically ill patients with AKI.The receiver operating characteristics(ROC)curve was used to evaluate the predictive ability of uNGAL,urinary NAG and uNGAL combined with urinary NAG in critically ill patients with AKI.Results Compared with control group,the levels of uNGAL,urinary NAG,urinary protein,urinary microalbuminuria and serum creatinine increased and the level of serum albumin decreased in AKI group(P＜0.01).The subgroup analysis indicated that the levels of uNGAL and urinary NAG increased with the severity of kidney injury in critically ill patients(P＜0.05).At the same time, compared with the survival group，the levels of uNGAL，urinary NAG，urinary microalbumin urinary protein and serum creatinine were significantly higher and the level of serum albumin was lower in death group(P＜0.05).Logistic regression 基金项目：国家自然科学基金面上资助项目（82072222）；国家自然科学基金青年资助项目（81902007）；中央高校基本科研业务费专项资金（3332019127）作者单位：天津医科大学总医院急诊医学科（邮编300052）作者简介：张艳（1988），女，硕士，主治医师，主要从事急性肾损伤的基础及临床研究。

小鼠肝Kupffer细胞分离方法探讨

小鼠肝Kupffer细胞分离方法探讨【摘要】目的探讨分离BALB/c小鼠肝Kupffer细胞(KC)的方法。

方法采用在体酶灌注和离体酶消化、不连续密度梯度离心、选择性贴壁三步法分离KC，并比较链霉蛋白酶、Ⅳ型胶原酶及联用链霉蛋白酶和Ⅳ型胶原酶等3种不同酶消化分离方法所得KC得率及纯度。

结果 3种不同酶消化分离方法细胞得率分别为(6.32±0.5)×106 g-1，(3.66±0.4)×106g-1，(10.3±0.7)×106 g-1；细胞纯度分别为(93.2±1.7)％，(90.7±1.5)％，(94.5±1.9)％。

结论联合链霉蛋白酶和Ⅳ型胶原酶在体灌注和离体消化是分离小鼠KC的较好方法。

【关键词】肝；枯否细胞；离心法，梯密度；小鼠，近交BABLc ；细胞分离肝Kupffer细胞（Kupffer cell，KC）为定居在肝窦内的巨噬细胞，约占全身单核巨噬细胞总数的80％～90％。

肝KC能吞噬、杀灭病原微生物，清除体内的内毒素，并具有抗原递呈、分泌细胞因子等免疫调节作用，同时影响肝细胞、贮脂细胞及内皮细胞的生物学功能[1]。

近期发现KC能诱导同种异体T淋巴细胞凋亡，在调节肝移植免疫耐受中发挥重要作用[2]。

如何获得较多数量和较高纯度的KC是研究其在机体中作用机制的首要条件。

而传统的分离方法往往因数量和纯度不足而影响实验结果。

本试验采用在体酶灌注和离体酶消化、不连续密度梯度、选择性贴壁三步法分离KC，探讨分离小鼠肝KC的较好方法。

1 材料与方法1.1 材料1.1.1实验动物 BABL/c小鼠，雄性，10～12周龄，清洁级，由四川大学华西医学中心试验动物中心提供（许可证号：046）。

试验前禁食12 h，自由饮水，随机分为3组。

1.1.2 主要试剂和仪器Ⅳ型胶原蛋白酶、DNAaseⅠ、Percoll及HEPES （美国Sigma公司）；兔抗人溶菌酶（lysozyme，美国DAKO公司）；链霉蛋白酶E（瑞士Roche公司）；RPMI��1640培养基（美国Gibco公司）。

高效液相色谱法-综述

Journal of Chromatography A,1217(2010)858–880Contents lists available at ScienceDirectJournal of ChromatographyAj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o maReviewThe challenges of the analysis of basic compounds by high performance liquid chromatography:Some possible approaches for improved separationsDavid V.McCalley ∗Centre for Research in Biomedicine,University of the West of England,Frenchay,Bristol BS161QY,UKa r t i c l e i n f o Article history:Available online 3December 2009Keywords:HPLCBasic compounds Stationary phases Reversed-phase HILICa b s t r a c tThis review considers some of the difﬁculties encountered with the analysis of ionised bases using reversed-phase chromatography,such as detrimental interaction with column silanol groups,and over-loading which both lead to poor peak shapes.Methods of overcoming these problems in reversed-phase (RP)separations,by judicious selection of the column and mobile phase conditions,are discussed.Hydrophilic interaction chromatography is considered as an alternative method for the separation of some basic compounds.©2009Elsevier B.V.All rights reserved.Contents 1.Introduction.........................................................................................................................................8592.Choice of column....................................................................................................................................8592.1.Column testing procedures..................................................................................................................8592.2.The Tanaka test and the Snyder hydrophobic subtraction parison of results with direct peak shape measurements...8602.3.Monolithic silica columns ...................................................................................................................8632.4.Slow column equilibration.Anion-exchange behaviour of alkylsilica RP columns e of column materials other than silica...................................................................................................8653.Choice of mobile phase..............................................................................................................................8663.1.Choice of modiﬁer ...........................................................................................................................8663.2.Choice of mobile phase pH.Problem of reduced retention of bases at low pH.............................................................8664.Overloading .........................................................................................................................................8674.1.Overview of the problem....................................................................................................................8674.2.Possible causes of overloading ..............................................................................................................8684.3.Effect of buffer anion on overload...........................................................................................................8704.4.Overloading on mixed-mode reversed-phase/cation-exchange columns..................................................................8714.5.Effect of buffer pH on overloading ..........................................................................................................8715.Temperature effects.................................................................................................................................8736.Hydrophilic interaction chromatography (HILIC)..................................................................................................8747.Concluding remarks.................................................................................................................................8787.1.Overloading..................................................................................................................................8787.2.Selection of mobile phase pH................................................................................................................8787.3.Quality and choice of column ...............................................................................................................8797.4.Temperature.................................................................................................................................8797.5.Alternative separation mechanisms—e.g.HILIC.............................................................................................879References...........................................................................................................................................879DOI of original article:10.1016/j.chroma.2009.11.067.∗Tel.:+441173282469;fax:+441173282904.E-mail address:david.mccalley@0021-9673/$–see front matter ©2009Elsevier B.V.All rights reserved.doi:10.1016/j.chroma.2009.11.068D.V.McCalley/J.Chromatogr.A1217(2010)858–8808591.IntroductionThe analysis of basic compounds by high performance liquid chromatography(HPLC)continues to be of interest,as over70% of pharmaceuticals are bases(with about20%being acids)[1–3].A large number of compounds of biomedical and biological signiﬁ-cance are also bases.Reversed-phase(RP)separations are by far the most common in liquid chromatography(LC),due to advantages that include ease of use with gradient elution,compatibility with aqueous samples,versatility of the retention mechanism allowing changes in the separation to be brought about by changes in pH, organic modiﬁer or additives,and long experience with the tech-nique,allowing the rapid establishment of suitable experimental conditions for the analysis of a given sample[4].Nevertheless,it has been recognised for a long time that the analysis of basic com-pounds poses particular difﬁculties in RP separations.Many of these problems are associated with the complex structure of the surface in silica-based RP packings,shown in Fig.1.The surface concentra-tion of silanols on bare silica is reported to be about8.0␮mol m−2 [5].C18ligands are too bulky to react completely with all silanols; thus,a maximum coverage of4–4.5␮mol m−2can be achieved.A further number of reactive silanols can be“endcapped”by reac-tion with smaller silylating agents such as trimethylchlorosilane, but as many as50%of the original silanol groups remain unreacted on a typical RP column.The average p K a of these silanol groups is around7.1,but their acidity can be enhanced by the presence of metal impurities in the silica.Some groups appear to be sufﬁ-ciently acidic that their ionisation cannot be entirely suppressed using acidic mobile phases with a pH within the stability limit of typical RP columns(2.5–7.5).Over this range of operational pH values,basic compounds are likely to be ionised,leading to ionic interactions with ionised silanol groups.BH++SiO−M+→SiO−BH++M+(1) where BH+represents the protonated base,and M+the mobile phase buffer cation.The problem of poor column efﬁciency(N)and exponentially tailing peaks shown by small quantities of bases is often attributed to this mixed mechanism process of hydropho-bic interaction and ion-exchange with the silanols.The slower sorption–desorption kinetics of silanol ion-exchange sites(kinetic tailing)with sample ions may be responsible[6],which will occur regardless of sample size.The simple existence of two retention processes cannot per se be the sole cause of tailing,as mixed-mode phases with carboxylic acid functions embedded within a hydrophobic chain can show excellent peak symmetry for bases[7]. However,the kinetics of interaction of such embedded groups,and the stereochemistry around the active site,could be completely dif-ferent from that of ionised silanols,which may be buried beneath the hydrophobic chains on classical C18phases.Instead of sim-ple ion-exchange sites,Neue et al.[8]have proposed the existence of strong synergistic sites with combined RP and ion-exchange properties.The overall retention for bases was described by the equation:k=k RP+k IX+k∗RP k∗IX(2) where k is the total retention,k RP is the hydrophobic contribu-tion,k IX is the ion-exchange contribution from surface silanols,and k∗RP k∗IX is a multiplicative contribution of both processes.These syn-ergistic sites could correspond to the subset of very high-energy sites with slow kinetics which have been long suspected to be the cause of exponential tailing for bases,as they appear to be domi-nant in the retention process.It was shown that this type of tailing is not responsive to small changes in sample load in RP–LC at low pH[6].This result might indicate that exponential tailing is not caused by overload of a small number of strong sites on the column. In contrast,overload often gives rise to right-angled triangle peak shapes when ionisation of silanols is suppressed in RP–LC when working at low pH.Overload tailing still occurs even for the most modern columns operated under conditions where there are no or a negligible number of ionised silanols on the column surface.It was recognised more than20years ago that bonded phases synthesised from pure silica(Type B phases)made from the hydrol-ysis of metal-free tetraalkoxysilanes resulted in reduced silanol acidity,and their use has considerably improved the analysis of bases[9].Only small contamination of such materials occurs dur-ing the processing of such packings,or from the water used in the hydrolysis.Nevertheless,some other features of the analysis of these solutes(such as overloading)remain problematic,and these issues have not been resolved by the use of high-purity silica.Already in1988,Snyder and co-workers[10]had reviewed the problems of analysis of basic solutes and had proposed some pos-sible solutions.The following recommendations were made: (a)Judicious selection of the column to reduce the number of avail-able acidic sites.(b)Reduction of the mobile phase pH to suppress ionisation of thesilanols.(c)Increasing the mobile phase pH above the analyte p K a,such thatthe analyte is unprotonated.(d)Addition of a silanol blocker such as triethylamine to the mobilephase to interact preferentially with ionised silanols.(e)Reduction of the sample concentration to alleviate the satura-tion of the acidic sites.Most of the arguments in this paper remain true more than20 years later,and these conclusions can be used as a simple guide for the chromatographer aiming to achieve the best separations for basic solutes.Perhaps only the use of silanol blocking agents has fallen somewhat out of favour,as these are less necessary with modern high-purity silica phases,and can also have some undesirable effects.Such effects include the generation of addi-tional background in HPLC–MS,the difﬁculty of removal from the stationary phase after use leading to permanent alteration of its properties,and even chemical reaction with some solute types.This topic,and some other well-known aspects of the chromatography of bases have been covered adequately in earlier reviews[11–13]. However,other features of the chromatography of these“difﬁcult”compounds are still extensively debated in the literature,for exam-ple,the problem of their ready overloading in RP separations.This review will concentrate on the latest research in these topics,while attempting to summarise brieﬂy previousﬁndings.Thus,it will con-sider RP column choice by use of evaluation data obtained from the Tanaka and the Snyder“hydrophobic subtraction”tests;current theories and the effect of overload for ionised solutes;the use of high pH to improve peak shape;whether temperature is a useful parameter in improving peak shape;andﬁnally whether other sep-aration mechanisms such as HILIC can provide a viable alternative to RP–LC for the analysis of bases.2.Choice of column2.1.Column testing proceduresThe selection of an appropriate RP column for the analysis of bases can be a daunting task,as now many hundreds are com-mercially available,with a considerable number recommended especially by their manufacturers for the analysis of basic solutes. Nevertheless,several databases are now available where a large number of different columns have been subjected to the same test procedure by the same group of workers on the same or similar instruments,allowing a useful and objective comparison of perfor-860 D.V.McCalley /J.Chromatogr.A 1217(2010)858–880Fig.1.Structures present on a typical RP monomeric-bonded silica (C8)endcapped with trimethylsilyl groups.After U.D.Neue,“Silica Gel and its derivatization for Liquid Chromatography”,in “Encyclopedia of Analytical Chemistry”,R.A.Meyers,Ed.,John Wiley &Sons,Ltd.,Chichester (2000)11450–11472.mance to be made.A question arises as to the validity of databases constructed by evaluation of only a few or even a single column of a given type,as to whether the results obtained may be truly repre-sentative of the performance of this brand,due to column to column and batch to batch variations.However,a careful study [14,15]has suggested that columns from major manufacturers actually show a rather high degree of reproducibility,probably resulting from the use of stringent quality control procedures.Indeed,the industry is likely to be self-regulating to a degree,as dissatisﬁed customers would switch to the use of more reproducible brands.Tight reten-tion speciﬁcations exist in the HPLC user environment,especially in the pharmaceutical industry,and changes in the column can jeop-ardise product release.However,it is possible that a manufacturer could be forced to change the sourcing of a production raw mate-rial,which might occur for example,if the column manufacturer does not make their own silica.Thus,under some circumstances,a recently purchased column may not behave in the same way as one tested several years beforehand.Nevertheless,we believe that such situations are rare,and in most cases,manufacturers strive to main-tain the reproducibility of their products over a long period of time,as many customers have established methods on a given brand of phase.It appears more common to introduce a new name or name variant of an existing phase to mark deﬁnitively such changes or improvements to the production process.Taking this factor,and the reasonable reproducibility of commercial columns into account,it seems that the results of tests on a particular brand of column would generally reﬂect the performance of that brand throughout the product lifetime.Both of the column evaluation methods described in detail below incorporate strongly basic compounds as test probes.In each test,their retention is monitored at low and intermediate pH val-ues.Columns which give relatively low retention of basic probes are also likely to give higher efﬁciency for basic solutes,as shown by correlation studies for at least one of the procedures (see below).2.2.The Tanaka test and the Snyder hydrophobic subtraction parison of results with direct peak shape measurementsWhile many different column testing methods have been devel-oped,two have become prominent and have the distinct advantage that databases of results for many hundreds,rather than just a few columns,are available.The Tanaka method [16]and the hydropho-bic subtraction procedure developed by Snyder et al.[17]both incorporate tests which allow a user to select phases that are likely to be suitable for the separation of basic compounds.We will consider here the Tanaka method as adapted and applied by Euerby and Petersson [18]to the evaluation of over 200commercial columns that can be compared on a freely available program from Advanced Chemistry Development [19].These databases appear to be updated periodically;for instance,the ACD database contains evaluations of recently introduced sub-2␮m phases.An alternative adaptation of the Tanaka procedure and its application to a large number of different stationary phases has also been made [20],and data are again freely available [21].A fourth testing scheme is that published by the US Pharmacopeia.This protocol is an adaptation of the work of Sander and Wise [22].For activity towards bases,this method uses the tailing factor of amitriptyline (the same probe as used in the Snyder–Dolan procedure).At the time of writing,the database contained fewer columns than the two major proce-dures (∼100)and will not be considered further here.However,data for both this procedure and the Snyder–Dolan (S–D)method are available on the USP website [23].In the Tanaka–Euerby (T–E)procedure,columns are tested by measurement of k for pentylbenzene as a measure of sur-face area and surface coverage;hydrophobic selectivity from the ratio of k (pentylbenzene)/k (butylbenzene);shape selectivity from k (triphenylene)/k (o-terphenyl);hydrogen bonding capac-ity from k (caffeine)/k (phenol)in unbuffered methanol–water;total ion-exchange capacity from k (benzylamine)/k (phenol in methanol–phosphate buffer pH 7.6;and acidic ion-exchange capacity from k (benzylamine)/k (phenol)in methanol–phosphate buffer pH 2.7.The latter three tests are of particular interest for the analysis of basic solutes.The program [19]allows the comparison of the similarities and differences between various columns,and per-mits the separate weighting of the various factors—for example,columns can be ranked according solely to their total ion capacity at pH 7.6if so desired.The S–D model recognises that hydrophobic retention is the dominant process in RP chromatography,and in the absence of other retention mechanisms,plots of log k for one column versus another should be a straight line.However,these other mechanisms give rise to scatter in the plots.Clearly,ion-exchange and hydrogen bonding are important contributors to the retention of basic solutes.The general equation for retention in theD.V.McCalley/J.Chromatogr.A1217(2010)858–880861Table1Evaluation of some selected RP columns by two different procedures.For details on the procedure,see text.Column name k pentylbz k(pentbz)/k(butbz)k(triphen)/k(terph)k(caff)/k(phen)k(bzm)/k(phen)2.7k(bzm)/k(phen)7.6Tanaka–Euerby procedureChromolith 4.22 1.24 1.310.480.120.63Discovery Amide 1.65 1.35 1.810.490.190.44Discovery C18 3.32 1.48 1.510.390.100.28Inertsil ODS-37.74 1.45 1.290.480.010.29Resolve C18 2.40 1.46 1.59 1.29 1.23 4.06Spherisorb ODS-2 3.00 1.51 1.560.590.230.76Symmetry C18 6.51 1.46 1.490.410.010.68Symmetry Shield RP18 4.66 1.41 2.220.270.040.20Xterra MS C18 3.52 1.42 1.260.420.100.35Xterra RP18 2.38 1.29 1.830.330.070.20H S A B C(2.8)C(7.0)Snyder procedureChromolith 1.0030.0290.008−0.0140.1030.187 Discovery Amide0.7200.013−0.6250.218−0.092−0.025 Discovery C180.9840.027−0.1280.0040.1760.153 Inertsil ODS-30.9900.022−0.146−0.023−0.474−0.334 Resolve C180.968−0.1270.335−0.046 1.921 2.144 Spherisorb ODS-20.962−0.0760.070.0340.908 1.263 Symmetry C18 1.0520.0630.018−0.021−0.3020.123 Symmetry Shield RP180.8500.027−0.4110.093−0.7280.136 Xterra MS C180.9840.012−0.143−0.0150.1330.051 Xterra RP180.757−0.043−0.4830.097−0.170−0.173model is:log˛=log k/log k(ethylbenzene)=Á Hhydrophobic − S∗steric resistance(to bulky interactions)+ˇ Acolumn H-bond acidity(non-ionised silanols)+˛ BH-bond basicity(from sorbed water)+Ä Cion interaction(ionised silanols)(3)Ethylbenzene is used as a non-polar reference solute.Greek letters represent empirical,eluent-and temperature-dependent proper-ties of the solute,which are relative to the values for ethylbenzene, for which all solute parameters are identically zero.The selection of the optimum probes for evaluation of each retention mode has been made from detailed studies.Bold capitals represent eluent-and temperature-independent properties of the column;these val-ues are relative to a hypothetical average Type B C18column.Any column which behaves identically to this hypothetical reference column will have H=1and all other values S*,A,B,C=0.The dataset of columns evaluated by this procedure is even larger than that for the T–E procedure and presently extends to at least400columns.In some versions of the program,different weightings can be assigned to each evaluation parameter,as in the Euerby procedure.Results for some RP columns selected from each database are shown in Table1.The T–E data show clearly that the older Type A bonded phases(Resolve C18and Spherisorb ODS-2)give higher retention of benzylamine relative to phenol at pH7.6(alpha values 4.06and0.76,respectively)compared with newer Type B phases based on highly pure silica(Discovery C18and Inertsil ODS-3, alpha values0.28and0.29,respectively).Similarly with the S–D method,values of C(7.0)for Resolve C18and Spherisorb ODS-2 are high(2.144and1.263,respectively)compared with Discov-ery C18and Inertsil ODS-3(0.153and−0.334,respectively.Values of alpha(benzylamine/phenol)at pH2.7and values of C(2.8)are also higher for the Type A compared with the Type B phases using both procedures,indicating general agreement between them. Snyder and co-workers[24]have correlated a published dataset of“silanol activity”for a number of RP columns(measured by the average plate number for amitriptyline and pyridine with methanol-phosphate buffer pH6.0)with values of C at pH6.0,inter-polated from C(2.8)and C(7.0).Columns with a highvalue of C(6.0) correlated with columns of high silanol activity,and those with low values of C(6.0)with low silanol activity.In a later study[6]95%of Type B columns(designated either on the basis of manufacturer claims,or on the date a column wasﬁrst sold)were shown to have C(2.8)≤0.25,while only11%of Type A columns satisﬁed this crite-rion.Tailing of basic solutes(as measured by the asymmetry factor A s)was minimal for columns with C(2.8)<0.25(i.e.Type B columns) and tended to increase for larger values of C(2.8).From Table1,the Type A phases Resolve C18and Spherisob-ODS-2,now identiﬁed as such due to values of C(2.8)≥0.25,also give the highest values of hydrogen bonding acidity(parameter A,0.335and0.07,respec-tively,determined from the retention of amide probe compounds). Similarly,these phases also gave the highest relative retention of caffeine/phenol in the Tanaka procedure(1.29and0.59,respec-tively).The data can also be used to compare the effect of other features,e.g.the performance of embedded polar group phases (EPG)and the equivalent conventional C18phase,manufactured on the same silica.EPG phases include columns with embedded amide groups within the hydrocarbon chain:or carbamate groups:EPG phases have been proposed to give better peak shapes for the analysis of bases[24,27].The incorporation of an EPG in XTerra RP18reduces somewhat the Tanaka alpha(benzylamine/phenol) 7.6parameter to0.20,compared with0.35for the XTerra MS C18 column.Similarly,the S–D C(7.0)parameter is reduced to−0.173 for the EPG compared with0.051for the conventional phase.It is862 D.V.McCalley /J.Chromatogr.A 1217(2010)858–880possible that the reduced retention of benzylamine and other bases may be caused by a layer of water that is adsorbed close to the surface of EPG phases,providing some deactivating effect for the silanol groups [25,26].Other authors have compared conventional and EPG phases bonded on the same type of silica,on the basis of peak shape measurements.It was found that on average,peak shapes were indeed improved on the latter phases [27].Neverthe-less,it appears that the EPG technology gives more improvement in performance with phases bonded on older impure silicas,rather than the modern Type B silicas [27].This result seems to be reﬂected in the somewhat inconclusive data from Table 1concerning the rel-ative retention of bases on conventional and EPG phases.Thus the Discovery EPG phase (amide)has a slightly larger value of the T–E alpha (benzylamine/phenol)7.6parameter (0.44)compared with the regular C18phase (0.28).In contrast,the S–D C (7.0)parameter is smaller on Discovery Amide (−0.025)compared with Discovery C18(0.153).Similarly,while the T–E procedure indicates a con-siderable lower value of alpha (benzylamine/phenol)at pH 7.6for Symmetry Shield (0.2)compared with Symmetry C18(0.68),the S–D C (7.0)parameter for the EPG phases is slightly greater (0.136)compared with the regular phase (0.123).Euerby and Petersson pointed out that the extra retentiveness of phenols on EPG phases might invalidate the results of tests for silanophilic activity which involve the use of such solutes.They therefore suggested substitut-ing benzyl alcohol for phenol in the Tanaka test.Benzyl alcohol has retention properties similar to those of phenol but does not show excess retention on EPG phases [28].These particular comparisons point to some possible differences in the compatibility of column evaluations from either method.The Hoogmartens group looked more generally at the compati-bility of results from the S–D method and their own adaptation of the Tanaka procedure [29],ﬁnding a rather poor overall correlation between the two approaches.In a previous paper,this group had demonstrated a good correlation between their own method and the Euerby results.This latter ﬁnding is perhaps not surprising,as both are based on the Tanaka method.The problem of compatibility of the S–D and Tanaka methods may well be in the different mobile phase conditions and different probe solutes used in these tests.The S–D procedure uses the retention of the strong bases amitriptyline and nortriptyline in acetonitrile–phosphate buffer to calculate the cation-exchange term C (2.8)and derives the value of C (7.0)from the C (2.8)results by multiplying by the ratio of the retention fac-tors of the quaternary amine berberine at pH 7.0and 2.8;the T–E benzylamine tests use methanol as the organic modiﬁer.Indeed the use of these different modiﬁers may explain the somewhat differ-ent evaluations of the EPG phases by either method.Even using the same mobile phase conditions,McCalley and Brereton [27,30–32]showed that peak shape data was not consistent between different basic probes.Thus,for example there was little correlation between A s for codeine and nortriptyline when using methanol–phosphate buffers at pH 3.0,whereas either of these solutes has been used as a single test compound to evaluate the relative silanol activity of different phases.One phase (Waters Symmetry Shield)gave,of 9highly inert RP columns,the highest N and lowest A s for nico-tine using acetonitrile–phosphate buffer at pH 7.0but the lowest efﬁciency for analysis of pyridine.Fig.2shows a principal compo-nents analysis (PCA)loadings plot for analysis of nine basic solutes on eight different RP columns using a mixture of methanol with a pH 3.0buffer.Lines can be drawn from the centre of the plot to each data point.Parameters that are opposed (i.e.appear at 180◦)measure equivalent but opposite trends.Thus N and A s values are opposed,with efﬁciency increasing as asymmetry decreases,as expected.Parameters that are at 90◦,like the asymmetry factors of pyridine and quinine,measure unrelated trends,and thus may be evaluating relatively different aspects of the detrimental inter-action of bases with the column surface.Conversely,the asymmetry parameters of nortriptyline and diphenhydramine have a smaller angle between them,and may be measuring more related proper-ties.It might therefore not be necessary to include both substances in a test mix for these particular mobile phase conditions.For over-all evaluation of column properties exploring different aspects of detrimental interactions,a test mix could include ﬁve compounds:codeine,quinine,amphetamine,nortriptyline and pyridine.The ranking of columns at pH 7using methanol was different from that at pH 7using acetonitrile;note that these correspond to the differ-ent modiﬁers of the T–E and S–D evaluation schemes,respectively.Snyder and co-workers [6]also observed that the tailing of basic (cationic)solutes on a given column appeared to be solute speciﬁc,ﬁnding that values of A s for the bases amitriptyline,nortriptyline,the quaternary compound berberine,and 4-n -hexylaniline corre-lated extremely poorly (r 2=0.01–0.19).The use of multiple basic test solutes and different mobile phase modiﬁers at different pH values would be a considerable task for the construction of these column evaluation databases.However,inclusion of a range of test compounds would undoubtedly improve the performance of these databases.It seems certain that these differences in test solutes and conditions contribute to the lack of correlation between the S–D and T–Etests.Fig.2.PCA loadings plots based on retention factor (k ),column efﬁciency (N ),Dorsey–Foley column efﬁciency (N df )and asymmetry factor (A s ).Data for eight different Type B reversed-phase columns and nine different probe compounds with methanol–phosphate buffer pH 3.0as mobile phase.See [30].。

辩论技巧：比较性辩题的比较标准

辩论技巧：比较性辩题的比较标准英文回答：When it comes to debating, especially on comparative topics, it's important to have clear and consistent standards for comparison. These standards can help ensure that the debate is fair and that both sides have an equal opportunity to present their arguments.One important standard for comparison is relevance. This means that the points being compared should be directly related to the topic at hand. For example, if the debate topic is about the benefits of online learning versus traditional classroom learning, the points of comparison should focus on factors such as accessibility, engagement, and effectiveness.Another important standard is objectivity. This means that the comparison should be based on factual evidence rather than personal opinions or biases. For instance, ifthe debate topic is about the impact of social media on mental health, the comparison should be based on scientific studies and data rather than individual anecdotes or beliefs.Furthermore, it's essential to consider the context when setting comparison standards. Different topics may require different criteria for comparison. For instance, if the debate is about the best form of exercise, the standards for comparison may include factors such as cardiovascular benefits, muscle engagement, and overall enjoyment. On the other hand, if the debate is about the most effective form of government, the standards for comparison may include factors such as citizen participation, economic stability, and social equality.In addition to relevance, objectivity, and context,it's also important to consider the practicality of the standards. This means that the standards should berealistic and achievable within the scope of the debate. For example, if the debate topic is about the best travel destination, the standards for comparison should take intoaccount factors such as cost, accessibility, and cultural experiences, rather than focusing solely on luxury or exclusivity.Ultimately, the standards for comparison in a debate should be designed to promote a meaningful and constructive discussion. By establishing clear and consistent criteria, both sides can engage in a fair and balanced exchange of ideas, leading to a more productive and insightful debate.中文回答：在辩论中，特别是在比较性的话题上，设立清晰和一致的比较标准是非常重要的。

少量AG1-X4阴离子交换树脂分离地质标样中的铁及铁同位素测定

48
2013 年
离子交换柱类型树脂体积上柱淋洗
收集 Fe
适用范围
表 1 Fe 离子交换分离条件
Table 1 Ion-exchange conditions for Fe
AG1-X4(微型柱)
AG MP-1(微型柱)
Geochimica ▌ Vol. 42 ▌ No. 1 ▌ pp. 46~52 ▌ Jan., 2013
第1期
唐索寒等: 少量 AG1-X4 阴离子交换树脂围更广[1–6]。早期 Fe 同位素分析采用热电离质谱法(TIMS)[7]
和双稀释剂热电离质谱法[8], 2000 年之后兴起发展的多接收等离子质谱法[9–10]和双稀释剂多接收等离子质谱法[11], 使得 Fe 同位素分析方法日趋成熟。近几年, 国内也建立了 Fe 的化学分离和同位素质谱分析方法 [12–13], 其中的化学分离方法是基于用 AG MP-1 阴离子交换树脂将样品中的 Cu、Fe、Zn 分离提纯, 测定它们的同位素组成, 为了将这三个元素彻底分离, 使用的是大交换柱(柱床 43×Φ6.8mm)多树脂量(1.6～1.8 mL)。然而对于很多研究者, 只需要分析 Fe 同位素, 所以有必要建立一个更为快速有效的 Fe 的分离方法, 用于 Fe 同位素测定。
元素含量: OPPIMA8300 等离子光谱分析仪(美国 PE 公司)。
1.3 岩石标准样品
BCR-2、BHVO-2 和 BIR-1a(玄武岩)、AGV-2 (安山岩 )、 W-2(辉绿岩 )、 GSP-2(花岗闪长岩 )、 COQ-1(碳酸盐岩)和 DTS-2b(橄榄岩), 为美国地质调查局 USGS 研制; JB-2(玄武岩)和 Jsd-1(沉积岩) 为日本地质调查所研制 ; GBW07105( 玄武岩 ) 和 GBW07267(黄铁矿)为中国地质科学院地球物理地球化学勘查研究所研制。

3种芝麻菜种子油脂理化特性及体外抗氧化活性比较

井豪宾，牛茵茵，肖祉婧，等.3种芝麻菜种子油脂理化特性及体外抗氧化活性比较[J].沈阳农业大学学报，2022，53（6）：685-692.沈阳农业大学学报，2022，53(6)：685-692Journal ofShenyang Agricultural University http ：//DOI:10.3969/j.issn.1000-1700.2022.06.006收稿日期：2022-09-08基金项目：国家自然科学基金项目(32260070)第一作者：井豪宾(1998-)，男，硕士研究生，从事粮食、油脂及植物蛋白工程研究，E-mail ：*******************.cn 通信作者：唐巧玉(1979-)，女，硕士，副教授，从事植物硒营养生理生化研究E-mail ：****************3种芝麻菜种子油脂理化特性及体外抗氧化活性比较井豪宾a,b,c ，牛茵茵b,c ，肖祉婧c ，肖晶c ，罗兴武c ，罗凯c ，周毅峰c ，唐巧玉a,b（湖北民族大学a.生物科学与技术学院，b.硒食品营养与健康智能技术湖北省工程研究中心,c.生物资源保护与利用湖北省重点实验室,湖北恩施445000）摘要：为提高芝麻菜的开发，选取3种常见的芝麻菜种子为原料，探究火箭生菜、花叶芝麻菜、小叶芝麻菜3种种子油脂理化特征及抗氧化活性，采用索氏提取法提取种子油脂，参照国标对其理化性质指标进行评价。

采用Folin-Ciocalteu 法测定油脂中总酚含量；利用GC-MS 法鉴定油脂组成并对3种脂肪酸成分进行分析；分析了3种油脂对1,1-二苯基-2-苦基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)和超氧阴离子自由基的清除率，并以此为评价指标研究3种油脂的体外抗氧化活性。

结果表明：3种芝麻菜种子出油率在20%～35%范围内，油脂酸价均符合国家标准；除火箭生菜外其他两种油脂过氧化值均符合国家标准；3种油脂的碘值相差较大，火箭生菜为(151.67±7.09)g·100g -1，花叶芝麻菜为(99.67±1.97)g·100g -1，小叶芝麻菜为(43.23±3.76)g·100g -1。

阳离子交换膜孔径的表征

阳离子交换膜孔径的表征摘要：离子交换膜的孔径参数对于电渗析应用起着关键性的影响，因为离子是通过膜孔渗透的，在利用电渗析分离不同含盐组分时，只有当分离组分的尺寸小于离子交换膜的孔径时，该组分才能在电场作用下迁移通过膜。

离子交换膜的电化学性能和实际应用性质很大程度上决定于膜孔径的大小。

因此评估膜孔径大小对于包括单价选择性膜、质子选择性膜、特种离子选择性膜等新膜产品的开发以及离子膜的工业应用具有重要意义。

本文通过测试不同阳离子膜在不同电解液中的面电阻来表征离子膜的孔径大小和致密程度。

测试了几种阳膜在氯化铵、四甲基氯化铵、四乙基氯化铵和四丙基氯化铵中的电阻，膜电阻随着铵盐离子半径的增加而增加，通过对比电阻值和铵盐离子半径，膜孔大小排序为：FKS < CMV/CMX < Fujifilm膜/异相膜 < TWEDC2，FKS的膜孔半径最小（约为2.5nm），TWEDC2的膜孔半径最大（约为4.4nm），其余膜孔半径介于2.8~3.5nm，其中CMX和CMV的孔径小于Fujifilm 膜和异相膜。

关键词：离子交换膜；电渗析；膜孔径；面电阻；电解液电渗析技术在工业废水脱盐/浓缩处理中发挥着重要作用。

该技术的核心是离子交换膜。

离子交换膜的孔径参数对于电渗析应用有关键性的影响，因为离子是通过膜孔渗透的；在利用电渗析分离不同含盐组分时，只有当分离组分的尺寸小于离子交换膜的孔径时，该组分才能在电场作用下迁移通过膜。

出于提高离子交换膜性能的实际需求，需要依据膜孔径大小的评估数据来确定膜性能和膜实际应用间的关系，离子交换膜的电化学性能和实际应用性质很大程度上决定于膜的孔径大小。

因此评估膜孔径大小对于包括单价选择性膜、质子选择性膜、特种离子选择性膜等新膜产品的开发以及离子膜的工业应用具有重要意义。

本论文建立了离子膜的孔径表征方法，即测量离子膜在不同离子半径电解质中的膜电阻，当电解质离子半径大于离子膜的孔径时，膜面电阻会明显升高，据此判断离子膜的孔径。

三体关联度量方式

*
fabrizio.buscemi@unimore.it
in the past years quantum discord has received increasing attention as a means to estimate the quantum correlations encoded in a system and potentially usable for applications in quantum information processing [15–19]. Indeed, it was shown theoretically [20], and later experimentally [21], that bipartite separable mixed states with nonzero discord may provide computational speedup compared to classical states in some quantum computation protocols. Under suitable conditions quantum discord is also found to be more robust than entanglement in noisy environments [17]. While the characterization of quantum discord in terms of the discrepancy between two quantum analogues of the classical mutual information has well been grasped and widely used in bipartite systems, its extensions in multipartite systems are still discussed and tackled with different approaches [22–27]. Recently, Giorgi et al. [26] introduced a measure of genuine total, classical, and quantum correlations relying on the use of relative entropy to quantify the distance between two density matrices. Like other proposed measures of correlations in quantum systems [23,24,27], the approach of Ref. [26] involves difﬁcult extremization procedures over operators or states which makes the calculations very hard. This justiﬁes the scarce number of works exploring the time evolution of quantum correlations in multipartite open systems. In this paper, we investigate the dynamics of the quantum correlations in a physical model consisting of three qubits, not interacting among each other, and subject to a classical random telegraph noise (RTN). Two different initial conﬁgurations are examined, namely GHZ and W Werner-type states. The inﬂuence of the classical environmental noise on the system is described by means of a stochastic Hamiltonian with a coupling term mimicking a random telegraph signal. The dynamics of the three qubits is evaluated by averaging the time-evolved states over the noise. Even if in similar systems of two qubits the time evolution of quantum correlations has

元素的形态分析3

（pg/mL）（pg/mL）（fg） 0.3 2.5 0.7 1 0.3 1 0.4 0.1 0.3 0.1 0.3 0.1 0.4 0.1 2 7 1 5 2 7 2
不同检测器（ICP-AES与ICP-MS）性能的比较
性能灵敏度分辨率 ICP-AES 高/(µg/L) 较高/(λ/△ λ) ICP-MS 超高/(ng/L) 最高可达10000的质量分辨率 /(质量分数) 覆盖面检测噪声线性响应范围宽的波长覆盖较低宽,4-5个数量级元素质量覆盖面,从1-250u 低极宽,6-8个数量级真正的同时多元素检测否
icpms不同接口的检出限比较元素超声雾化电热蒸发元素超声雾化电热蒸发pgmlpgmlfgpgmlpgmlfgtb0301la1501dy2503ce1101ho0701pr0801nd3804tm0301sm430510ybeu0803lu0401gd400612不同检测器icpaes与icpms性能的比较性能icpaesicpms灵敏度超高ngl分辨率较高最高可达10000的质量分辨率质量分数覆盖面宽的波长覆盖元素质量覆盖面从1250u检测噪声较低线性响应范围宽45个数量级极宽68个数量级能否多元素同时检测多元素同时或顺序检测真正的同时多元素检测能否要求校正背景用于形态分析的光谱检测器的适应性比较分析方法灵敏度连续操作成本faas代表差
(1)八甲基环四硅氧烷 (2)二氯八甲基环四硅氧烷
四、HPLC与原子光谱/质谱联用技术及元素形态分析 1、概述 HPLC-ICP-AES/MS联用技术是元素形态分析的强有力工具，因为它反映了高选择性分离与高灵敏度检测的结合。接口是一个关键因素，甚至涉及形态方法的成败！基于这点，文献中有关接口技术的报道非常多，接口技术已成为痕量分析/形态分析中的研究热点之一。一个理想的HPLC-ICP-AES/MS接口应该具备以下几点：（1）产生的气溶胶的平均粒径应很小，且分布范围窄；

酸解法制备纤维素纳米晶体水解残液的糖酸分离

酸解法制备纤维素纳米晶体水解残液的糖酸分离王帅;刘鹏涛;侯佳玲【摘要】硫酸法制备纤维素纳米晶体(CNC)的水解残液中含有大量的硫酸、一些未充分水解的纤维素片段以及以单体和寡聚形式存在的糖,直接丢弃不仅会污染环境,更是对资源的一种极大浪费.通过向水解残液中加入硫酸(质量分数80％)的方法,调节水解残液中的硫酸浓度,并通过水浴加热使残液中未充分水解的物质转化为葡萄糖;然后用阴离子交换膜将水解残液中的硫酸和葡萄糖分离,再将分离后的液体用旋转蒸发仪浓缩,以提高硫酸和葡萄糖的浓度.研究结果表明,调节水解残液中硫酸质量分数为56％,在45℃水浴中反应3h,水解残液中葡萄糖含量达到最大值13.73g/L;处理后的水解残液通过2次阴离子交换膜过滤,硫酸的回收率达到90.31％,浓缩可得到10.06 mol/L的浓硫酸和36 g/L的葡萄糖溶液.回收得到的硫酸和副产品葡萄糖溶液可分别用于CNC的制备和用作生物发酵的碳源.%There were lots of sulfuric acid,some of not fully hydrolyzed cellulose fragments,monomer sugars and oligo in the waste liquid from preparing cellulose nanocrystals (CNC) by sulfuric acid hydrolysis process.If the waste liquid was discarded directly without recycling,and it could cause environmental pollution and a great waste of resource.This paper studied the method for recovering both sulfuric acid and glucose.The concentration of sulfuric acid in the waste liquid was adjusted by adding 80％ (wt) sulfuric acid,then the materials not fully hydrolyzed were converted to glucose by heating in water bath.The sulfuric acid and glucose were separated by anion exchange membrane,then concentrated by using rotary evaporating and concentrating system.Results showed thatglucose concentration in the waste liquid reached maximum when sulfuric acid concentration was 56％(wt) and heating at 45℃ for 3 h in w ater bath.The recovery rate of sulfuric acid was 90.31％ after the liquid was treated twice by anion exchange membrane,10.06 mol/L sulfuric acid and 36 g/L glucose solution were obtained after being concentrated.【期刊名称】《中国造纸学报》【年(卷),期】2017(032)003【总页数】5页(P27-31)【关键词】纤维素纳米晶体;水解残液;阴离子交换膜;葡萄糖;硫酸【作者】王帅;刘鹏涛;侯佳玲【作者单位】天津市制浆造纸重点实验室,天津科技大学造纸学院,天津,300457;天津市制浆造纸重点实验室,天津科技大学造纸学院,天津,300457;天津市制浆造纸重点实验室,天津科技大学造纸学院,天津,300457【正文语种】中文【中图分类】TQ353.9;X793纤维素纳米晶体(CNC)是由纤维素派生而成的具有多种优异性能的生物聚合物[1]，可应用于复合增强、催化、光电材料、酶固定化、抗菌和医用材料、生物传感仪、荧光探针和药物释放等方面，是最有潜力的材料之一[2-3]。