DOCSLIB.ORG

The LC Handbook Guide to LC Columns and Method Development Contents

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The LC Handbook Guide to LC Columns and Method Development Contents Agilent CrossLab combines the innovative laboratory services, software, and consumables competencies of Agilent Technologies and provides a direct connection to a global team of scientific and technical experts who deliver vital, actionable insights at every level of the lab environment. Our insights maximize performance, reduce complexity, and drive improved economic, operational, and scientific outcomes. Only Agilent CrossLab offers the unique combination of innovative products and comprehensive solutions to generate immediate results and lasting impact. We partner with our customers to create new opportunities across the lab, around the world, and every step of the way. More information at www.agilent.de/crosslab Learn more The LC Handbook – Guide to LC Columns and Method Development www.agilent.com/chem/lccolumns Buy online www.agilent.com/chem/store Find an Agilent customer center or authorized distributor www.agilent.com/chem/contactus U.S. and Canada 1-800-227-9770 [email protected] Europe [email protected] Asia Pacific [email protected] The LC India [email protected] Handbook Information, descriptions and specifications in this publication are subject to change without notice. Agilent Technologies shall not Guide to LC Columns and be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance or use of this material. Method Development © Agilent Technologies, Inc. 2016 Published in USA, February 1, 2016 Publication Number 5990-7595EN The LC Handbook Guide to LC Columns and Method Development Contents Introduction 4 Selecting your HPLC column 27 HPLC mode 28 Essential chromatography Column selection basics: conventional columns 29 concepts 5 • High Performance Liquid Efficiency (N) 6 Chromatography (HPLC) columns 30 Retention Factor (k) 7 • UHPLC columns 31 Selectivity or separation factor (α) 7 • Superficially porous particle columns 31 • Columns for LC/MS 32 Resolution (Rs) 8 Pressure 9 • Columns for Gel Permeation van Deemter Curves 10 Chromatography (GPC), Size Exclusion Chromatography (SEC), and Gel The gradient equation 10 Filtration Chromatography (GFC) 33 LC Instrumentation Fundamentals 12 • Columns for biocharacterization 33 Column characteristics 34 The pump – the heart of an LC system 13 • Silica 34 • Pressure range 13 • Bonded phases 34 • Power ranges of Agilent 1200 Infinity Series pumps 14 • Polymers 34 • Solvent mixing 15 • Pore size 35 • Delay volume 17 • Particle size 35 • Extra column volume 17 • Column dimensions 36 The autosampler – the brain behind Cartridge column systems 37 workflow automation logistics 18 • Fixed-loop and flow-through autosamplers 18 Keys to performance: column • Injection precision and accuracy 20 configurations and settings 40 • Flexibility and sample capacity 20 The importance of reducing extra-column • Injection cycle time 20 volume 41 • Carryover 21 Preparing the perfect fitting connection 42 • Novel sampling approaches and Fitting connection requirements 42 automation 22 Nonadjustable metallic fittings 42 The column thermostat – the muscles to Adjustable finger-tight fittings 44 generate heat 22 Agilent A-Line fittings 44 Detector - the organ of perception 23 Robustness over 200 reconnections 46 Detector sensitivity 25 Compatibility with different column brands 46 • Linear range 25 Sample injections 46 • Data rate 26 Setting the data collection rate 47 2 Dwell volume and its impact on chromatography 48 • Troubleshooting example: drifting baseline 75 • Measuring your system’s dwell volume 50 • Troubleshooting example: broadening • Evaluating the impact of dwell volume 52 or splitting caused by high pH 77 • Dwell volume and analysis time 53 • Troubleshooting example: mobile phase modifiers and selectivity 77 Chelating compounds 53 Optimizing your chromatographic conditions pH and mobile phase modifiers 54 for reversed-phase chromatography 79 Working with gradients 55 • Isocratic optimization 80 Optimizing column re-equilibration 56 • Gradient optimization 83 Column aging 58 Polymeric columns for reversed-phase • Loss of bonded phase 58 chromatography 85 Cleaning a reversed-phase silica column 58 A step-by-step guide for 'hands-on' isocratic Cleaning a normal phase silica column 59 method development in reversed-phase Cleaning a reversed-phase polymeric column 59 chromatography 86 Tips for transferring methods from Method development 61 conventional columns to high efficiency columns 88 Method development: where to start 62 Automated method development tools 90 • Mode Selection 62 Method development for other HPLC modes 92 • Choosing the column and packing • HILIC 92 dimensions 65 • Normal phase chromatography 94 • Choosing the stationary phase 66 • Ion-exchange chromatography 96 Method development for reversed-phase • Gel permeation chromatography/size chromatography 66 exclusion chromatography 97 Selection of stationary phase for reversed- LC/MS Instrumentation phase chromatography 66 Fundamentals 98 Selection of mobile phase solvents for What is LC/MS 98 reversed-phase chromatography 69 What information does LC/MS provide 99 • Working with mobile phases 69 Types of LC/MS instruments 100 • Troubleshooting mobile phases and mobile phase modifiers 69 • Single-quadrupole instruments 100 • Mixing mobile phases 70 • Triple-quadrupole instruments 100 • Degassing mobile phases 70 • Time-of-flight and quadrupole time- of-flight instruments 100 Managing your pH with mobile phase modifiers 70 Uniform field ion mobility 101 • Common buffers for UV detectors 72 MS/MS – breaking down a compound for • Considerations for LC/MS 73 extra specificity 101 • Troubleshooting issues with mobile phase modifiers 75 Continued on next page 3 Ionization of compounds 102 Column care and storage 117 • Electrospray ionization 102 • Maximizing column lifetime 117 • Atmospheric pressure chemical ionization 102 • Care in storage 119 • Atmospheric pressure photo ionization 102 • Unblocking a column 119 Quickly determining when a column is going Tips for developing methods with LC/MS 103 bad 119 • Solvent selection for ESI-MS 103 Ensuring method reproducibility around the world 120 • Solvent selection for APCI-MS and APPI-MS 104 • Increasing method robustness 120 • Solvent selection for ion pair • A reminder about dwell volume chromatography with MS 104 implications 121 Sample preparation 105 Quick troubleshooting reference 122 • Matrix components 105 Tips for effective troubleshooting 122 • Concentration issuess 105 Ion suppression considerations 105 Useful references 128 Considerations for using LC/MS with high- USP designations 128 efficiency columns 106 Solvent miscibility 134 • Improvements in sensitivity 106 UV cutoffs for mobile phase modifiers 137 • MS scan rates 107 Solid Phase Extraction sorbents 138 • System Dispersion 107 SPE sorbent conditions 139 LC/MS software 108 Other suggested reading 140 • Types of scans – comparing TIC, EIC Other Agilent resources 140 and SIM 108 • Personal compound databases and Glossary 141 libraries 109 Protecting your Index 169 chromatographic results 111 Agilent Products and Scalability 112 ordering information 174 The importance of using high-quality-grade solvents 115 Special considerations for UHPLC 115 Inline filters 116 • Low-volume inline filters 116 Guard columns 117 Solvent-saturation columns 117 Column inlet frits 117 4 Introduction Where to begin? Liquid chromatography is a vast and complex subject, but one for which we never lose our interest. Chromatographers around the world are using HPLC techniques to ensure the safety of our food and water, develop life-saving pharmaceutical products, protect our environment, guard public health, and that’s just the start of it. The more you know about chromatography, the more you can get done with this amazing technology. Today, you have more choices of columns and packing materials to suit an ever expanding range of uses. Agilent now offers more than 2,000 column choices covering the broadest array of applications and conditions. This increases your opportunities to select the most appropriate column for your needs. As part of our commitment to helping you get the best results from your liquid chromatography, we have compiled this handy guide to choosing LC columns, with plenty of tips and tricks to make your job easier and more productive. In addition, we’ve drawn on more than 40 years of experience to provide suggestions for overcoming some of the common problems that can occur with columns and fittings in everyday use. The guide covers the main columns used in LC, with particular emphasis on reversed-phase high performance liquid chromatography. How to use this guide: • Sections are color-coded for your easy reference. • The glossary in the back is fairly comprehensive. It’s intended to be a good resource, although we have not touched on every glossary term in the rest of the book, due to space considerations. • This book primarily focuses on reversed-phase HPLC although we highlight other techniques elsewhere in the book. 5 Essential chromatography concepts We all remember the feeling we had in school as we learned math, wondering how it would actually come into practical use. Scientists have to learn more math than many professionals, and this section reminds us why. Here, we will briefly review the equations and theory behind many of the concepts that drive chromatography. Understanding these concepts will help you to get the best results, and to troubleshoot if you encounter problems. We start with
Load more

Recommended publications
  • J . Org. Chem., Vol. 43, No. 14, 1978 2923 A
    Notes J. Org. Chem., Vol. 43, No. 14, 1978 2923 (11) Potassium ferricyanide has previously been used to convert vic-l,2-di- carboxylate groups to double bonds. See, for example, L. F. Fieser and M. J. Haddadin, J. Am. Chem. SOC., 86, 2392 (1964). The oxidative dide- carboxylation of 1,2dicarboxyiic acids is, of course, a well-known process. See inter alia (a) C. A. Grob, M. Ohta, and A. Weiss, Helv. Chirn. Acta, 41, 191 1 [ 1958); and (b) E. N. Cain, R. Vukov, and S. Masamune, J. Chern. SOC. D, 98 (1969). 1 I I L <40 26- 40- 63- 40 63 200 Rapid Chromatographic Technique for Preparative Figure 2. Silica gel particle size6 (pm):(0) rh: (0) r/(w/2). Separations with Moderate Resolution W. Clark Still,* Michael Kahn, and Abhijit Mitra Departm(7nt o/ Chemistry, Columbia Uniuersity, 1Veu York, Neu; York 10027 ReceiLied January 26, 1978 We wish to describe a simple absorption chromatography technique for the routine purification of organic compounds. 0 1.0 2 0 3.0 4.0 Large scale preparative separations are traditionally carried Figure 3. Eluant flow rate (in./min). out by tedious long column chromatography. Although the results are sometimes satisfactory, the technique is always time consuming and frequently gives poor recovery due to band tailing. These problems are especially acute when sam- ples of greater than 1 or 2 g must be separated. In recent years 41 several preparative systems have evolved which reduce sep- aration times to 1-3 h and allow the resolution of components having Al?f 1 0.05 on analytical TLC.
    [Show full text]
  • Assessment of Energetic Heterogeneity of Reversed-Phase
    Seton Hall University eRepository @ Seton Hall Seton Hall University Dissertations and Theses Seton Hall University Dissertations and Theses (ETDs) Spring 5-15-2018 Assessment of Energetic Heterogeneity of Reversed-Phase Surfaces Using Excess Adsorption Isotherms for HPLC Column Characterization Leih-Shan Yeung [email protected] Follow this and additional works at: https://scholarship.shu.edu/dissertations Part of the Analytical Chemistry Commons Recommended Citation Yeung, Leih-Shan, "Assessment of Energetic Heterogeneity of Reversed-Phase Surfaces Using Excess Adsorption Isotherms for HPLC Column Characterization" (2018). Seton Hall University Dissertations and Theses (ETDs). 2527. https://scholarship.shu.edu/dissertations/2527 Assessment of Energetic Heterogeneity of Reversed- Phase Surfaces Using Excess Adsorption Isotherms for HPLC Column Characterization By: Leih-Shan Yeung Dissertation submitted to the Department of Chemistry and Biochemistry of Seton Hall University In partial fulfilment of the requirements for the degree of Doctor of Philosophy In Chemistry May 2018 South Orange, New Jersey © 2018 (Leih Shan Yeung) We certify that we have read this dissertation and that, in our opinion, it is adequate in scientific scope and quality as a dissertation for the degree of Doctor of Philosophy. Approved Research Mentor Nicholas H. Snow, Ph.D. Member of Dissertation Committee Alexander Fadeev, Ph.D. Member of Dissertation Committee Cecilia Marzabadi, Ph.D. Chair, Department of Chemistry and Biochemistry Abstract This research is to explore a more general column categorization method using the test attributes in alignment with the common mobile phase components. As we know, the primary driving force for solute retention on a reversed-phase surface is hydrophobic interaction, thus hydrophobicity of the column will directly affect the analyte retention.
    [Show full text]
  • Environmental Quality: in Situ Air Sparging
    EM 200-1-19 31 December 2013 Environmental Quality IN-SITU AIR SPARGING ENGINEER MANUAL AVAILABILITY Electronic copies of this and other U.S. Army Corps of Engineers (USACE) publications are available on the Internet at http://www.publications.usace.army.mil/ This site is the only repository for all official USACE regulations, circulars, manuals, and other documents originating from HQUSACE. Publications are provided in portable document format (PDF). This document is intended solely as guidance. The statutory provisions and promulgated regulations described in this document contain legally binding requirements. This document is not a legally enforceable regulation itself, nor does it alter or substitute for those legal provisions and regulations it describes. Thus, it does not impose any legally binding requirements. This guidance does not confer legal rights or impose legal obligations upon any member of the public. While every effort has been made to ensure the accuracy of the discussion in this document, the obligations of the regulated community are determined by statutes, regulations, or other legally binding requirements. In the event of a conflict between the discussion in this document and any applicable statute or regulation, this document would not be controlling. This document may not apply to a particular situation based upon site- specific circumstances. USACE retains the discretion to adopt approaches on a case-by-case basis that differ from those described in this guidance where appropriate and legally consistent. This document may be revised periodically without public notice. DEPARTMENT OF THE ARMY EM 200-1-19 U.S. Army Corps of Engineers CEMP-CE Washington, D.C.
    [Show full text]
  • Synthesis and Applications of Monolithic HPLC Columns
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2005 Synthesis and Applications of Monolithic HPLC Columns Chengdu Liang University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Chemistry Commons Recommended Citation Liang, Chengdu, "Synthesis and Applications of Monolithic HPLC Columns. " PhD diss., University of Tennessee, 2005. https://trace.tennessee.edu/utk_graddiss/2233 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Chengdu Liang entitled "Synthesis and Applications of Monolithic HPLC Columns." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Chemistry. Georges A Guiochon, Major Professor We have read this dissertation and recommend its acceptance: Sheng Dai, Craig E Barnes, Michael J Sepaniak, Bin Hu Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Chengdu Liang entitled, “Synthesis and applications of monolithic HPLC columns.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Chemistry.
    [Show full text]
  • Comprehensive Two-Dimensional Liquid Chromatography - Practical Impacts of Theoretical Considerations
    Cent. Eur. J. Chem. • 10(3) • 2012 • 844-875 DOI: 10.2478/s11532-012-0036-z Central European Journal of Chemistry Comprehensive Two-Dimensional Liquid Chromatography - practical impacts of theoretical considerations. A review. Review Article Pavel Jandera Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice 532 10, Czech Republic Received 24 October 2011; Accepted 31 January 2012 Abstract: A theory of comprehensive two-dimensional separations by liquid chromatographic techniques is overviewed. It includes heart-cutting and comprehensive two-dimensional separation modes, with attention to basic concepts of two-dimensional separations: resolution, peak capacity, efficiency, orthogonality and selectivity. Particular attention is paid to the effects of sample structure on the retention and advantages of a multi-dimensional HPLC for separation of complex samples according to structural correlations. Optimization of 2D separation systems, including correct selection of columns, flow-rate, fraction volumes and mobile phase, is discussed. Benefits of simultaneous programmed elution in both dimensions of LCxLC comprehensive separations are shown. Experimental setup, modulation of the fraction collection and transfer from the first to the second dimension, compatibility of mobile phases in comprehensive LCxLC, 2D asymmetry and shifts in retention under changing second-dimension elution conditions, are addressed. Illustrative practical examples of comprehensive LCxLC separations are shown. Keywords:
    [Show full text]
  • Monolithic Stationary Phases for Capillary Electrochromatography Based on Synthetic Polymers: Designs and Applications Frantisek Svec, Eric C
    Reviews Monolithic Stationary Phases for Capillary Electrochromatography Based on Synthetic Polymers: Designs and Applications Frantisek Svec, Eric C. Peters1), David Sy´kora, Cong Yu, Jean M. J. Fre´chet* Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA Ms received: September 29, 1999; accepted: November 3, 1999 Key Words: Capillary electrochromatography; monolithic columns; synthetic polymers; stationary phase Summary Monolithic materials have quickly become a well-established station- the use of a solid stationary phase and a separation mechan- ary phase format in the field of capillary electrochromatography ism characteristic of HPLC based on specific interactions of (CEC). Both the simplicity of their in situ preparation method and solutes with a stationary phase. Therefore CEC is most com- the large variety of readily available chemistries make the monolithic monly implemented by means typical of both HPLC (packed separation media an attractive alternative to capillary columns packed with particulate materials. This review summarizes the con- columns) and HPCE (use of electrophoretic instrumentation). tributions of numerous groups working in this rapidly growing area, To date, both columns and instrumentation developed specifi- with a focus on monolithic capillary columns prepared from syn- cally for CEC remain scarce. thetic polymers. Various approaches employed for the preparation of the monoliths are detailed, and where available, the material proper- Although numerous groups around the world prepare CEC ties of the resulting monolithic capillary columns are shown. Their columns using a variety of approaches, the vast majority of chromatographic performance is demonstrated by numerous separa- these efforts mimic in one way or another standard HPLC tions of different analyte mixtures in variety of modes.
    [Show full text]
  • Producing Nitrogen Via Pressure Swing Adsorption
    Reactions and Separations Producing Nitrogen via Pressure Swing Adsorption Svetlana Ivanova Pressure swing adsorption (PSA) can be a Robert Lewis Air Products cost-effective method of onsite nitrogen generation for a wide range of purity and flow requirements. itrogen gas is a staple of the chemical industry. effective, and convenient for chemical processors. Multiple Because it is an inert gas, nitrogen is suitable for a nitrogen technologies and supply modes now exist to meet a Nwide range of applications covering various aspects range of specifications, including purity, usage pattern, por- of chemical manufacturing, processing, handling, and tability, footprint, and power consumption. Choosing among shipping. Due to its low reactivity, nitrogen is an excellent supply options can be a challenge. Onsite nitrogen genera- blanketing and purging gas that can be used to protect valu- tors, such as pressure swing adsorption (PSA) or membrane able products from harmful contaminants. It also enables the systems, can be more cost-effective than traditional cryo- safe storage and use of flammable compounds, and can help genic distillation or stored liquid nitrogen, particularly if an prevent combustible dust explosions. Nitrogen gas can be extremely high purity (e.g., 99.9999%) is not required. used to remove contaminants from process streams through methods such as stripping and sparging. Generating nitrogen gas Because of the widespread and growing use of nitrogen Industrial nitrogen gas can be produced by either in the chemical process industries (CPI), industrial gas com- cryogenic fractional distillation of liquefied air, or separa- panies have been continually improving methods of nitrogen tion of gaseous air using adsorption or permeation.
    [Show full text]
  • Identification Criteria for Qualitative Assays Incorporating Column Chromatography and Mass Spectrometry
    WADA Technical Document – TD2010IDCR Document Number: TD2010IDCR Version Number: 1.0 Written by: WADA Laboratory Committee Approved by: WADA Executive Committee Approval Date: 08 May, 2010 Effective Date: 01 September, 2010 IDENTIFICATION CRITERIA FOR QUALITATIVE ASSAYS INCORPORATING COLUMN CHROMATOGRAPHY AND MASS SPECTROMETRY The ability of a method to identify a compound is a function of the entire procedure: sample preparation; chromatographic separation; mass analysis; and data assessment. Any description of the method for purposes of documentation should include all parts of the method. The appropriate analytical characteristics shall be documented for a particular assay. The Laboratory shall establish criteria for identification of a compound. 1.0 Sample Preparation The purpose of the sample preparation and chromatographic separation is to present a relatively pure chemical component from the sample to the mass spectrometer. The sample purification step can significantly change both the performance of the chromatographic system and the mass spectrometer. For example, a change in extraction solvent can selectively remove interferences and matrix components that might otherwise co-elute with the compound of interest. In addition, selective preparation procedures such as immunoaffinity extraction or fractions collected from high performance liquid chromatography separation can provide a solution that is nearly devoid of any other compounds. 2.0 Chromatographic Separation 2.1 Gas Chromatography • For capillary gas chromatography, the retention time (RT) of the analyte shall not differ by more than two (2) percent or ±0.1 minutes (whichever is smaller) from that of the same substance in a spiked urine sample, Reference Collection sample, or Reference Material analyzed contemporaneously; • Alternatively, the laboratory may choose to use relative retention time (RRT) as an acceptance criterion, where the retention time of the peak of interest is measured relative to a chromatographic reference compound (CRC).
    [Show full text]
  • Column Chromatography Methods of Analysis In
    The Journal of Undergraduate Neuroscience Education (JUNE), Spring 2005, 3(2):A36-A41 ARTICLE Column Chromatography Analysis of Brain Tissue: An Advanced Laboratory Exercise for Neuroscience Majors William H. Church Chemistry Department and Neuroscience Program, Trinity College, Hartford, Connecticut 06106 Neurochemical analysis of discrete brain structures in to useful laboratory techniques such as standard solution experimental animals provides important information on preparation and calibration curve construction, centrifugation, synthesis, release, and metabolism changes following quantitative pipetting, and data evaluation and graphical behavioral or pharmacological experimental manipulations. presentation. Typically, only students that participate in Quantitation of neurotransmitters and their metabolites independent neuroscience research are familiar with these following unilateral drug injections can be carried out using types of quantitative skills. The usefulness of this type of standard chromatographic equipment typically found in most experimental design for understanding behavioral or undergraduate analytical laboratories. This article describes pharmacological effects on neurotransmitter systems is an experiment done in a six session (four hours each) emphasized through a final report requiring a comprehensive component of a neuroscience research methods course. The literature search. experiment provides advanced neuroscience students experience in brain structure dissection, sample preparation, and quantitation of catecholamines
    [Show full text]
  • Chapter 15 Liquid Chromatography
    Chapter 15 Liquid Chromatography Problem 15.1: Albuterol (a drug used to fight asthma) has a lipid:water KOW (see “Profile—Other Applications of Partition Coefficients”) value of 0.019. How many grams of albuterol would remain in the aqueous phase if 0.001 grams of albuterol initially in a 100 mL aqueous solution were allowed to come into equilibrium with 100 mL of octanol? C (aq) ⇌ C (org) Initial (g) 0.001 g 0 Change -y +y Equil 0.001-y y 푦 100 푚퐿 퐾푂푊 = 0.019 = (0.001−푦) ⁄100푚퐿 1.9 x 10-5 – 0.019y = y 1.019y = 1.9 x 10-5 y = 1.864x10-5 g albuterol would be in the octanol layer Problem 15.2: (a) Repeat Problem 15.1, but instead of extracting the albuterol with 100 mL of octanol, extract the albuterol solution five successive times with only 20 mL of octanol per extraction. After each step, use the albuterol remaining in the aqueous phase from the previous step as the starting quantity for the next step. (Note: the total volume of octanol is the same in both extractions). (b) Compare the final concentration of aqueous albuterol to that obtained in Problem 15.1. Discuss how the extraction results changed by conducting five smaller extractions instead of one large one. What is the downside to the multiple smaller volume method in this exercise compared to the single larger volume method in Problem 15.1? 푦 (a) Each step will have this general form of calculation: 퐾 = 20 푚퐿 푂푊 (푔푖푛푖푡−푦) ⁄100푚퐿 (퐾 )(푔 ) so we can calculate the g transferred to octanol from 푦 = 푂푊 푖푛푖푡 5+퐾푂푊 We can use a spreadsheet to calculate the successive steps: First extraction:
    [Show full text]
  • Triple-Detection Size-Exclusion Chromatography of Membrane Proteins
    Triple-Detection Size-Exclusion Chromatography of Membrane Proteins vom Fachbereich Biologie der Technischen Universit¨atKaiserslautern zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Doctor rerum naturalium; Dr. rer. nat.) genehmigte Dissertation von Frau Dipl.-Biophys. Katharina Gimpl Vorsitzende: Prof. Dr. rer. nat. Nicole Frankenberg-Dinkel 1. Gutachter: Prof. Dr. rer. nat. Sandro Keller 2. Gutachter: Prof. Dr. rer. nat. Rolf Diller Wissenschaftliche Aussprache: 08. April 2016 D 386 Abstract Membrane proteins are generally soluble only in the presence of detergent micelles or other membrane-mimetic systems, which renders the determination of the protein's mo- lar mass or oligomeric state difficult. Moreover, the amount of bound detergent varies drastically among different proteins and detergents. However, the type of detergent and its concentration have a great influence on the protein's structure, stability, and func- tionality and the success of structural and functional investigations and crystallographic trials. Size-exclusion chromatography, which is commonly used to determine the molar mass of water-soluble proteins, is not suitable for detergent-solubilised proteins because the protein{detergent complex has a different conformation and, thus, commonly exhibits a different migration behaviour than globular standard proteins. Thus, calibration curves obtained with standard proteins are not useful for membrane-protein analysis. However, the combination of size-exclusion chromatography with ultraviolet absorbance,
    [Show full text]
  • Recent Trends in the Application of Chromatographic Techniques in the Analysis of Luteolin and Its Derivatives
    biomolecules Review Recent Trends in the Application of Chromatographic Techniques in the Analysis of Luteolin and Its Derivatives Aleksandra Maria Juszczak 1 , Marijana Zovko-Konˇci´c 2 and Michał Tomczyk 1,* 1 Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Białystok, ul. Mickiewicza 2a, 15-230 Białystok, Poland; [email protected] 2 Department of Pharmacognosy, University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovaˇci´ca1, 10000 Zagreb, Croatia; [email protected] * Correspondence: [email protected]; Tel.: +48-85-748-5694 Received: 1 October 2019; Accepted: 8 November 2019; Published: 12 November 2019 Abstract: Luteolin is a flavonoid often found in various medicinal plants that exhibits multiple biological effects such as antioxidant, anti-inflammatory and immunomodulatory activity. Commercially available medicinal plants and their preparations containing luteolin are often used in the treatment of hypertension, inflammatory diseases, and even cancer. However, to establish the quality of such preparations, appropriate analytical methods should be used. Therefore, the present paper provides the first comprehensive review of the current analytical methods that were developed and validated for the quantitative determination of luteolin and its C- and O-derivatives including orientin, isoorientin, luteolin 7-O-glucoside and others. It provides a systematic overview of chromatographic analytical techniques including thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC) and counter-current chromatography (CCC), as well as the conditions used in the determination of luteolin and its derivatives in plant material. Keywords: luteolin; hyphenated techniques; chromatography 1. Introduction Luteolin (Figure1) is a yellow dye commonly found in fresh plants.
    [Show full text]