Issue 3 • 2012
AnalytixPolymer Reference Materials
• Polymer Reference Materials • Certified Reference Materials
for Genotoxics • LC/MS Test Mixtures • Rapid Screening for Micro
organisms • HPLC Buffers • Ionic Liquids in MALDIMS
Applications • Water Content in Aldehydes
and Ketones
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Polymer Reference Materials
Dear Reader, Cotton shirts, laptops, children’s toys, plastic bags, tires, pencils … All of these diverse items in our everyday lives have something in common – they all consist of polymers, either synthetic ones such as polyolefin, polyurethane, polypropylene or polyacrylate, or natural ones such as cellulose, starch, or caoutchouc. The molecular weight and the molecular weight distribution of polymers are crucial parameters that determine their specific properties. Therefore, we can assume that most of these polymers that surround us have undergone a rigid quality control with special focus on their molecular weight distribution.
The analytical technique of choice is gel permeation chromatography (GPC). The diversity of application fields for this technique is almost as huge as the diversity of the polymers that exist. Its use is not only important in the plastic and textile industry, but also in both the food and pharma industries for testing the properties of excipients of pharmaceuticals.
To ensure the accuracy of these measurements, the use of reliable analytical standards or certified reference materials is crucial. This is important because you can only really trust your analytical results if you can trust the quality of your reference materials. The featured article of this issue of Analytix (page 4) gives an overview of the wide variety of polymer standards for the calibration of GPC instruments available at SigmaAldrich.
To gain insight into our comprehensive portfolio of analytical standards and reagents, I recommend that you visit our webpage at sigma-aldrich/analytical and hope you enjoy this issue of Analytix.
Best regards,
Dr. Matthias Nold Product Manager Analytical Standards [email protected]. Matthias Nold
Product Manager Analytical Standards
Analytix is published five times per year by Sigma-Aldrich Chemie GmbH, MarCom Europe, Industriestrasse 25, CH-9471 Buchs SG, Switzerland Publisher: Sigma-Aldrich Marketing Communications Europe Publication director: Ingo Haag, PhD Editor: Isabell Davies-Falch
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Chromatography
14 Reversed-phase HPLC Buffers High quality buffers (solutions, solids or
concentrates)
15 High-Purity Headspace Grade N,N-Dimethylacetamide in the Analysis of Residual Solvents
17 New Solvents and Additives Exclusively Designed for UHPLC
LCMS Ultra CHROMASOLV®
Spectroscopy
18 Second Generation Ionic Liquid Matrices for MALDI-MS
Titration
19 Volumetric Concentrates for Titration by Sigma-Aldrich
Prepare Acid, Base and Salt Standard Solutions with FIXANAL® Ampoules
21 Determination of Water Content in Aldehyde and Ketone Samples
HYDRANAL®K Reagent Line for Karl Fischer Titration
Featured Article
4 Applications and Quality Requirements of Polymer Reference Materials
Standards
7 Certified Reference Materials for Genotoxic Sulfonic Acid Esters
8 Prescreened, In-Stock Chemicals Pesticides, PAHs, PCBs, VOCs, and More
8 Quality Antioxidant Standards in Convenient Kit
9 Maximize Sample Recovery with Center Drain (CD™) Vials
10 Is Your LC/MS System All Right? Mass Calibration and System Test Standards
Microbiology
12 PCR & DNA was Yesterday; the Future is RNA HybriScan®, a rapid and innovative screening
method for microorganisms based on the detection of rRNA.
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Applications and Quality Requirements of Polymer Reference Materials
The use of polymer reference standards for the calibration of GPC/SEC instruments with concentration detectors or viscometers is a wellknown procedure [1]. In addition, many other instances exist where polymer reference standards are used routinely. All these applications require well characterized reference materials. In this paper, we will discuss quality requirements for polymer standards as well as their scope of application.
Types of Reference MaterialsIn general, polymers are statistically distributed polymer chains. Controlled polymerization processes minimize the chain length distribution. They cannot be described by a single molar mass but by various molar mass average values.
Polymer reference materials are precisely characterized polymers in terms of molar mass average values, such as the molar mass average number (Mn) and the molar mass average weight (Mw). Besides this, the molar mass on the peak maximum (Mp) is important for narrow molar mass standards. Another significant parameter is the polydispersity index PDI (Mw/Mn) which describes the width of the molar mass distribution. We make a differentiation between narrow molar mass distribution (PDI <1.2) and broad molar mass distribution (PDI>1.5).
The reference materials can be based on a single molecule species (homopolymer), on two (copolymer) or on three different monomeric molecules (terpolymer). Copolymers and terpolymers are structured as block copolymers or statistical polymers.
Polymer reference standards can show a welldefined structure, e.g. stars, combs, hyperbranched polymers, or even a precisely defined stereo structure (e.g. tacticity). Specific end groups are also possible.
Polymer reference materials are available as single standards or as calibration kits with wellcharacterized standards of different molar masses. Due to the different complexity of requirements, reference materials are very often designed and characterized according to the needs of the individual user.
Quality Requirements for Polymer Reference MaterialsEspecially in a regulated environment, particular attention is usually paid to the measurement results. This also applies to the molar mass results from GPC/SEC analysis. If possible, the information in the analysis certificates should be well proven and include the measurement uncertainty. The traceability of certificate information is important, i.e. the question of whether the measurements are traceable to generally accepted reference values or at least to the values of an absolute method. Of key importance is information on shelf life and stability as well as longterm and timely quality control by the manufacturer over the entire sales and shelf life of the materials. The reference materials described meet the challenge of the demand for highquality reference standards with accurate measurements. Constant quality control of our different polymer batches available for sale is standard practice. In addition to the classic GPC/SEC reference standards (molar mass values from GPC/SEC measurements, calibration results based on light scattering), more elaborate reference materials are also available. The difference here refers to quality criteria in terms of peak purity, polydispersity, number and type of characterization methods used for molecular weight determination and the application area. Table 1 shows the methods used for the characterization of polymer reference materials.
T. Hofe, H.-U. Ehmcke, M. Nold [email protected]
Classification of Polymer Standards
Characterization Methods Available as Individual Standard
Available as Kit
GPC organic and aqueous GPC DIN certified GPC + absolute method (NMR, light scattering, viscometry or
vapour pressure osmometry (VPO)
ERM GPC + scattering + viscosity and physical constants, Roundrobin test
LS / Visco Validation GPC+light scattering + viscosity (NMR, light scattering, viscometry or vapour pressure osmometry (VPO)
Maldi Validation GPC + MALDI + absolute method ReadyCal GPC
Table 1 Characterization of polymer reference materials
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ReadyCal KitsPSS ReadyCal Standards (Figure 1) are polymer cocktails preweighted into autosampler vials. Each vial has three or four polymers of the same type with different molar masses. Carefully selected molar masses are placed into each vial so that the resulting chromatograms are baseline separated (if a proper column combination is used).
A ReadyCal Kit allows you to prepare a fast and reproducible eight to twelvepoint calibration curve
without the inconvenience of weighing samples. Just add solvent directly into the autosampler vial, let it stand for two hours, shake gently, and load into your autosampler for analysis. Each kit contains 30 autosampler vials (for at least ten calibration curves) that are color coded for your convenience.
DIN (German Institute for Standards) Certified StandardsPolymers characterized in the respective solvent (water or THF) according to GPC/SEC DIN requirements are called the DIN standards. In addition to GPC/SEC measurements (relative method), the molar mass of each polymer is determined by an absolute method such as light scattering detection MALS/MALLS (Mw), NMR, vapour pressure osmometry VPO (Mn) or MALDI (Mw, Mn). The results and the experimental conditions are also specified. DIN standards are available as individual standards or kits.
Round-Robin Tested Reference MaterialsThe premium reference materials are the European Reference Materials (ERM) of the German Federal Institute for Material Research and Testing (BAM) in Berlin. These ERM are roundrobin tested. The tests were coordinated by BAM and performed by selected laboratories under standard
ized conditions. In addition to the GPC/SEC experiments, the roundrobin tests also cover viscosity and lightscattering measurements. Thus, the molar mass results of these three methods are independent from laboratory and operator. The results were statistically evaluated and besides the average values, the measurement uncertainties as well as the standard deviation of the roundrobin test results were also quantified. In addition, the report also includes the results from NMR and IR measurements. Figure 2 shows the certificate of an ERM.
Validation kitsValidation kits allow the specific testing of complex systems and detectors as well as the determination of specific system and detector constants.
A light scattering/viscometry validation kit consists of narrow and broad distribution polymer standards (aqueous applications: dextran; organic applications: polystyrene, PMMA). The normal molar mass information, Mp from GPC/SEC and the MarkHouwink constants K and α from the viscosity measurements, the molar mass average values Mw and Mn from GPC/SEC, MALLS and viscometry are also provided. Thus, online viscosity and lightscattering detectors can be checked. With the help of the information provided, complex GPCMALLSviscosity detector couplings can be validated and corrected, if necessary. For example, using the K and α constants, it is possible to check and correct the interdetector offset of the viscosity detector compared to the concentration detector (RI or UV).
MALDI kits are available as homologous polymers of different molecular weights or as a mix of different polar polymers. In addition to enabling the measurement of the resolution of the spectrometer, they also help select the appropriate matrix for a particular class of polymers. The certificates contain the comparison of the molar mass information from a relative method with two absolute methods based on MALDI, NMR, VPO or MALS/MALLS. In addition, the MALDI spectrum, and the molecular weight distribution MWD from GPC/SEC, are provided.
Application for Polymer Reference StandardsSome of the practical uses of polymer reference materials are listed below.
Homopolymer Standards with Narrow Molar Mass Distribution • The most widely used application for homopolymer
standards with narrow molar mass distribution is the calibration of a GPC/SEC system by narrow standard calibration. The GPC/SEC calibration is performed to correlate the elution volume to a molar mass. Therefore, solutions of different standards are injected into the chromatograph. To create the calibration curve, the logarithm of the molar mass is plotted against the molar mass [1]. A typical calibration curve is shown in Figure 3.
• Universal calibration allows the conversion of existing calibrations to other types of polymers. The method is based on the assumption that the property which determines the elution behavior in GPC is the hydrodynamic volume Vh [1].
(continued on page 6)
Figure 1 PSS ReadyCal kit
Figure 2 Analysis certificate of European Reference Material (ERM)
Certificate ERM®-FA001 All following pages are an integral part of the certificate.
Page 1 of 4
CERTIFICATE OF ANALYSIS
ERM®-FA001
POLYSTYRENE
Molar mass Mw
1)
Weight averaged molar mass Mw
Certified value 3) [g/mol]
Uncertainty 4)
[g/mol]
87600 2245
Viscosity 2)
Intrinsic viscosity [η]
Certified value 3) [mL/g]
Uncertainty 4)
[mL/g]
42.37 0.83
1) obtained by laser light scattering
2) obtained by viscometry using an UBBELOHDE viscometer according to DIN 51562 – 1
3) Unweighted mean value of the means of accepted sets of data, each set being obtained in a different laboratory. The certified value is traceable to the International System of units (SI).
4) The certified uncertainty is the expanded uncertainty estimated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) with a coverage factor k = 2, corresponding to a level of confidence of about 95 %. The certified uncertainty value is traceable to the International System of units (SI).
This certificate is valid for five years after purchase. This validity may be extended as further evidence of stability becomes available.
Sales date:
The minimum amount of sample to be used is 10 mg.
Accepted as an ERM®, Berlin,
Latest revision Berlin, 2008-05-29 BAM Berlin Department I Analytical Chemistry; Reference Materials 12200 Berlin, Germany
BAM Berlin Division I.3 Structural Analysis 12200 Berlin, Germany
Prof. Dr. U. Panne Dr. A. Thuenemann (Head of Department) (Head of Division)
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• The qualification and validation of instrumentation used for the analysis of natural or synthetic polymers and biopolymers. This includes:
– The GPC/SEC system including all detectors (RI,UV, ELSD, lightscattering detector/viscometer)
– MALDToF instruments
– Detectors for dn/dc determination in batch mode
• Normalization of MALLS/MALS (Multiangle {Laser} Light Scattering) detectors to obtain a precise determination of the radius of gyration, which describes the size of the molecule in solution.
• Inverse GPC/SEC allows the determination of the average pore size as well as pore size distribution of a material. The test material is filled in a column, which is installed in a GPC/SEC system, and flushed with eluent. Then the reference standards are injected. The elution volume and the molar mass of the reference material enables the calculation of the pore size parameter mentioned [2].
• To determine structure property relationships.
Homopolymer Standards with Broad Molar Mass Distribution • Broad standard calibration allows the precise analysis
of polymers even if narrow standards are not available for the specific polymer. It requires the existence of a base calibration curve and one or more broad polymer samples with given Mw, Mn or intrinsic viscosity [η] [1].
• Allow the detection of column mismatch. This is a shoulder or side peak that does not result from the physical reality of a molar mass distribution but from uneven or dislocation of pore size distributions [3].
• Characterization of membranes by sieve curve determination. The membrane to be characterized is used to filter a stock solution of a broad molar mass/molecular size distribution reference standard. The stock solution, the filtrate, and optionally the retained fraction (retentate) are analyzed with a GPC system. The comparison of these results allows the determination of important membrane properties such as average pore size, pore size distribution, molar mass cutoff, membrane selectivity, retention efficiency, prep accessibility, membrane stability or hydrophobic properties (see Figure 4). The sieve curves can help to identify lowquality membranes and establish quality parameters for working membranes [4].
Polymers with dedicated properties are used for determination of physical properties if precise molar mass distribution, structure, or composition distribution is a need.
Summary The versatile structure of polymers, and complex analytical problems, all require a variety of reliable and wellcharacterized reference materials to assure the quality of analytical results. This includes not only the analysis itself but also the operational check of the analytical instruments used. Polymer reference standards are well accepted in various fields of operation such as QC, R&D and in the world of science, as well as in the world of industry and production. They help to gain a deeper insight into the structure and property relations of macromolecules. Please visit the SigmaAldrich website at sigma-aldrich.com/polymerstandards to find the extensive range of polymer standards and certified reference materials.
AuthorsProf. Dr. Thorsten Hofe and Dr. HansUlrich Ehmcke PSS Polymer Standards Service GmbH In der Dalheimer Wiese 5, 55120 Mainz/Germany
References[1] Yau, W.W., Kirkland, J.J., D.D. Bly, Modern Size-Exclusion Liquid
Chromatography, John Wiley & Sons, New York, Chichester, Brisbane, Toronto, 1979.
[2] Dauwe, C., and Marme, S., Quality Assurance with Inverse GPC, GIT Laboratory Journal, 3, 106 (2002).
[3] Hofe, T., Beware of Mismatch, The Column, 4,21 (2008).[4] Kilz, P., Sieve Curves – Membrane Characterization Using
GPC/SEC, PSS Streamliner, 1,1 (2007).
Figure 3 Typical GPC/SEC calibration curve using narrow standards
Figure 4 Comparison of three different sieve curves obtained by a filtration experiment and GPC/SEC measurements. A stock solution has been prepared from a broad polymer standard. The analysis of the filtrate and the stock solution by GPC/SEC allows the creation of sieve curves.
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Certified Reference Materials for Genotoxic Sulfonic Acid Esters
Sulfonic acid derivatives, such as methanesulfonic acid (MSA), are frequently used in pharmaceutical chemistry. Mesylate salts of aminecontaining drug substances may have advantages over the more commonly used hydrochloride salts, such as higher solubility and bioavailability and the absence of an ion effect [1].
However, there are severe concerns about contamination with alkyl sulfonate ester traces, either as side products resulting from the crystallization and recrystallization process using methanol or ethanol as a solvent or as impurities of the MSA starting material. These sulfonic acid esters are known to have genotoxic and carcinogenic effects [2].
Therefore, not only the final pharmaceutical product, but also the alkyl sulfonic acids used as starting material for the salt formation must be meticulously tested for the presence of these compounds.
The new SAFC Pharma® Grade Product line provides GMP compliant building blocks and reagents for use in the pharmaceutical production (sigma-aldrich.com/pharmagrade). To support our quality control of these products, TraceCERT® organic CRMs have been developed for the methyl and ethyl esters of methanesulfonic acid and ethanesulfonic acid, as well as for other pharma grade products such as LMethionine sulfoximine and methyl laureate.
The specifications of the methanesulfonic acid pharma grade product include limits of 5ppm (GC) for ethyl methanesulfonate as well as for methyl methanesulfonate. For calibration of these GC measurements, the corresponding TraceCERT CRMs have been used. The CRMs we use for our internal quality control are also available for our customers as TraceCERT catalogue products.
Organic TraceCERT products are highquality certified reference materials (CRMs) manufactured according to ISO/IEC 17025 and ISO Guide 34 with direct traceability to NIST SRM. Traceability is achieved by using highperformance quantitative NMR (HPqNMR®) as a relative primary method. The big advantage of this method is that, unlike most analytical techniques, the signals of the integrals are not dependent on the structure of the compound. There
fore, different compounds can be quantitatively compared with high accuracy. Detailed technical articles about the organic TraceCERT certification concept have been published in previous issues of Analytix [3].
Currently almost 100 organic TraceCERT products are available, ranging from amino acids, natural products, PAHs, fatty acids/FAME, pesticides to pharmaceuticals. Table 1 shows recent product additions relevant for the quality control of the SAFC Pharma Grade Products. A complete and uptodate product list can be found on our website sigma-aldrich.com/organiccrm together with technical articles and example certificates.
Cat. No. Brand Product Package Size
30934 Fluka® Ethyl ethanesulfonate 120 mg
72945 Fluka Ethyl methanesulfonate 120 mg
50490 Fluka Methyl ethanesulfonate 120 mg
78697 Fluka Methyl methanesulfonate 120 mg
91016 Fluka LMethionine sulfoximine 100 mg
07041 Fluka Methyl laurate 100 mg
Table 1 TraceCERT Organic CRMs
Cat. No. Brand Product Package Size
11317 SigmaAldrich Methanesulfonic Acid 1 kg
76078 SigmaAldrich LMethionine sulfoximine 1 g; 5 g
39937 SigmaAldrich Methyl Laurate 250 g
Table 2 Examples of SAFC Pharma Grade Products (GMP)
References[1] Snodin D.J; Regulatory Toxicology and Pharmacology,
45 (2006); 79 –90.[2] Eder E; Kutt W; Deindinger C; Annals Chem. Biol. Interact.
137 (2001), 89 –99.[3] Analytix 03/2010; 01/2011; 03/2011; 04/2011; 01/2012.
Matthias Nold [email protected] Detterbeck [email protected]
H3O OCH3
OOSH3O CH3O
O
O
SH3C
OCH3
O
O
SCH3O
O
O
SH3C
NH
OS
OHO
H3C
H2N
H
Figure 2 Chemical structures of the sulfonic acid esters
Figure 1 Chemical structure of L-Methionine sulfoximine and methyl laurate
CH3(CH2)9CH2 OCH3
O
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Prescreened, In-Stock ChemicalsPesticides, PAHs, PCBs, VOCs, and More
Quality Antioxidant Standards in Convenient Kit
What do these compounds have in common?They are examples of the thousands of prescreened, instock chemicals available through the SigmaAldrich custom standards group. We can formulate, test, and package custom standard solutions to meet your needs for all your chromatographic applications. Our custom standard chemists will gladly discuss stability and solubility concerns with you, and make suggestions where needed to improve the quality of your purchase.
You can rely on Sigma-Aldrich custom standard solutions to include: • Raw materials and solvents screened for identity
and purity
• Your choice of gravimetric, qualitative, and quantitative testing
• Packaging choices from ampuls to bottles
• Manufacturing processes following the guidelines of ISO 9001/2000
• Proper handling of light and/or oxygensensitive chemicals
• Documentation and Material Safety Data Sheets
Antioxidants are added to food and other products to prevent rancidity. Although the mechanisms are not clearly understood, the antioxidants react with free radicals and peroxides slowing the rancidity. Certain other additives greatly enhance the effectiveness of antioxidants. Metal scavenger and chelating agents, such as citric acid and citrates, tie up the trace metals and greatly reduce their catalytic activity. Synergism between antioxidants has also been noted, and many commercial antioxidant mixes are formulated to contain mixtures of the antioxidants. The most common of these mixtures contain both butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT).
In order to ensure consistent product quality, these additives must be monitored. In the past, food analysts had to go to multiple vendors to obtain quality standards of all the antioxidants they may be required to monitor. SigmaAldrich offers an antioxidant standards kit containing several of the antioxidants listed in Association of Official Analytical Chemists Method 983.15: Phenolic Antioxidants, Fats, and Butter
• Free technical support
• Strict adherence to all shipping regulations
If you are interested in a customized standard, please email us at [email protected], or complete the online custom standards quote request form, available 24/7, at our website sigma-aldrich.com/standards
Oil plus ethoxyquin, an additional antioxidant commonly used in spices and other food products, and in cosmetics. Each antioxidant has been evaluated for purity, then packaged neat under nitrogen. A certificate of analysis for each antioxidant is included with the kit.
Reference[1] Official Methods of Analysis (17th Ed.), Method 983.15.
Association of Official Analytical Chemists, Arlington, VA USA (1996).
Description Cat. No.
Phenolic Antioxidant Kit500 mg each, individually packagedtertButylhydroquinone (PG)Ethoxyquin2,3tertButyl4hydroxyanisol (BHA)2,6Ditertbutyl4hydroxymethylphenol (IONOX 100)3,5Ditertbutyl4hydroxytoluene (BHT)Lauryl gallateNordihydroguaiaretic acid (NDG)Octyl gallatePropyl gallate
40048U
Vicki Yearick [email protected]
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Maximize Sample Recovery with Center Drain (CD™) Vials
Features and Benefits: • Limited volume injections with less than 4 μL dead
volume
• Eliminates bent needle problem associated with limited volume inserts
• Will withstand temperatures from 70 °C to 160 °C
• Concentrate samples in the same vial that you use in autosampler
• Solid glass base offers better heat sink for concentrating samples
• Made from Type 1 Borosilicate glass
The patented Center Draining or CD Vial has a unique conical interior bottom that promotes draining of the vial’s contents into the center bottom of the vial, where virtually all of it can be extracted. The production process ensures consistency of quality and precision in the CD Vials. The conical well, located in the exact center of the vial bottom, features a smooth
transition from sidewall into the well, and the bottom thickness dimension is the same in each vial.
In comparison tests against other manufacturers’ conical highrecovery vials, the CD Vial allows maximum extraction (<4 μL dead volume), while the competitors’ vials allow the contents to hang up in the transition area, leaving several microliters of liquid still in the vials after extraction. As shown in Figure 1, there is a significant amount of sample remaining in the competitor’s vial on the right, while the CD Vial has yielded almost total recovery of the liquid in the vial.
Below is a list of the many applications for which these vials can be used. To learn more, email Technical Service at [email protected]
Applications • Library sample storage
• Lyophilization
• Cetrifugation
• Micro mixing
• Extraction
• Concentrations
• Derivatization
• Hybridization
• Anaerobic reactions
• Homogenization
• Smallscale reactions
• Catalytic hydrogenation
Ordering InformationDescription Cat. No.
Certified Center Drain CD Vials
12 x 32 mm, 1.5 mL, large opening, Pk/100
Screw top vial kits with blue cap and septa, 9 mm thread
Clear glass, PTFE/silicone septa 29307U
Clear glass, PTFE/silicone/PTFE septa 29308U
Clear glass, preslit PTFE/silicone septa 29309U
Amber glass, PTFE/silicone/PTFE septa 29313U
Amber glass, preslit PTFE/silicone septa 29314U
Snap top kits with caps and septa
Clear glass, blue cap with PTFE septa 29301U
Clear glass, blue cap with PTFE/silicone septa 29302U
Clear glass, blue cap with preslit PTFE/silicone septa 29303U
Crimp top vials (vial only)
Clear glass 29298UscriTr
Figure 1 Superior sample extraction from CD Vial compared to competitor vial
Trademark on CD owned by QIS, Inc.
Vicki Yearick [email protected]
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163.060 267.089
371.107
423.112475.116
527.133
578.153
630.178
682.177
734.167
786.173
838.198
994.198
+MS, 9.8-13.2min #(319-372)
148.907
200.907252.964
305.014357.044
409.073
461.121513.145
565.181
721.266
772.296824.316
876.342928.357
980.358
1032.381
1084.399
1136.403
-MS, 15.3-17.0min #(405-432)0
1
2
3
4
55x10
Intens.
0
2
4
6
8
4x10
200 400 600 800 1000 1200 m/z
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Is Your LC/MS System All Right? Mass Calibration and System Test Standards
System Test StandardsModern HPLC or UHPLC systems are very complex systems and should work properly with less attention during daily routine work. Annual maintenance and IQ/OQ/PQ verifications guarantee compliance with hardware specifications. But this includes only the HPLC and MS instruments themselves and is not related to the chromatographic application during daily routine work. System test standards help to monitor the present performance of a chromatographic system including different types of detectors and possible performance losses caused by intense usage of a column, high matrix load, long duty time of lamps, etc. Additionally, the system test standards may indicate the cause of a failure and shorten the time for troubleshooting.
Mass Calibration and System Tuning StandardsMost of the mass spectrometers have single quadrupole mass analyzers with an m/z resolution of +/ 0.5 Da, which is good enough for the differentiation of single isotopes. More complex spectrometers are equipped with additional quadrupole mass selectors and collision cells (triple quadrupoles), ion traps, timeofflight mass or FTmass analyzers (including Orbi Traps). These kinds of system need a regular optimization of ion transfer, ion storage, and mass scan calibration to guarantee a precise and sensitive detection and fragmentation of analytes. Mass calibration standards are needed depending on the chosen mass range. The determination of molecular formulas using highresolution and
precision spectrometers particularly depends on an exact mass calibration in a range from 50 –1000 Da.
Going Hand in Hand – Calibrating and Testing LC/UV/CAD/MS SystemsBefore starting LCMS data acquisitions, most of the mass spectrometers have to be calibrated to insure the mass precision, accuracy, and highest sensitivity of MS/MS isolation (ion traps) and fragmentation. Highresolution timeofflight spectrometers especially need to be regularly calibrated several times a day if maximum precision is needed. Salt solutions and polymer solutions generate regular m/z patterns in a broad m/z range. Lithium and sodium formate solutions are especially ideal for the calibration of m/z ranges up to 1500 Da, which is suitable for LCMS analysis of most small molecules using electrospray ionization. Another convenient feature of alkali formate clusters is the ability to use both ESI modes in one step. Figure 1 shows the mass spectra of lithium formate clusters (ESI +/).
HPLC Performance and MS Sensitivity TestingAlthough well designed and tested, HPLC/MS and even UHPLCsuitable systems contain several potential error sources. The connection to a mass spectrometer increases the possibility of a malfunction or loss of performance. There are several ways to test each component of the LCMS system, but this is a timeconsuming process primarily useful after extensive maintenance.
Rudolf Köhling, Namtso Reichlin [email protected]
Figure 1 ESI LCMS spectra of lithium formate solution (Fluka®) in positive (upper) and negative ion mode (bottom). The regular pattern of the [Li(x+1)(HCOO)x]+ and [Lix(HCOO)(x+1)] cluster can be used to calibrate a mass range up to 1200 m/z, which covers most of the small molecules.
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Figure 3 Acquired and calculated mass spectrum of reserpine. The difference of the peak position is only 0.7 ppm. The resolution is 11000 in standard resolution mode. This agrees with the specification of the manufacturer and indicates an optimum setup.
Figure 2 Influence of an aged column on the performance of a separation. 5 µl of a 5 ppm reserpine standard is injected and monitored over a column lifetime. Peak height and symmetry decreases. UHPLC separations especially react sensitively to instrumental errors (extra volume, valve malfunction, clogging, or performance loss in the stationary phase).
0 1 2 3 4 5 6 7 8 9 Time [min]0.0
0.2
0.4
0.6
0.8
1.0 5x10Intens.
609.2811
610.2845
611.2881
+MS, 5.4-5.4min #(418-420)
609.2807
610.2840
611.2873
C 33 H 41 N 2 O 9 ,609.280
1
2
3
4 4x10Intens.
0
1
2
3
4 4x10
609.0 609.5 610.0 610.5 611.0 611.5 612.0 612.5 613.0 m/z
On the other hand, daily routine work only allows for short interruptions to complete short, robust tests to monitor the performance of the UHPLC and mass spectrometer. The main focus lies on the sensitivity of the ion source and the efficiency of the chromatographic separation. Injecting a system test standard can identify a lack of performance of the HPLC system including the column by monitoring peak height, peak width, signalnoise ratio, and symmetry. If these parameters are regularly recorded on a control chart, then it is possible to observe changes immediately. Figure 2 shows the effect of a chromatography malfunction on the peak shape and position of the test compound reserpine. Impurities in the solvent lead to the signal suppression in MS.
Caffeine, reserpine, digoxin or other small molecules, peptides and proteins are suitable compounds to test both HPLC and MS. Spectral data gives additional information about the status of a detector. Figure 3 shows the acquired and calculated mass spectrum of reserpine, which is used to check the resolution of the spectrometer and the precision of calibration, e.g. with lithium formate solution. Caffeine is
also used for this purpose and can be applied for MS detection (low mass range) or UV detection. The UV spectrum of caffeine is useful to check the adjustment and resolution of the UV detector and the performance of the UV/VIS lamps [1].
Most of these standards are used to specify the performance of an instrument, which is useful for comparing the results of the tests with the instrument specifications.
Reference[1] J. Emmert, Labor & More, 5 (2008), 56-57.
Cat. No. Brand Description
01886 Fluka® Lithium Formate Solution, Suitable for LCMS, 10 mM LiOH in isopropanol/water 1:1 (+0.2% HCOOH)
97574 Fluka Sodium Formate Solution, Suitable for LCMS, 10 mM NaOH in isopropanol/water 1:1 (+0.2% HCOOH)
43530 Fluka Reserpine Standard for LCMS, analytical standard, for LCMS
56396 Fluka Caffeine, certified reference material, TraceCERT®
MSCAL11KT
Sigma ProteoMass Peptide and Protein MALDIMS Calibration Kit
D6003 Fluka Digoxin, analytical standard
new column used column with corrupted
stationary phase
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sigma-aldrich.com/hybriscan
Mic
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olog
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Food and beverages have to be of the highest quality to survive in a competitive market. Spoilage causing organisms or pathogens are problems which can cause huge financial loss and serious image damage. Fast and reliable systems need to be in place in order to avoid microbial problems. Many laboratories still use conventional standard based cultivation methods, which are often very time consuming and take two to seven days for the product to be released to the market.
HybriScan kits are rapid test systems that identify contaminants and lead to a faster product release. A variety of applications have been developed for HybriScan including the detection of bacteria and yeast in food and beverages. The robustness of the HybriScan assay enables it, in contrast to other rapid test systems, to detect contaminants under beneficial microorganisms or in a difficult matrix. Furthermore, the HybriScan test system is a perfect tool for microbiological control of dispensing equipment. Without a preenrichment procedure, results within two hours are possible.
Specificity is achieved by targeting conserved or unique rRNA sequences. A biotinlabeled capture probe is used to immobilize the target sequence on a solid support plate (streptavidincoated microtiter plate). A digoxigeninlabeled detection probe provides an enzymelinked optical signal readout. Detection results from application of antiDIGhorseradish peroxidase Fab fragments. The bound complex is visualized by horseradish peroxidase substrate TMB (3,3’,5,5’tetramethylbenzidine). Photometric data are measured at 450 nm and compared with standard solutions.
Comparison of HybriScan and other rapid test systemsPerforming quality control using the standard cultivation based method takes a long time. In recent years many companies have developed rapid test systems to hasten this procedure. For quality control of food and beverages, some main technologies are available:
• HybriScan (sandwich hybridization)
• PCR (Polymerase Chain Reaction)
• VIT (Vermicon Identification Technology)
• ELISA (Enzymelinked Immunosorbent Assay)
PCR & DNA were Yesterday; the Future is RNAHybriScan®, a rapid and innovative screening method for microorganisms based on the detection of rRNA.
Jvo Siegrist, Product Manager Microbiology [email protected]
A comparison of these different technologies is given in Table 1. Comparing HybriScan to PCR, ELISA or VITtechnology demonstrates that the benefits of this rapid test system are:
• Fast and costefficient analysis
• Inexpensive readout technology
• High sensitivity and specificity
• Low cross reactions
By using two different probes for detection of microbial RNA, falsepositive results are almost impossible. Comparison of the HybriScan test with a cultivationbased analytical method displays the equivalent results within the limits of microbiological sample variability (see Figure 2).
Figure 1 Bacterial ribosomal subunit consists of protein and rRNA
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20000
1 2 3 4 5 6 7 8 9 10 11 12 13 14
cells
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HybriScan®-CFU
sigma-aldrich.com/hybriscan
Mic
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The HybriScan® system can be used as a versatile microbiological detection and identification method. There is a wide variety of applications for using this innovative rapid test system with food, beverages, water and other specimens. It is possible to detect both a single species and a defined group of microorganisms.
Further information about the HybriScan method can be found at sigma-aldrich.com/hybriscan
Cultivation-based method
PCR ELISA VIT HybriScan
Method cultivationbased method with optical or microscopic readout
PCR/realtime PCR Immunological Assay fluorescence microscopy
sandwich hybridization and photometrical signal read out
Sample preparation enrichment enrichment and lysis enrichment enrichment, immobilisation
enrichment and lysis
Time 3 to 7 days 3 hours to 2 days 3 hours to 2 days 2 days 3 hours to 2 days
Costs per test €1 approx. €12 approx. €3 approx. €15 approx. €3 approx.
Detection limit (cfu) 1 1–5 x 103 103 – 105 103 1–5 x 103 bacteria 1–3 x 102 yeasts
Devices None PCR cycler microplate reader fluorescence microscope
microplate reader
Advantages high sensitivity, relatively inexpensive
high sensitivity, quantitative analysis
differentiation of serotypes or subspecies, cost efficient analysis
simple detection technology setup, detects only living cells (RNA)
rapid and sensitive, qualitative and quantitative detection of only living cells, costefficient analysis
Disadvantages time consuming, no detection of nonculturable microbes, labor expensive
expensive devices needed, no discrimination between live and dead cells, not officially accepted
low sensitivity, low specificity, higher crossreactivity
time consuming, low sample throughput, expensive, not automatable, difficult data analysis, not officially accepted
no differentiation of serotypes or subspecies, limited probe design (rRNA target), not officially accepted
Table 1 Comparison of different technologies for detection of bacteria
Figure 2 Comparison of total count of bacteria with the HybriScan test and the plate count agar
Table 2 Range of HybriScan detection and identification kits (for more details see sigma-aldrich.com/hybriscan)
Detection Kits for Applications/Groups Cat. No.
Beer Spoiling Organisms 62533
Beverage Spoiling Organisms 68301
Total Bacterial Count 02349
Waste Water Microthrix parvicella 04447
Waste Water Total Bacteria Count 78436
Yeasts 61397
Detection Kits for Genus & Species
Campylobacter 56917
Cronobacter spp. 12838
E. coli 96343
Lactobacilli 62533
Legionella 16593
Legionella pneumophila 07190
Listeria 55661
Listeria monocytogenes 49699
Salmonella 55662
Identification Kits Cat. No.
Brettanomyces 79742
Candida albicans 19503
Lactobacillus brevis 75724
Lactobacillus buchneri 80065
Lactobacillus lindneri 86827
Legionella pneumophila 49417
Leuconostoc 77007
Listeria monocytogenes 49712
Megasphaera 42875
Pectinatus cerevisiiphilus 33018
Pectinatus frisingensis 73582
Pediococcus damnosus 67289
14
sigma-aldrich.com/icsigma-aldrich.com/hplc
Buffer’s effect on detection: The choice of buffer is also dependent upon means of detection. For traditional UV detection, the buffer needs to be effectively transparent in this region especially, critical for gradient separations. Buffers listed in Table 1 have low enough absorption below 220 nm.
Phosphoric acid and its sodium or potassium salts are the most common buffer systems for reversedphase HPLC. Phosphonate buffers can be replaced with sulfonate buffers when analyzing organophosphate compounds. With the growth in popularity of LCMS, volatile buffer systems, such as TFA, acetate, formate, and ammonia, are frequently used due to compatibility with mass spectral (MS) detection. In regard to the issue of suppression of ionization, formate and acetate are ideal choices for positiveion mode detection. TFA, however, can negatively impact detector response even in positiveion mode [4, 5], while it strongly suppresses ionization with negative ion mode. Acetic acid is good for negativeion mode. LCMS applications further limit buffer selection and buffer concentration.
References[1] McMaster, M.C. HPLC A Practical User’s Guide, VCH Publishers,
Inc.: New York, NY, 1994; 85. [2] Poole, C.F. and Poole, S.K. Chromatography Today, Elsevier
Science: Amsterdam, Netherlands, 1991; 431. [3] Analytix, Five-part series on Mobile Phase Additives for LC-
MS, Issue 3, 2006 (sigma-ldrich.com/analytix).[4] Temesi, D., Law, B., 1999, The Effect of LC Eluent Composition
on MS Response Using Electrospray Ionization, LC-GC, 17:626.[5] Apffel, A. et. al. 1995. Enhanced Sensitivity for Peptide
Mapping with Electrospray Liquid Chromatography-Mass Spectrometry in the Presence of Signal Suppression Due to Trifluoroacetic Acid-Containing Mobile Phases, J. Chrom. A. 712:177.
Consideration of the effects of pH on analyte retention, type of buffer to use, and its concentration, solubility in the organic modifier and its effect on detection are important in reversedphase chromatography (RPC) method development of ionic analytes. An improper choice of buffer, in terms of buffering species, ionic strength and pH, can result in poor or irreproducible retention and tailing in reversephase separation of polar and ionizable compounds.
Problems, such as partial ionization of the analyte and strong interaction between analytes and residual silanoles or other active sites on the stationary phases, can be overcome by proper mobile phase buffering (maintaining the pH within a narrow range) and choosing the right ionic species and its concentration (ionic strength) in the mobile phase [1–2]. In sensitive LCMS separations that depend heavily on the correct choice of acid, base, buffering species and other additives [3], a buffer must be chosen based on its ability to maintain, and not suppress analyte ionization in the MS interface.
Buffer selectionThe typical pH range for reversedphase on silicabased packing is pH 2 to 8. Choice of buffer is typically governed by the desired pH. It is important that the buffer has a pKa close to the desired pH since buffers control pH best at their pKa. A rule of thumb is to choose a buffer with a pKa value <2 units of the desired mobile phase pH (see Table 1).
Buffer pKa (25°C) Useful pH Range
TFA 0.5 <1.5
Sulfonate 1.8 <12.8
Phosphate 2.1 1.13.1
Chloroacetate 2.9 1.93.9
Formate 3.8 2.84.8
Acetate 4.8 3.85.8
Sulfonate 6.9 5.97.9
Phosphate 7.2 6.28.2
Ammonia 9.2 8.210.2
Phosphate 12.3 11.313.3
Table 1 HPLC buffers, pKa values and useful pH range
Buffer concentration: Generally, a buffer concentration of 10–50 mM is adequate for small molecules.
Buffer solubility: A general rule is no more than 50% organic should be used with a buffer. This will depend on the specific buffer as well as its concentration.
Reversed-phase HPLC BuffersHighquality buffers (solutions, solids or concentrates)
14Ch
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Shyam Verma, Market Segment Manager [email protected]
HPLC-grade buffers and additives from Sigma-Aldrich/Fluka®Cat. No. Description Package Size
17836 Ammonium acetate 50 g, 250 g
17843 Ammonium formate 50 g, 250 g
17837 Ammonium hydroxide solution in water 100 mL, 1 L
17842 Ammonium phosphate monobasic 250 g
17839 Ammonium trifluoroacetate 10 g, 50 g
17835 Potassium phosphate dibasic anhydrous 250 g
17841 Sodium formate 50 g, 250 g
71633 Sodium phosphate dibasic dehydrate 250 g
17844 Sodium phosphate monobasic anhydrous 50 g, 250 g
17840 Sodium trifluoroacetate 10 g
09746 Trifluoroacetic acid:Triethylamine 2M:1M 500 mL
09747 Trifluoroacetic acid:Triethylamine 2M:2M 100 mL
For a complete list of HPLC buffers and additives, please refer to our online product catalog sigma-aldrich.com/hplc
15
sigma-aldrich.com/gc-hs
Chro
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ogra
phy
Compound ICH Solvent Class ICH Limit (ppm)
Acetone 3 5000
Acetonitrile 2 410
Dichloromethane 2 600
Diethyl ether 3 5000
Dimethylformamide 2 880
1,4Dioxane 2 380
Ethanol 3 5000
Ethyl acetate 3 5000
Hexane 2 290
Heptane 3 5000
lsopropanol 3 5000
lsopropyl acetate 3 5000
Methanol 2 3000
Methyl t butyl ether 3 5000
Methycyclohexane 2 1180
2Methyltetrahydrofuran Not Classified Not Classified
Tetrahydrofuran 2 720
Toluene 2 890
Triethylamine Not Classified Not Classified
Table 1 Residual solvents analyzed
ResultsThe comparison of the highpurity GC headspace grade DMA to the conventional grade (highpurity) DMA showed the superior quality of the GC headspace grade solvent. Figure 1 demonstrates that the GC headspace grade DMA is cleaner and free from potentially interfering peaks.
Contributed articleMelissa P Grella, Jessica Hoover, and Mark ShapiroPharmaCore, Inc, High Point, NC 27265, USA
Residual solvents in pharmaceuticals are defined as organic volatile impurities (OVIs) that are remnants of solvents used or produced in the manufacture of drug substances and excipients. Residual solvents are classified as: Class 1 solvents (solvents to be avoided – known or suspected human carcinogens and environmental hazards), Class 2 solvents (solvents to be limited – nongenotoxic animal carcinogens and suspected of other significant but reversible toxicities), and Class 3 solvents (solvents with low toxic potential and no healthbased exposure limit is needed). The solvents are not completely removed by practical manufacturing techniques. Testing is, therefore, done to ensure that these solvents are not above concentration limits listed by USP and in the ICH guidelines [1, 2].
Static headspace GC (HSGC) is a commonly used technique in the analysis of OVIs. This technique concentrates volatile analytes and allows their analysis free from sample matrix. Samples to be analyzed by HSGC must be dissolved in a suitable solvent. N,NDimethylacetamide (DMA) is a useful and commonly used solvent for headspace analysis of OVIs in pharmaceutical products.
ExperimentalFor the comparison of the different purity grades of DMA, blank samples were prepared by pipetting 1 mL of solvent into a 10 mL headspace vial. The blanks were analyzed by GC headspace. For the different analytical methods used, the highpurity GCHS grade DMA from SigmaAldrich (Product no. 44901) was used. Residual solvent stock standards were prepared using a positive displacement pipettor to add each residual solvent being tested. The stock standards were diluted in DMA to prepare working standard solutions. The concentration of each solvent in the working standard was calculated using the known density of each solvent. The working standard solutions were aliquoted into headspace vials and analyzed in the sequence along with blanks of the DMA by GC headspace. Table 1 lists the various residual solvents that were analyzed by the different methods and their ICH limits. The chromatograms for the residual solvents analyzed in each method are shown in various figures.
High-Purity Headspace Grade N,N-Dimethylacetamide in the Analysis of Residual Solvents
Shyam Verma, Market Segment Manager [email protected]
Figure 1 DMA comparison: headspace grade vs synthetic grade
The results for the different methods using the GCHS grade DMA, comparing the blank and standard solution containing various residual solvents are presented in Figures 2 and 3. The corresponding chromatographic and headspace conditions are listed in Tables 2 and 3.
0 5 10 15 20 25min.
A = Fluka® #44901B = Other HighQuality DMA
AAABBB
DMA
pA
80
60
40
20
0
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sigma-aldrich.com/icsigma-aldrich.com/gc-hs
Chro
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ogra
phy
Headspace Parameters
Temperatures: Oven 90 °C
Loop 105 °C
Transfer Line 110 °C
Times: Vial Equilibration 15 min
Pressurization 0.2 min
Loop Fill 0.2 min
Loop Equilibration 0.05 min
Pressures: ViaI 14 psi
Loop Volume 1.0 mL
lnject Time 0.5 min
GC Parameters
Column Equivalent to USP G43 phase, 105 m x 0.32 mm I.D. x 1.5 µm
lnjector and Detector Temperatures
250 °C, 300 °C (FID)
Oven Program 35 °C (10 min.), 3 °C/min. to 65 °C, 20 °C/min. to 190 °C (13 min.)
Carrier Helium, 2.1 mL/min. (constant pressure)
lnjection Headspace, split ratio 24.1:1
Table 2 Example Method 1
Headspace Parameters
Temperatures: Oven 90 °C
Loop 105 °C
Transfer Line 110 °C
Times: Vial Equilibration 15 min
Pressurization 0.2 min
Loop Fill 0.2 min
Loop Equilibration 0.05 min
Pressures: ViaI 14 psi
Loop Volume 1.0 mL
lnject Time 0.5 min
GC Parameters
Column Equivalent to USP G43 phase, 105 m x 0.32 mm I.D. x 1.5 µm
lnjector and Detector Temperatures
250 °C, 300 °C (FID)
Oven Program 35 °C (10 min.), 3 °C/min. to 65 °C, 20 °C/min. to 190 °C (12 min.)
Carrier Helium, 2.1 mL/min. (constant pressure)
lnjection Headspace, split ratio 24.1:1
Table 3 Example Method 2
DMA Blank
1 23
4 5
6 7 8 9
10
11
12
5 10 15 20 25 30 35 min
5 10 15 20 25 30 35 min
DMA Blank
12
3
4567
8 15
13
10 1216
17,1821,22
23,24
25
19
9
20
11
26
5 10 15 20 25 30 35 min
5 10 15 20 25 30 35 min
14
Figure 2 Example Method 1
Figure 3 Example Method 2
• This conclusion is supported by considering that multiple GC oven ramp rates and column chemistries were evaluated in support of multiple and various residual solvents and their associated levels.
In each case, there were no interference peaks resulting from the DMA irksome solvent peaks that could interfere with proper detection of target analytes.
References[1] United States Pharmacopoeia (USP), 31st Edition (2008), <467>
Residual Solvents.[2] ICH Guidelines for Industry, Q3C Impurities: Residual Solvents,
US Dept. of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), ICH December, 1997.
In each case, the method was used for the residual solvents analysis of an earlystage active pharmaceutical ingredient. The GCHS grade DMA blank chromatograms were clean and free of interfering peaks. The content of each residual solvent was determined with ease. Particularly, the low responding solvents or low ICH limit class solvents were detected with no background interference with the highpurity DMA.
• The data in this presentation clearly demonstrate the superior performance of the highpurity GC headspace grade solvent used (DMA).
1. Diethyl ether2 Acetone3. Isopropanol4. Acetonitrile5. Dichloromethane6. Methyl tbutyl ether
7. nHexane8. Ethyl acetate9. nHeptane10. 1,4Dioxane11. Toluene12. Dimethylformamide
1. Diethyl ether2 Acetone3. Isopropanol4. Hexane isomer 15. Acetonitrile6. Dichloromethane7. Hexane isomer 28. Methyl tbutyl ether9. Hexane
10. Hexane isomer 311. Ethyl acetate12. Heptane isomer 113. Heptane isomer 214. Heptane isomer 315. Heptane isomer 416. Heptane isomer 517. Heptane isomer 618. Heptane isomer 7
19. 2Methyl THF/Heptane20. Heptane isomer 821. Heptane isomer 922. Heptane isomer 1023. Heptane isomer1124. 1,4Dioxane25. Toluene26. Dimethylformamide
DMA Blank
DMA Blank
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Chro
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sigma-aldrich.com/uhplc-ms
Michael Jeitziner, Market Segment Manager [email protected] Shyam Verma, Market Segment Manager [email protected]
New Solvents and Additives Exclusively Designed for UHPLCLCMS Ultra CHROMASOLV®
Recent innovations in HPLC and mass spectrometry (MS) have escalated the limits of speed (throughput), efficiency and sensitivity. UHPLC in combination with very sensitive detection systems sets new limits for the purity of solvents and the additives used in the mobile phase. Solvents used for mobile phase, sample preparation, and sample dissolution are critical components of the system. Their influence
on background noise and baseline stability, column lifetime and system integrity cannot be overlooked. For the UHPLC system to provide reliable data and high performance, and eliminate system downtime, it is critical to use solvents that are as carefully developed, prepared, and tested as other components of the system.
SigmaAldrich offers the new LCMS Ultra CHROMASOLV product line that provides an outstanding quality for the ultrapure mobile phase. These solvents offer advantages such as:
• Extremely small drift in UHPLC gradient analysis
• Positive and negative ion mode testing for all MS applications
• Tested specific to UHPLC applications
• Most narrow and consistent specifications
UHPLC suitability of these solvents is achieved by considering various parameters of the preparation process far beyond filtration. The new LCMS Ultra CHROMASOLV grade solvents are exclusively tested for high performance with UHPLC gradient separations plus UV, positive and negative mode MS detection. Fluka’s new LCMS Ultra CHROMASOLV grade solvents are tested in situ under demanding UHPLC conditions, and various detection modes.
For additional information and to order, visit sigma-aldrich.com/uhplc-ms
Cat. No. Brand Name Description Package Size
14261 Fluka® Acetonitrile LCMS Ultra CHROMASOLV, ≥99.9%, tested for UHPLCMS 1 L, 2 L
14262 Fluka Methanol LCMS Ultra CHROMASOLV, ≥99.9%, tested for UHPLCMS 1 L, 2 L
14263 Fluka Water LCMS Ultra CHROMASOLV, tested for UHPLCMS 1 L, 2 L
14264 Fluka Trifluoroacetic acid LCMS Ultra eluent additive, ≥ 99.0% suitable for UHPLCMS 1 mL, 2 mL
14265 Fluka Formic acid LCMS Ultra eluent additive, ≥ 98% suitable for UHPLCMS 1 mL, 2 mL
14266 Fluka Ammonium formate LCMS Ultra eluent additive, suitable for UHPLCMS 25 g
14267 Fluka Ammonium acetate LCMS Ultra eluent additive, suitable for UHPLCMS 25 g
Table 1 New LC-MS Ultra CHROMASOLV solvents and LC-MS Ultra eluent additives
18
sigma-aldrich.com/maldi
Second Generation Ionic Liquid Matrices for MALDI-MS
Matthias Drexler, Product Manager, Analytical Reagents [email protected]
Salts with a melting point below 100 °C are commonly referred to as ionic liquids. Due to their unique properties, they have increasingly gained interest over the last few decades and have been used successfully in many applications. Examples include their use as solvents for organic reactions and homogeneous catalysis, in liquidliquid extractions, as ion pair reagents, and as stationary phases for gas chromatography.
In 2001, Armstrong et al. first demonstrated that ionic liquids may also be applied as matrices in matrixassisted laser desorption/ionization mass spectrometry [1]. These ionic liquid matrices (ILMs) offer advantages over the classic solid matrices, most importantly, the increased homogeneity of the sample and subsequently, a higher shottoshot reproducibility and improved quantification during the MALDI analysis. The need for a timeconsuming search for a “hot spot” on the sample plate is reduced, thus enabling better automated analyses. Other benefits of ILMs include their extremely low vapor pressure and overall easier sample preparation, as acidic additives such as trifluoroacetic acid are not necessary.
In general, the anionic part of the ILM is based on classic MALDI matrix substances, such as αcyano4hydroxycinnamic acid (CHCA) or ferulic acid, and the cationic part consisting of ammonium or imidazolium moieties commonly used in ionic liquids. Both ionic parts of an ILM can be varied in order to improve the properties and suitability for certain classes of analytes. Armstrong et al. were able to show in 2009 that the secondgeneration matrix N,Ndiisopropylethylammonium 4hydroxy3methoxycinnamate (DIEAF, Fluka® 94155) is especially fit for analyses of carbohydrates while N,Ndiisopropylethylammonium αcyano4hydroxycinnamate (DIEACHCA, Fluka 18211) and NtertButylNisopropylNmethylammonium acyano4hydroxycinnamate (IMTBACHCA, Fluka 94190) have proven to be best choices for protein and peptide samples in a considerable mass range of 1000 to 270,000 Da [2, 3]. In comparison with firstgeneration ILMs (such as BACHCA and DEACHCA), these new matrices perform even better with respect to analyte signal strength.
Overall, the development of ILMs offers a valuable tool for analyses with MALDIMS—a method where the success is very much dependent on choosing the right matrix substance suitable for the analyte.
Spec
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copy
Cat. No. Brand Description Abbrev. Gen. Suitable for Package Sizes
18211 Fluka αCyano4hydroxycinnamic acid NethylN,Ndiisopropylammonium salt
DIEACHCA 2nd Peptides, proteins, biodegradable polymers
10 x 10 mg
94190 Fluka αCyano4hydroxycinnamic acid NtertbutylNisopropylNmethylammonium salt
IMTBACHCA 2nd Peptides, proteins 10 x 10 mg
94155 Fluka Ferulic acid NethylN,Ndiisopropylammonium salt
DIEAF 2nd Carbohydrates 10 x 10 mg
67336 Fluka αCyano4hydroxycinnamic acid butylamine salt
BACHCA 1st Peptides, proteins, polymers
100 mg, 1 g
55341 Fluka αCyano4hydroxycinnamic acid diethylammonium salt
DEACHCA 1st Peptides, proteins, polymers
1 g
Product Table Ionic liquid matrices (ILMs) for MALDI-MS. Please find the complete product list at sigma-aldrich.com/maldi
HOCN
O
O
NH3
BA-CHCA67336
H3C
HOCN
O
O
NH2
DEA-CHCA55341
H3C CH3
HOCN
O
O
HN CH3
CH3
CH3H3C
H3C
DIEA-CHCA18211
HO
O
O
HN
H3CCH3
CH3
CH3H3CDIEA-F94155
CH3
OCH3HOCN
O
O
HN
H3CCH3
CH3
CH3H3C
IMTBA-CHCA94190
CH3
References[1] D. W. Armstrong, L. Zhang, L. He, M. L. Gross, Ionic Liquids as
Matrixes for Matrix-Assisted Laser Desorption/ionization Mass Spectrometry, Anal. Chem. 2001, 73, 3679 –3686.
[2] A. Berthod, J. A. Crank, K. L. Rundlett, D. W. Armstrong, A Second-Generation Ionic Liquid Matrix-Assisted Laser Desorption/Ionization Matrix for Effective Mass Spectrometric
Analysis of Biodegradable Polymers, Rapid Commun. Mass Spectrom. 2009, 23, 3409 –3422.
[3] J. A. Crank, D. W. Armstrong, Towards a Second Generation of Ionic Liquid Matrices (ILMs) for MALDI-MS of Peptides, Proteins, and Carbohydrates, J. Am. Chem. Soc. 2009, 20, 1790 –1800.
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Titr
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sigma-aldrich.com/fixanal
Volumetric Concentrates for Titration by Sigma-Aldrich Prepare Acid, Base and Salt Standard Solutions with FIXANAL® Ampoules
Andrea Felgner, Market Segment Manager [email protected]
Our volumetric concentrates, packaged in convenient FIXANAL ampoules, allow our customers to achieve specific concentrations of high accuracy. This is accomplished with speed and simplicity due to the design features of the innovative FIXANAL ampoules. The volumetric concentrate contains an exact amount of the prescribed substance. By diluting the entire content of the ampoule by a determined volume, the analyst
has complete control over the final solution concentration. The analyst’s confidence is aided by knowing for certain that the absolute amount of the titration substance has been tested against a certified reference material.
distilled and degassed; otherwise sluggish endpoints or changes to the titer may occur.
Certain concentrates with sensitive chemical properties require black pigmented PE ampoules or brown glass ampoules. All FIXANAL concentrates are calibrated against independently produced reference standards. All chemicals and test equipment are examined according to the rules of DIN EN ISO 9001 and DIN EN ISO 14001.
Quality Assurance, Packaging and Handling • FIXANAL ampoules are subject to strict production and
quality control specifications
• A special ampoulesealing process guarantees titer to be accurate within specified shelf life
• Ampoules are supplied with selfadhesive labels that can easily be attached to a storage vessel
• Ampoules are made of highresistance polyethylene (PE), natural or blackpigmented, or glass, depending on the content
• PE ampoules have an integrated rinsing funnel for perforating the membrane (see Figure 1); the integrated deflector accelerates complete rinsing of the concentrate from the ampoule
• Glass ampoules do not have the twistopening mechanism; instead, a glass rod is used for piercing both ends of the ampoule (see Figure 2)
• The water used to fill up to volume should be distilled and degassed; otherwise, sluggish endpoints or changes of titer may occur
• Concentrates of iodine, containing iodideiodatemixtures, have to be treated with an equivalent amount of acid (+ 1% excess) before bringing them up to volume, in order to react to free iodine
(continued on page 20)
Product Characteristics: • Contains exact amount of substance (e.g. 1 mol)
• Titer precision 1.000 ± 0.2%
• Economical and spacesaving
• Final concentration is userspecified
For convenience, the ampoules are supplied with selfadhesive labels that can easily be attached to a storage vessel by the enduser. Most ampoules are made of highresistance polyethylene and have an attached funnel. To open a FIXANAL ampoule, simply twist the top of the ampoule to activate flow; a specially developed rinsing funnel facilitates the perforation of the sealing membrane. An integrated deflector accelerates the complete rinsing of the concentrate. The water used for dilution should be
Figure 1 PE ampoulesPlace the ampoule on the volumetric flask. Fix the lower part and rotate the upper part in the direction of the arrow (maximum of 2 turns). Remove the funnel, turn it upside down and place it firmly on the ampoule so that the membrane opens. Hold the ampoule at an angle and rotate it while thoroughly rinsing it. Fill the volumetric flask up to the mark at 20 °C and mix thoroughly.
Figure 2 Glass ampoulesPlace the ampoule on the volumetric flask. Carefully pierce both ends using a glass rod. Rinse and remove glass rod, then rinse ampoule. Fill the volumetric flask up to the mark at 20 °C and mix thoroughly.
20Ti
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ion
Special Offer – The following FIXANAL® volumetric concentrates are available with HUGE SAVINGS OF 35% OFF:
Cat. No. Brand Description382101EA Fluka® Sodium hydroxide concentrate, for 1 L standard solution
c(NaOH) = 0.1 mol/L (0.1 N)
382001EA Fluka Sodium thiosulfate concentrate, for 1 L standard solution c(Na2S2O3) = 0.1 mol/L (0.1 N)
382171EA Fluka Sodium hydroxide concentrate, for 1 L standard solution c(NaOH) = 0.5 mol/L (0.5 N)
380201EA Fluka Ammonium thiocyanate concentrate, for 1 L standard solution c(NH4SCN) = 0.1 mol/L (0.1 N)
320431EA Fluka Sulfuric acid concentrate, for 1 L standard solution c(H2SO4) = 0.05 mol/L (0.1 N)
382821EA Fluka Hydrochloric acid concentrate, for 1 L standard solution c(HCl) = 1.0 mol/L (1.0 N)
380731EA Fluka Potassium hydroxide concentrate, for 1 L standard solution c(KOH) = 1.0 mol/L (1.0 N)
320471EA Fluka Zinc sulfate concentrate, for 1 L standard solution c(ZnSO4) = 0.1 mol/L
380611EA Fluka Iodide Iodate concentrate, for 1 L standard solution c(I2) = 0.05 mol/L (0.1 N)
380401EAR Fluka Bromine concentrate, for 1 L standard solution c(Br2) = 0.05 mol/L
320421EA Fluka Nitric acid concentrate, for 1 L standard solution c(HNO3) = 0.1 mol/L (0.1 N)
382851EA Fluka Hydrochloric acid concentrate, for 1 L standard solution c(HCl) = 0.5 mol/L (0.5 N)
382151EAR Fluka Sodium hydroxide concentrate, for 1 L standard solution c(NaOH) = 1.0 mol/L (1.0 N)
380701EAR Fluka Potassium hydroxide concentrate, for 1 L standard solution c(KOH) = 0.1 mol/L (0.1 N)
381101EA Fluka Potassium iodide concentrate, for 1 L standard solution c(KI) = 0.1 mol/L
380021EA Fluka Ammonium iron(II) sulfate concentrate, for 1 L standard solution c((NH4)2Fe(SO4)2) = 0.06 mol/L, for COD determination
381001EAR Fluka Potassium dichromate concentrate, for 1 L standard solution c(K2Cr2O7) = 1/60 mol/L (0.1 N)
320461EA Fluka Perchloric acid concentrate, for 1 L standard solution c(HClO4) = 0.1 mol/L (0.1 N), for titration in nonaqueous liquids
382831EA Fluka Hydrochloric acid concentrate, for 10 L standard solution c(HCl) = 1 mol/L (1 N)
380651EA Fluka Iodide Iodate concentrate, for 1 L standard solution c(I2) = 0.005 mol/L (0.01 N)
381501EAR Fluka Sodium (meta)arsenite concentrate, for 1 L standard solution c(NaAsO2) = 0.05 mol/L
382261EA Fluka Sodium hydroxide concentrate, for 1 L standard solution c(NaOH) = 0.025 mol/L (0.025 N)
382141EA Fluka Sodium hydroxide concentrate, for 10 L standard solution c(NaOH) = 1 mol/L (1 N)
320441EA Fluka Sulfuric acid concentrate, for 10 L standard solution c(H2SO4) = 0.5 mol/L (1.0 N)
320381EA Fluka Sodium chloride concentrate, for 1 L standard solution c(NaCl) = 0.1 mol/L (0.1 N)
382941EA Fluka Sulfuric acid concentrate, for 1 L standard solution c(H2SO4) = 0.5 mol/L (1.0 N)
381401EA Fluka Potassium thiocyanate concentrate, for 1 L standard solution c(KSCN) = 0.1 mol/L
380301EAR Fluka Barium chloride concentrate, for 1 L standard solution c(BaCl2) = 0.05 mol/L (0.1 N)
382951EA Fluka Sulfuric acid concentrate, for 1 L standard solution c(H2SO4) = 0.25 mol/L (0.5 N)
320361EA Fluka Potassium bromide concentrate, for 1 L standard solution c(KBr) = 0.1 mol/L
To take advantage of this offer, please use Promotion Code SCZ. Offer valid until July 31, 2012.
Special
Offer
35% Off
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C
O
+ 2 CH3OH
H+
C
OCH3
OCH3 + H2O
C
O
R H
+ SO2 +H2O + NR' CR
OH
SO3 HNR'
H
(NR' = base)
sigma-aldrich.com/hydranal
Determination of Water Content in Aldehyde and Ketone SamplesHYDRANAL®K Reagent Line for Karl Fischer Titration
Andrea Felgner, Market Segment Manager [email protected] Wendt, HYDRANAL Technical Service [email protected]
The HYDRANALK reagent product line is designed to meet the needs of the analytical chemist by providing accurate water content determination using Karl Fischer (KF) titration of aldehyde and ketone samples. HYDRANALComposite 5K, as a replacement for methanolcontaining titrating agents, prevents side reactions that occur during KF titration of aldehydes or ketones. These side reactions can compromise the water content determination. HYDRANALComposite 5K is a special formulation consisting of imidazole, sulfur dioxide, and iodine dissolved in diethylene glycol monoethyl ether. Three Fluka®branded working media for use with HYDRANALComposite 5K are supplied by SigmaAldrich:
(1) HYDRANALKetoSolver (free of halogenated hydrocarbons)
(2) HYDRANALMedium K(3) HYDRANALWorking Medium K (very toxic /excellent
dissolving properties)
Suppression of Side Reactions that lead to Erroneous Water Content DeterminationsThe HYDRANALK line of reagents does not contain the conventional methanol component. Methanol has been omitted in order to prevent nucleophilic addition with aldehydes and ketones which leads to the formation of water (Figure 1). Water produced by this side reaction would cause elevated water content results and vanishing end points. The HYDRANALK reagents are also designed to guard against bisulfite addition to aldehydes (Figure 2).
(continued on page 22)
This second side reaction consumes a portion of the original water content from the sample and results in erroneously low water content measurements. HYDRANALComposite 5K is a variant of HYDRANALComposite 5 with a slightly slower reaction speed to maximize suppression of the bisulfite addition side reaction.
HYDRANALK reagents are also suitable for some amines, siloxanes and other titrations requiring methanolfree working media. HYDRANALMedium K, HYDRANALWorking Medium K, and HYDRANALKetoSolver can each act as the solvent for the determination of water in any substance where methanol can interfere with the titration and therefore must be avoided. The general utility of these reagents renders a universal applicability for KF titrations; they are also suited for easytohandle samples that do not interfere with the KF reaction.
Distinctives of the HYDRANAL-K Working MediaAll these Flukabranded working media are free of methanol, but each possesses unique properties for optimization with particular samples. HYDRANALKetoSolver is primarily 1methoxy2propanol and is entirely free of halogenated hydrocarbons. It is used with HYDRANALComposite 5K for the KF titration of aldehydes and highly reactive ketones like cyclohexanone, trifluoroacetone, and diacetyl. For certain less reactive ketones, HYDRANALComposite 5 can be used as the titrating agent.
Figure 1 Aldehydes and ketones undergo nucleophilic addition of methanol resulting in formation of acetal or ketal and water
Figure 2 Bisulfite addition reaction consuming water
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HYDRANAL®Working Medium K has exceptional solvating power but must be designated as toxic because it contains 2chloroethanol. This hazardous component is omitted from HYDRANALMedium K and replaced with less toxic alcohols in order to lower the toxicity rating; HYDRANALMedium K contains a percentage of chloroform. Not only is the toxicity level lowered, but the revised formulation of HYDRANALMedium K provides performance advantages with very reactive aldehydes such as propionaldehyde, butyraldehyde and crotonaldehyde. Capacity and accuracy are also improved for other compounds such as salicylaldehyde, acetylacetone, 2,4dihydroxyacetophenone, and 2benzolpyridine.
Replacement of HYDRANAL®-Working Medium K with HYDRANAL-Medium KEssentially, HYDRANALMedium K is a fullfledged substitute for HYDRANALWorking Medium K, providing the same sample capacity, speed and accuracy. In addition, HYDRANALMedium K offers important application, safety and transportation benefits. It not only improves workplace safety, but also reduces the amount of packaging material required for shipment and the associated handling and disposal of that packaging material.
The SigmaAldrich laboratories compared HYDRANALWorking Medium K and HYDRANALMedium K and have reported these findings:
• Comparable sample capacity in 30 mL medium
• Comparable results for water content and standard deviation
• Comparable titration speed
• Comparable accuracy of recovery rate of added water after sample titration
Coulometric KF Titration in KetonesFor the coulometric water determination in ketones, SigmaAldrich offers specially designed HYDRANALCoulomat reagents: Coulomat AK and Coulomat CGK.
HYDRANALCoulomat AK is an anolytic reagent for the coulometric determination of water in ketones. It contains imidazole, sulfur dioxide, and iodide dissolved in a suitable solvent mixture and has a capacity of approximately 100 mg of water per 100 mL. HYDRANALCoulomat AK can also be used as a single reagent for coulometry without diaphragm. HYDRANALCoulomat CGK is the corresponding catholytic reagent. It does not contain halogenated hydrocarbons. The water capacity of 5 mL HYDRANALCoulomat CGK is 100 mg.
The total sample volume used with one charge of coulometric reagents should not exceed 20 mL (as liquid sample or as dissolved solid sample). Because of ongoing side reactions, larger sample amounts can lead to high instrument drift, causing vanishing end points and erroneous results of the titration.
Application Example: Determination of Water in AcetoneThe water content in acetone can be determined using both volumetric and coulometric KF titration techniques:
• Volumetric titration Add 30 mL HYDRANALMedium K to the titration vessel and titrate to dryness with HYDRANALComposite 5 K. Add approximately 5 mL of the acetone sample, weighed by difference, to the vessel. Titrate the water content with HYDRANALComposite 5 K.
• Coulometric titration Add 100 mL HYDRANALCoulomat AK to the anolyte compartment of the titration vessel and 5 mL HYDRANALCoulomat CGK to the catholyte compartment. After starting the titrator, the cell is dried automatically. When the drift is stable, add approximately 0.5 g of the sample, weighed by difference, to the cell.
Benefits of HYDRANAL-Medium K over HYDRANAL-Working Medium K • Reduced toxicity for improved workplace safety
while providing equal reactivity
• Performance advantages with very reactive aldehydes
• Improved capacity and accuracy
• Less waste of packaging material
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General Recommendations for KF Titration in Aldehydes and Ketones • Aldehydes
Shortchain aldehydes show a strong tendency to form acetals (along with water formation). Aromatic aldehydes tend to undergo the bisulfite addition. To overcome these potential interferences, we recommend using relatively small samples and titrating rapidly to suppress the formation of acetals and bisulfite compounds. Coulometry is not advised, but if it is necessary, the sample volume should be very small. For example, for water determination in aromatic aldehydes, the sample volume must be less than 0.5 mL. Aliphatic aldehydes, such as acetaldehyde, are highly reactive and tend to rapidly form acetals. Only volumetric titration with ethanolbased Kreagents is recommended and the amount of sample should be kept to a minimum.
• Ketones Ketones have a tendency to form ketals while at the same time forming water. Cyclohexanone and acetone react rapidly, while longchain ketones and aromaticsubstituted ketones show slower reaction rates. Reactive ketones are titrated with ethanolbased Kreagents.
Substance Application Number
1,2Cyclohexanedione 306
2Dimethylaminomethyl cyclohexanone (DMC base) 518
Butanal (NButyraldehyde and isoButyraldehyde) 248
Crospovidone (Crosslinked homopolymer of 1vinylpyrrolidone2on)
472
Dexpanthenol 507
Diacetyl (2,3Butanedione) 496
Ethosuximide (3Ethyl3methyl2,5pyrrolidindion) 510
Glutardialdehyde 50% 273
Glyoxal solution 40% 267
Glyoxylic acid methylester methylhemiacetal 261
Hexafluoroacetone sesquihydrate 154
NFormylmorpholine (4Morpholine carboxaldehyde) 392
N,NDimethyl formamide 424
Table 1 Available KF application reports for selected aldehydes and ketones
Cat. No. Brand Description Package Size
34816 Fluka® HYDRANALComposite 5K 500 mL, 1 L, 2.5 L
34805 Fluka HYDRANALComposite 5 500 mL, 1 L, 2.5 L
34738 Fluka HYDRANALKetoSolver 500 mL, 1 L
34698 Fluka HYDRANALMedium K 1 L
34817 Fluka HYDRANALWorking Medium K 1 L
34820 Fluka HYDRANALCoulomat AK 500 mL
34821 Fluka HYDRANALCoulomat CGK 50 mL
Table 2 Selected HYDRANAL products (complete list can be found on Sigma-Aldrich website)
To obtain application reports and more information on HYDRANALK reagents as well as on our other highquality HYDRANAL reagents for pyridinefree water determination by KF titration, please visit our website sigma-aldrich.com/hydranal or contact our HYDRANAL laboratories:
Europe and Global MarketMr. Thomas WendtTechnical Service HYDRANALWunstorfer Straße 40D30926 Seelze, GermanyTel.: +49 (0) 5137 8238353Fax: +49 (0) 5137 8238698EMail: [email protected]
USA and CanadaMr. Doug ClarkHYDRANAL Technical Center545 S. Ewing AveSt. Louis MO 63103, USAToll free: +1 800 4937262 (USA and Canada)Fax: +1 314 2866699EMail: [email protected]
The coulometric determination is possible by using HYDRANAL®Coulomat AK and Coulomat CGK reagents. The sample size for coulometric measurements should not exceed 1 mL; for very reactive ketones like cyclohexanone, a maximum of 0.5 mL per sample is recommended.
Order/Customer Service (800) 325-3010 • Fax (800) 325-5052 Technical Service (800) 325-5832 • sigma-aldrich.com/techserviceDevelopment/Custom Manufacturing Inquiries (800) 244-1173Safety-related Information sigma-aldrich.com/safetycenter
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Date: 06/2012Sams Code: OMC