DOCSLIB.ORG

High-Performance Liquid Chromatography Method for Rapid Assessment of Viral Particle Number in Crude Adenoviral Lysates of Mixed Serotype

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
High-Performance Liquid Chromatography Method for Rapid Assessment of Viral Particle Number in Crude Adenoviral Lysates of Mixed Serotype Gene Therapy (2007) 14, 180–184 & 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 www.nature.com/gt SHORT COMMUNICATION High-performance liquid chromatography method for rapid assessment of viral particle number in crude adenoviral lysates of mixed serotype I Kuhn, B Larsen, C Gross and T Hermiston Berlex Laboratories, Gene Therapy Department, Richmond, CA, USA Accurate adenovirus (Ad) quantification requires labor- and resulting in efficient retention of viral particles of all time-intensive viral stock purification. While crude viral serotypes, while non-viral components of crude infected lysates can be titered by plaque assay, this cell-based cultures remain largely in the flow-through. The high- assay is neither rapid nor accurate. Consequently, a method performance liquid chromatography-QSXL method allows for quantification of crude, unpurified viral culture lysates rapid, accurate adenoviral quantification in crude lysates as is needed. Given growing interest in alternative Ad well as identification of the various serotypes present in serotypes (different from well-studied and characterized mixed-serotype crude lysates. We also report on conditions serotype Ad5) for basic research and for therapeutic that efficiently strip and regenerate the column, extending its applications, such a method should also apply to alternative functional life. serotypes. Using a Q Sepharose XL (QSXL) column- Gene Therapy (2007) 14, 180–184. doi:10.1038/sj.gt.3302851; based method, we describe a robust quantification method published online 5 October 2006 Keywords: adenovirus quantification; HPLC; crude viral lysate Efficient and effective development of oncolytic viruses When running viruses in a series, the peak area of CsCl- and virotherapies requires rapid assessment of the purified viral standards analyzed at the end of, or late in, relative potencies of a large variety of viral candidates. a sequence of viral samples was often reduced more than As these viruses may differ in cellular tropisms, these 50% relative to the same standard analyzed at the start of potencies must be assessed on a per viral particle basis. the sequence. Peak areas for viral standards returned to Additionally, development of these agents may require their maxima when column compaction was reversed by rapid identification of the variety of viruses present in allowing the column to rest for at least 24 h, by backflow, any given mixed-serotype lysate without viral purifica- or by repacking the column. The tendency of the tion before quantification. This report describes a relatively soft TMAE matrix to compact, compromising comprehensive method that simultaneously accom- viral particle recovery, increased for adenoviral particles plishes these objectives. in crude culture lysates. In spite of sample centrifugation Methods have been explored to improve the speed and filtration, crude lysate samples clogged the column, and accuracy of adenoviral particle quantification, many increasing column backpressure and promoting TMAE of them based on chromatographic approaches. For bead compression and/or column compaction. The peak example, Shabram et al.1 described a method for areas of purified viral standards analyzed between crude quantification of purified adenoviral particles based on viral culture lysate samples showed that the TMAE Resource Q anion-exchange resin. The Shabram method column was rapidly compromised, requiring extensive was improved by use of an alternate anion-exchange stripping and salt regeneration combined with backflow resin, Fractogel EMD TMAE-650(S) (TMAE), which or column repacking before the peak area of the viral allowed resolution of RNA and viral peaks (E Lehmberg, quantification standard was restored. It is worth noting personal communication). This enhanced resolution that when samples of pure BSA (bovine serum albumin) was especially important for adenovirus (Ad)5 analyses standards were run on compressed TMAE columns (i.e. as this viral serotype is strongly retained on anion those that appeared to exclude viral particles), the exchange, and was formerly difficult to resolve from protein peak areas were not affected. We hypothesize RNA and transfer RNA. However, despite the ability to that when the TMAE beads are compressed, the viral resolve RNA from viral particles, TMAE is not ideal. particles, due to their large size, cannot access sufficient anion-exchange sites to be retained on the resin and so appear in the column flow-through (data not shown) Correspondence: Dr I Kuhn, Gene Therapy Department, Berlex rather than eluting at their known retention time. On the Laboratories, 2600 Hilltop Drive, Richmond, CA 94806, USA. E-mail: [email protected] other hand, we theorize that BSA protein standard and Received 11 May 2006; revised 19 July 2006; accepted 20 July 2006; multimers are sufficiently small to access sites, and published online 5 October 2006 their retention times remain unaffected by column HPLC method for rapid assessment of viral particle number I Kuhn et al 181 Figure 1 QSXL chromatograms of pure Ad stocks. HPLC separa- tions were performed using an Agilent 1100 HPLC that included a well-plate autosampler, quaternary pump and multiwavelength detector. An in-line conductivity meter was used to follow actual salt conditions. Samples and buffers were at ambient temperature, and the column compartment was controlled at 221C. Pharmacia HR 5/5 columns were packed with 1.25 ml bed volume of QSXL (GE Healthcare, no. 17-5072-01) anion-exchange media. The mobile phases were Buffer A, 20 mM Tris/HCl, pH 7.5, 0.1 M NaCl, and Buffer B, 20 mM Tris/HCl, pH 7.5, 1 M NaCl. The flow rate was 1 ml/min, except during the strip method at which time flow rate decreased to 0.02 ml/min. Typical sample injection volume was 0.1 ml; up to 1 ml sample volume of very low titer viral stocks was loaded. A 0.5 mm prefilter was included upstream of the column to extend the life of the column, and this prefilter was changed after no more than 15 crude samples were injected onto the column. The column was equilibrated for 4 column volumes (CV) with Buffer A. After sample loading, the column was washed with 4 CV of Buffer A, followed by sample separation with a linear salt gradient of 0–55% Buffer B over 15 CV, 2 CV of 70% Buffer B, then a second gradient of 70–100% Buffer B over 2 CV, which constituted the first salt regeneration of the column. The column was then re- equilibrated with 5 CV of Buffer A before stripping. The column was stripped (cleaned of any remaining nucleic acids) by loading 1 CV of 0.5 N NaOH, reducing the pump flow rate to 0.02 ml/min for 10 min, followed by 3 CV of Buffer A at 1 ml/min flow rate. This strip step was followed by a salt regeneration step of 3 CV of 100% Buffer B. The column was then re-equilibrated by 10 CV of Buffer A before loading the next sample. Prior to using QSXL, columns for viral particle separations were packed with Fractogel EMD TMAE- 650(S) (EM Science, Gibbstown, NJ, USA) (TMAE), and the buffers and gradients used were those specified by Shabram et al.1 All adenovirus serotypes (Ad3, Ad5, Ad9, Ad11p, Ad16, Ad35) were obtained from the ATCC (American Type Culture Collections). Viral compression adversely affecting viral peak areas. These stocks for HPLC viral titer analysis were prepared by infection of experiences with TMAE led us to test the use of an A549, 293, HT-29 or PC-3 cells. All cells were obtained from the alternate, more rigid column matrix. ATCC, and were grown to approximately 70–80% confluency in the 2 ATCC-recommended culture media before infection. Cells were One such method, reported by Blanche et al., allowed infected at multiplicity of infection (MOI) of 1–10 in Dulbecco’s reproducible quantification of virus of several serotypes modified Eagle’s media Gibco (Invitrogen, Frederick, MD) supple- in crude lysates by employing Q Sepharose XL (QSXL). mented with 2% FBS (Hyclone, Logan, Utah), 2 mM glutamine and We tested the QSXL-based method for the analysis of gentimicin. Full cytopathic effect (CPE) was generally reached by crude viral lysate samples, but found that contaminants 48–72 h post-infection, at which time the viral lysates were eluting at the beginning of the salt gradient obscured harvested. Cells and supernatants were harvested, cells ruptured by 1–3 cycles of freeze–thaw, cell debris pelleted by a 5 min adenoviral serotypes, such as Ad3, Ad9 and Ad16, that centrifugation at 3000 r.p.m. in a Beckman Allegra 6R centrifuge and elute in a similar region of the gradient. To improve the viral stock supernatants collected. These crude viral lysates were sensitivity and precision, we sought to eliminate con- filtered through 0.45 mm filters before loading on the QSXL columns. taminating peaks from the chromatogram. We tested For preparation of pure viral stocks, virus was purified from the alternative starting buffer conditions in which the salt crude lysates using the cesium chloride buoyant density method as described by Tollefson et al.3 Viral HPLC peaks are identified after concentration was increased in 20 mM increments from QSXL chromatography due to their distinctively narrow shape, 0.01 M NaCl to 0.1 M NaCl (data not shown). The their 260 nm/280 nm ratio of 1.3370.06 and the distinctive serotype majority of non-viral culture components that bound retention times. The Ad5 chromatogram is that of our viral standard under the no-salt buffer conditions (and co-eluting early for quantification, an Ad5 stock that was purified, then analyzed by in the salt gradient with early-eluting viral serotypes) reversed-phase HPLC to determine the exact number of viral 4 were not retained when 0.1 M NaCl salt was added to the particles per ml.
Load more

Recommended publications
  • Chemistry Grade Level 10 Units 1-15
    COPPELL ISD SUBJECT YEAR AT A GLANCE ​ ​ ​ ​​ ​ ​​ ​ ​ ​ ​ ​ ​ GRADE HEMISTRY UNITS C LEVEL 1-15 10 Program Transfer Goals ​ ​ ​ ​ ● Ask questions, recognize and define problems, and propose solutions. ​ ​​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ● Safely and ethically collect, analyze, and evaluate appropriate data. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ● Utilize, create, and analyze models to understand the world. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ● Make valid claims and informed decisions based on scientific evidence. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ● Effectively communicate scientific reasoning to a target audience. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ PACING 1st 9 Weeks 2nd 9 Weeks 3rd 9 Weeks 4th 9 Weeks ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Unit 1 Unit 2 Unit 3 Unit 4 Unit 5 Unit 6 Unit Unit Unit Unit Unit Unit Unit Unit Unit ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 7 8 9 10 11 12 13 14 15 1.5 wks 2 wks 1.5 wks 2 wks 3 wks 5.5 wks ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 1.5 2 2.5 2 wks 2 2 2 wks 1.5 1.5 ​ ​ ​ ​ wks wks wks wks wks wks wks Assurances for a Guaranteed and Viable Curriculum ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Adherence to this scope and sequence affords every member of the learning community clarity on the knowledge and skills ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ on which each learner should demonstrate proficiency. In order to deliver a guaranteed and viable curriculum, our team ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ commits to and ensures the following understandings: ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Shared Accountability: Responding
    [Show full text]
  • Chapter 9 Titrimetric Methods 413
    Chapter 9 Titrimetric Methods Chapter Overview Section 9A Overview of Titrimetry Section 9B Acid–Base Titrations Section 9C Complexation Titrations Section 9D Redox Titrations Section 9E Precipitation Titrations Section 9F Key Terms Section 9G Chapter Summary Section 9H Problems Section 9I Solutions to Practice Exercises Titrimetry, in which volume serves as the analytical signal, made its first appearance as an analytical method in the early eighteenth century. Titrimetric methods were not well received by the analytical chemists of that era because they could not duplicate the accuracy and precision of a gravimetric analysis. Not surprisingly, few standard texts from the 1700s and 1800s include titrimetric methods of analysis. Precipitation gravimetry developed as an analytical method without a general theory of precipitation. An empirical relationship between a precipitate’s mass and the mass of analyte— what analytical chemists call a gravimetric factor—was determined experimentally by taking a known mass of analyte through the procedure. Today, we recognize this as an early example of an external standardization. Gravimetric factors were not calculated using the stoichiometry of a precipitation reaction because chemical formulas and atomic weights were not yet available! Unlike gravimetry, the development and acceptance of titrimetry required a deeper understanding of stoichiometry, of thermodynamics, and of chemical equilibria. By the 1900s, the accuracy and precision of titrimetric methods were comparable to that of gravimetric methods, establishing titrimetry as an accepted analytical technique. 411 412 Analytical Chemistry 2.0 9A Overview of Titrimetry We are deliberately avoiding the term In titrimetry we add a reagent, called the titrant, to a solution contain- analyte at this point in our introduction ing another reagent, called the titrand, and allow them to react.
    [Show full text]
  • Titration Endpoint Challenge
    Analytical and Bioanalytical Chemistry (2019) 411:1–2 https://doi.org/10.1007/s00216-018-1430-y ANALYTICAL CHALLENGE Titration endpoint challenge Diego Alejandro Ahumada Forigua1 & Juris Meija2 Published online: 1 January 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2018 We would like to invite you to participate in the Analytical practical aspects related to the sources of uncertainty of Challenge, a series of puzzles to entertain and challenge our this technique. readers. This special feature of “Analytical and Bioanalytical Origins of titrimetry date back to 1690s, when Wilhelm Chemistry” has established itself as a truly unique quiz series, Homberg (1652–1715) published the first report related to with a new scientific puzzle published every three months. an acidity measurement [1]. Several decades later Claude Readers can access the complete collection of published prob- Geoffroy (1729–1753) used this method to determine the lems with their solutions on the ABC homepage at http://www. strength of vinegar by adding small amounts of potassium springer.com/abc. Test your knowledge and tease your wits in carbonate until the no further effervescence was observed diverse areas of analytical and bioanalytical chemistry by [2]. William Lewis (1708–1781), who is also considered one viewing this collection. of the early pioneers of titration, recognized the difficulty in In the present challenge, titration is the topic. And please determining the endpoint of the titration through the process note that there is a prize to be won (a Springer book of your of cessation of effervescence, so he suggested the use of color choice up to a value of €100).
    [Show full text]
  • Analytical Chemistry Laboratory Manual 2
    ANALYTICAL CHEMISTRY LABORATORY MANUAL 2 Ankara University Faculty of Pharmacy Department of Analytical Chemistry Analytical Chemistry Practices Contents INTRODUCTION TO QUANTITATIVE ANALYSIS ......................................................................... 2 VOLUMETRIC ANALYSIS .............................................................................................................. 2 Volumetric Analysis Calculations ................................................................................................... 3 Dilution Factor ................................................................................................................................ 4 Standard Solutions ........................................................................................................................... 5 Primary standard .............................................................................................................................. 5 Characteristics of Quantitative Reaction ......................................................................................... 5 Preparation of 1 L 0.1 M HCl Solution ........................................................................................... 6 Preparation of 1 L 0.1 M NaOH Solution ....................................................................................... 6 NEUTRALIZATION TITRATIONS ...................................................................................................... 7 STANDARDIZATION OF 0.1 N NaOH SOLUTION ......................................................................
    [Show full text]
  • Comparison of Vitamin C Content in Citrus Fruits by Titration and High Performance Liquid Chromatography (HPLC) Methods
    International Food Research Journal 24(2): 726-733 (April 2017) Journal homepage: http://www.ifrj.upm.edu.my Comparison of vitamin C content in citrus fruits by titration and high performance liquid chromatography (HPLC) methods 1Fatin Najwa, R. and 1,2*Azrina, A. 1Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, 43400 UPM Serdang, Selangor, Malaysia 2Research Centre of Excellence, Nutrition and Non-communicable Diseases, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Article history Abstract Received: 29 June 2015 Vitamin C is one of the essential vitamins for human and animal. Many methods were Received in revised form: developed for the determination of vitamin C such as spectrophotometry, electrophoresis, 23 March 2016 titration, and high performance liquid chromatography (HPLC). This study aims to compare Accepted: 4 April 2016 vitamin C content of citrus fruits (orange, grapefruit, lemon, lime, kaffir lime and musk lime) using indophenol titration and HPLC-PDA methods. In the titration method, orange has the highest vitamin C content (58.30 mg/100g) followed by grapefruit (49.15 mg/100g), lemon Keywords (43.96 mg/100g), kaffir lime (37.24 mg/100g), lime (27.78 mg/100g) and musk lime (18.62 Vitamin C mg/100g). While, in the HPLC method orange also leads with the highest vitamin C content Ascorbic acid (43.61 mg/100g) followed by lemon (31.33 mg/100g), grapefruit (26.40 mg/100g), lime (22.36 Citrus fruits mg/100g), kaffir lime (21.58 mg/100g) and musk lime (16.78 mg/100g).
    [Show full text]
  • Determination and Identification of Fundamental Amino Acids by Thermometric Titration and Nuclear Magnetic Resonance Spectroscopy
    Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1967 Determination and Identification of Fundamental Amino Acids by Thermometric Titration and Nuclear Magnetic Resonance Spectroscopy. William Yong-chien Wu Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Wu, William Yong-chien, "Determination and Identification of Fundamental Amino Acids by Thermometric Titration and Nuclear Magnetic Resonance Spectroscopy." (1967). LSU Historical Dissertations and Theses. 1274. https://digitalcommons.lsu.edu/gradschool_disstheses/1274 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. This dissertation has been microfilmed exactly as received 67-8805 WU, William Yong-Chien, 1939- DETERMINATION AND IDENTIFICATION OF FUNDAMENTAL AMINO ACIDS BY THERMOMETRIC TITRATION AND NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY. Louisiana State University and Agricultural and Mechanical College, Ph.D., 1967 Chemistry, analytical University Microfilms, Inc., Ann Arbor, Michigan Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DETERMINATION AND IDENTIFICATION OF FUNDAMENTAL AMINO ACIDS BY THERMOMETRIC TITRATION AND NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Chemistry by William Yong-Chien Wu B.A., Hardin Simmons University, I96I January, I967 Reproduced with permission of the copyright owner.
    [Show full text]
  • Determination of the Chemical Concentrations
    American Journal of Analytical Chemistry, 2014, 5, 205-210 Published Online February 2014 (http://www.scirp.org/journal/ajac) http://dx.doi.org/10.4236/ajac.2014.53025 Online Process Control of Alkaline Texturing Baths: Determination of the Chemical Concentrations Martin Zimmer, Katrin Krieg, Jochen Rentsch Department PV Production Technology and Quality Assurance, Fraunhofer Institute for Solar Energy Systems, Freiburg, Germany Email: [email protected] Received December 16, 2013; revised January 20, 2014; accepted January 28, 2014 Copyright © 2014 Martin Zimmer et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor- dance of the Creative Commons Attribution License all Copyrights © 2014 are reserved for SCIRP and the owner of the intellectual property Martin Zimmer et al. All Copyright © 2014 are guarded by law and by SCIRP as a guardian. ABSTRACT Almost every monocrystalline silicon solar cell design includes a wet chemical process step for the alkaline tex- turing of the wafer surface in order to reduce the reflection of the front side. The alkaline texturing solution contains hydroxide, an organic additive usually 2-propanol and as a reaction product silicate. The hydroxide is consumed due to the reaction whereas 2-propanol evaporates during the process. Therefore, the correct reple- nishment for both components is required in order to achieve constant processing conditions. This may be sim- plified by using analytical methods for controlling the main components of the alkaline bath. This study gives an overview for a successful analytical method of the main components of an alkaline texturing bath by titration, HPLC, surface tension and NIR spectrometry.
    [Show full text]
  • The Birth of Titrimetry: William Lewis and the Analysis of American Potashes
    66 Bull. Hist. Chem., VOLUME 26, Number 1 (2001) THE BIRTH OF TITRIMETRY: WILLIAM LEWIS AND THE ANALYSIS OF AMERICAN POTASHES Frederick G. Page, University of Leicester. William Lewis (1708- In order to place 1781), was a physician, author, Lewis’s work in the context and an experimental chemist. of the early beginnings of Sometime after 1730 he gave the industrial revolution in public lectures in London on Britain, Sivin (4) has used a chemistry and the improve- chronological argument; he ment of pharmacy and manu- cites Ashton’s suggestion facturing arts (1). With a grow- (5) that 1782 was the begin- ing reputation as a chemical ning of the industrial revo- experimentalist he was elected lution because in that year F.R.S., on October 31,1745, most statistics indicated a and was then living in Dover sharp increase in industrial Street, London. In 1747 he production. But, as Sivin moved to Kingston-upon- argues, it seems reasonable Thames, where he set up a to assume that by the time well-equipped laboratory and such early statistics became presumably continued in medi- available, those industries cal practice. From about 1750 that had created salable until his death in 1781 Lewis products had already be- employed Alexander Chisholm come established and were as his assistant in chemical and no longer in their early literary works (2). These im- years of founding. On this proved the knowledge and basis the industrial revolu- practice of pharmacy, but as a tion must have begun ear- practical consulting chemist lier. By the middle of the Lewis has received little bio- eighteenth century the so- graphical recognition.
    [Show full text]
  • High Performance Liquid Chromatographic Assay of Chlorpropamide, Stability Study and Its Application to Pharmaceuticals and Urine Analysis
    Open Access Austin Journal of Analytical and Pharmaceutical Chemistry Research Article High Performance Liquid Chromatographic Assay of Chlorpropamide, Stability Study and its Application to Pharmaceuticals and Urine Analysis Basavaiah K1* and Rajendraprasad N2 1Department of Chemistry, University of Mysore, Mysuru, Abstract Karnataka, India Chlorproamide (CLP) is a sulphonyl-urea derivative used in the treatment 2PG Department of Chemistry, JSS College of Arts, of type 2 diabetes mellitus. A rapid, sensitive and specific HPLC method with Commerce & Science, B N Road, Mysuru, Karnataka, uv detection is described for the determination of CLP in bulk and tablet forms. India The assay was performed on an Inertsil ODS 3V (150mm × 4.6mm; 5µm *Corresponding author: Basavaiah K, Department particle size) column using a mixture of phosphate buffer (pH 4.5), methanol of Chemistry, University of Mysore, Manasagangothri, and acetonitrile (30:63:7v/v/v) as mobile phase at a flow rate of 1mLmin-1 and Mysuru, Karnataka, India with uv detection at 254nm. The column temperature was 30ᴼC and injection volume was 20µL. The retention behaviour of CLP as a function of mobile phase Received: February 27, 2017; Accepted: March 16, pH, composition and flow rate was carefully studied, and chromatographic 2017; Published: March 22, 2017 conditions, yielding a symmetric peak with highest number of theoretical plates, were optimized. The calibration curve was linear (r = 0.9999) over the concentration range 0.5 – 300 µgmL-1. The limits of detection (LOD) and quantification (LOQ) were found to be 0.1 and 0.3 µgmL-1, respectively. Both intra-day and inter-day precisions determined at three concentration levels were below 1.0% and the respective accuracies expressed as %RE were ≤1.10%.
    [Show full text]
  • Isothermal Titration Calorimetry and Differential Scanning Calorimetry As Complementary Tools to Investigate the Energetics of Biomolecular Recognition
    JOURNAL OF MOLECULAR RECOGNITION J. Mol. Recognit. 1999;12:3–18 Review Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition Ilian Jelesarov* and Hans Rudolf Bosshard Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland The principles of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) are reviewed together with the basic thermodynamic formalism on which the two techniques are based. Although ITC is particularly suitable to follow the energetics of an association reaction between biomolecules, the combination of ITC and DSC provides a more comprehensive description of the thermodynamics of an associating system. The reason is that the parameters DG, DH, DS, and DCp obtained from ITC are global properties of the system under study. They may be composed to varying degrees of contributions from the binding reaction proper, from conformational changes of the component molecules during association, and from changes in molecule/solvent interactions and in the state of protonation. Copyright # 1999 John Wiley & Sons, Ltd. Keywords: isothermal titration calorimetry; differential scanning calorimetry Received 1 June 1998; accepted 15 June 1998 Introduction ways to rationalize structure in terms of energetics, a task that still is enormously difficult inspite of some very Specific binding is fundamental to the molecular organiza- promising theoretical developments and of the steady tion of living matter. Virtually all biological phenomena accumulation of experimental results. Theoretical concepts depend in one way or another on molecular recognition, have developed in the tradition of physical-organic which either is intermolecular as in ligand binding to a chemistry.
    [Show full text]
  • CSUS Department of Chemistry Experiment 4: Practice Titration Chem
    CSUS Department of Chemistry Experiment 4: Practice Titration Chem. 1A Experiment 4: Practice Titration A "titration" is a common laboratory method of quantitative chemical analysis that is used to determine the unknown concentration of a known analyte. Titrations belong to class of analytical techniques known as "volumetric analysis". Since volumes can be precisely delivered and measured using standard laboratory equipment, titration techniques can yield both accurate and precise measurements if care is taken by the analyst. Accuracy is defined as the closeness of a result (usually the average of several measurements) to a known accepted value. Precision relates the closeness of the measurements themselves. In any analytical experiment, one strives for both accuracy and precision to validate the results. In this experiment you will be graded on both your accuracy (how close you are to the actual value) and the precision of your results. (how reproducible they are) In a titration an analyst titrates a solution of unknown concentration with a solution of known concentration (aka a standard solution). The stoichiometry of the reaction between the standard and the analyte is known. Using a calibrated "buret" to add the standard solution, it is possible to determine with accuracy the amount of analyte present in the unknown solution when the titration endpoint is reached. The endpoint of a titration is that point at which the titration is complete. The endpoint is generally signaled by an indicator (see below) that was added to the analyte solution. This is ideally the same volume or very close to as the equivalence point—the volume of added titrant at which the number of moles of titrant is equal to the number of moles of analyte, or some multiple thereof.
    [Show full text]
  • Complexometric Titration and Atomic Emission Spectroscopy) That Allow Us to Quantify the Amount of Each Metal Present Individually
    Title Quantitative Analysis of a Solution Containing Cobalt and Copper Name Manraj Gill (Lab partner: Tanner Adams) Abstract In this series of experiments, we determine the concentrations of two metal species, copper and cobalt, in a mixture of the two by initially using an ion exchange column to separate them from the mixture. We consequently use two different techniques (complexometric titration and atomic emission spectroscopy) that allow us to quantify the amount of each metal present individually. This step-by-step analysis not only goes through the basis for the experimental approach but also is a breakdown of the measurements that leads us to the final results: the Co:Cu ratio in the unknown mixture. Purpose In this series of three experiments (Parts I – III), we aim to initially separate a solution containing a mixture of Cobalt, Co (II), and Copper, Cu (II), ions in an unknown ratio and in unknown concentrations. The end goal of the entire experiment is to determine how much Co and Cu were originally present in the sample we obtained in Part I. But to do so, as explained in further detail in the ‘Theory and Methods’ section below, we initially separate the two ions (Part I) based on their unique chemical properties and then we attempt to quantify the separated ions through two different methods (Parts II, III). The overall purpose is an analysis of the efficiency of the separation and quantification approaches used to measure the constituent compounds in a mixture. This “efficiency” can be determined by comparing the obtained measurements to the known amounts that were used to make the solution.
    [Show full text]