CRM
Guide 34/ISO 17025
Karl Fischer Titration
Reagents
Chromatography
100
What phrases inform you that your standard is a certified reference material (CRM)?
The following words/phrases are required on the Certificate of Analysis: 1. Metrological Traceability 2. Uncertainty 3. Homogeneity 2015 BERM Poster Presentation 2015 Uncertainty Presentation In-house Sara Allison A Certified Reference Material is defined in ISO Guide 31:2000 as a reference material characterized by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of a specified property, its associated uncertainty, and a statement of metrological traceability.
100
Where can you locate the scope of accreditation?
Log in to Sigma Aldrich. Bottom of page click on customer support/general information/ISO certificates/location desired. Accreditation is sorted by standard and location. Click on the pdf to view what accreditation is available by location.
100
What does Karl Fischer measure?
Karl Fisher (KF) titration is a method for measuring water content in all types of substances. Water can affect product quality, texture, shelf life, chemical stability and reactivity. KF is composed of iodine, sulfur dioxide, pyridine and methanol.
100
Why would you choose a derivatizing reagent?
1. Increase or decrease the volatility of the compound (GC) or Compound soluble in mobile phase (LC). 2. Improve chromatography (peak resolution and symmetry). 3. Increase detectability 4. Improve anaalytical efficiency 5. Improve stability of compound Example of poor chromatography and illustrate improvement with reagent. Bulletin
100
What considerations should you make in preparing your HPLC mobile phase? LCMS mobile phase?
1. Quality- HPLC grade for HPLC or LCMS grade for LCMS. This grade contains no impurities to produce spurious peaks in a chromatogram baseline. 2. Water- should be ultra pure or LCMS grade. Deionized water often contains trace levels of organic compounds. 3. Buffers- should be prepared freshly. This insures that the buffer pH is unaffected by prolong storage and that there is no microbial growth present. 4. Filtration- this removes may particulate matter that may cause blockages. Pump plungers, seals, and check valves will perform better and lifetime will be maximized. 5. Degassing- this removes all dissolved gasses. If degassing is not done, air bubbles can form in the high-pressure system resulting in problems with system instability, spurious baseline peaks, etc.
200
What is the importance of stability testing?
The stability study is designed so it yields sufficient data for answering long term stability/transport/shelf life of the CRM at the defined storage temperature. QC and R&D have a stability program. R&D is determinng stability by an accelerated protocol and QC is checking the actual stability. 2015 Poster Presentation at BERM (T415118) ISO Guide 34 and Guide 35 define Stabililty
200
What does ISO Guide 34 define?
ISO Guide 34 outlines the quality system requirements under which a CRM is produced including production, planning and control, maintenance of a suitable environment, starting material selection and processing, assignment of a property value, their uncertainty and traceability, assessment of homogeneity and stability, assurance of adequate packaging and storage and issuing certificates or documents.
200
What is the difference between volumetric and coulometric Karl Fischer titration?
The amount of water content detected. Volumetric- is used for high water content (>1% water) and the titrant is added directly to the sample by a burette. Coulometric- suitable for samples with low water content (<1%) and with this method the titrant is generated electrochemically in the titration cell.
200
What is the purpose of a derivatizing reagent?
1. Derivatizing reagents chemically modify compounds to produce new compounds which have properties that are suitable for GC or LC analysis. Note: a. It should not cause any rearrangements or structural alterations of compounds during formation of the derivative. b. It should produce a stable derivative. Supelco Bulletin 909A 2015 BERM Presentation (T415122)
200
Why add an internal standard to an analysis? Double Jeopardy Question: what is the difference between an internal standard and surrogate standard
Chromatography protocols typically utilize the internal standard method of quantification. Internal standards gauge the method’s ability to extract target analytes from the sample matrix, and are used to monitor and adjust instrument fluctuations. Pentafluorobenzene, 1,4-difluorobenzene, 1,4-dichlorobenzene-d4 and chlorobenzene-d5 are examples of internal standards. Surrogate standards are used to monitor the entire analytical process from sample preparation to instrument performance. Toluene-d8, 4-bromofluorobenzene and dibromofluorobenzene are examples of surrogate standards. Tuning solutions are utilized to “tune” or optimize MS detector parameters. This maximizes the accuracy of mass and ion abundance measurements. Some tuning solutions also contain compounds used to assess injection port inertness and GC column performance. Our tuning solutions are formulated as specified in particular EPA methods and are important tools for the qualification of GC-MS systems.
300
What does the CRM uncertainty mean?
The uncertainty indicates the range where the true property value can be expected with a certain probability. This information can directly be used, if the material is used for calibration. When the material is used to estimate method bias, the uncertainty range for the result obtained in the laboratory of the user should overlap with the uncertainty range given on the certificate. if this is true, the method applied by the user for determining the property is unbiased. Fishbone diagram Examples of uncertainty reported: homogeneity, pipettes, glassware, equipment, stability, etc. 2015 In-house presentation Sara Allison
300
What is the advantage of proficiency testing?
Perioidic participation in proficiency tests demonstrates technical capability. Supelco's QC and R&D personnel undergo proficiency tests. 2015 Poster Presented at BERM
300
What is the Merck equivalent for the Hydranal line?
Currently the Merck equivalent to Hydranal in the US is EMD Millipore Apura. Apura® means aqua pure analysis. Apura® reagents and standards are distinguished by excellent quality. The measuring results are therefore always reliable and accurate. Merk uses only high-quality and approved raw materials. This ensures batch-to-batch consistency and comparable analytical results. Customers benefit from our high scientific standards and Merk's long international experience. Merck Millipore in particular places great importance on safety. For this reason, the Apura® Karl Fischer reagents contain no toxic components or, if this is not possible, they are limited to a minimum. Our international sales network and local offices ensure safe delivery and service – worldwide. Whenever your customers need help, Merck Millipore can supply it via their application and development laboratories. They can advise on applications, offer technical support, and assist in validation projects. Our quality control is carried out according to the most stringent standards. Analyses are carried out using recognized international standards.
300
Can you name three (3) types of derivarization?
GC derivatives: Alkylation- the general process is esterification, reduces molecular polarity by replacing active hydrogens with an alkyl group (acidic hydrogens- carboxylic acids and phenols). Acylation- introduction of a acyl group,reduces the polarity of amno, hydroxyl, and thiol groups and adds halogenated functionalities for ECD (carbohydrates and amno acids). Silyation- introduction of a silyl group into a molecule, derivatives are more volatile, less stable, and more thermally stable (Alcohol, Phenol, Carboxyl, Ammine, Amide, Hydroxyl). Always include a reagent blank with your compound assays.
300
What happens if the sample volume injected into a GC is too large?
Backflash can occur and result in spurious chromatogram peaks and repeatability problems. Injection volumes should take into account the temperature of the inlet, type of solvent being used, and injection type (direct injection, split, etc). There can be considerable difference in the gas volumes generated by different solvents when a sample vaporizes after injection.
400
How can you use the data obtained on the CofA ?
Customer can: -Validate a method of measurement. This is to see if the observed value and the certified value agree with the uncertainty. -Verify a specific measurement procedure. This will show if the observed value and the certified value agree within the uncertainty under the specific laboratory conditions. -Control the accuracy of measurements within the uncertainty. -Calibrate a measurement device for a specific type of measurement. This is to establish the relation between a 'signal' and the quantity intended to be measured (usually a concentration). In the simplest case, a calibration factor and its uncertainty is calculated from the observed value and the certified value. -Proficiency testing. The use of a well characterized test sample, unknown by the PT participants, enables their performance to be monitored against certified values instead of the consensus values. -Develop new methods of measurement. -Estimate measurement uncertainty.
400
How does a CRM manufacturer validate instruments?
Instrument Qualification (IQ) - demonstrates that the equipment was delivered as designed and specified and that it was installed in a suitable location. Operational Qualification (OQ) - demonstrates that the equipment operates according to specification in the location in which it was installed. Performance Qualification (PQ) - demonstrates that the equipment consistently performs its routine function.
400
How do we use Karl Fischer at Supelco?
We use KF at Supelco to determine water content for some raw materials that go into our CRM finished goods. Water content is combined with other raw material tests to provide a certified purity value for the raw material. Raws that undergo KF:
400
How do you determine the amount of reagent to add to a sample?
The goal is 100% yield but this rarely happens. ● Make sure the reaction is complete. ● Use more than one reaction temperature and time. ● Watch out for possible side reactions with additional functional groups on the compound or sample matrix. ● Determine the thermal stability of the derivative. ● The reaction should proceed rapidly with simple and easy methodology. ● Use reagents and reactions that present no unusual hazards. ● The derivatized form of the analyte should be readily extractable. ● The derivative can be chromatographed accurately for trace analysis or separated from interfering compounds.
400
Why use validated methods to test CRM's?
Using validated methods is a requirement of ISO/IEC 17025. In addition, validating a method reduces uncertainty and provides you with consistent, reliable, and accurate data. It can also assist you judging the quality and consistency of analytical data. R&D validates methods with Linearity (3 points), Reproducibility (36 injections) and Stability (10 days at 40C).
500
What should be included on a CRM certificate?
1. Name and address of manufacturer 2. Description and material name 3. Product part number 4. Intended use 5. Instructions for correct use 6. Storage conditions (long term stability and transportation) 7. Certified property value w/their uncertainty (all stages of process) 8. Method used to obtain values 9. Hazardous information 10.Estimation of uncertainty measurement & statement regarding homogeneity/traceability 11. Date of certification and expiration 12. Method of preparation Example CofA ISO Guide 31 2015 Berm Poster Presentation (T415119)
500
What are the restrictions with using the certifying body logo (symbol) (i.e. ANAB)?
We can use the symbol in association with only those products that fall under our scope of accreditation at our location. We cannot misrepresent our accreditation status or we risk suspension or termination of accreditation.
500
What sample size would you use with Karl Fischer?
The larger the expected water content, the smaller the sample size needed. For example, using coulometric water determination with an expected water content of 1% water, your recommended sample size would be 0.2 grams vs an expected water content of 0.1% with a recommended sample size of 1 gram.
500
What can be a common problem when using Silyl reagents?
Silyl derivatives tend to be moisture sensitive. Moisture will decompose both TMS reagents and derivatives. The use of excess derivatization reagent and solvent can help minimize problems of moisture interference or other sample impurities. Water can hnder the reaction and/or hydrolyze the derivative, reducing the yield of derivative for analysis. Sodium sulfate can be added to the reaction mixture to trap water present in the samlle. Injection ports. A stainless steel injection port frequently yields erratic and isrreproducible results. The problem may not become apparent until after several weeks of use. Column selection- there are some specially tested capillary columns for evaluating underivatized and derivatized analytes.
500
What happens when you increase or decrease temperature when running HPLC? Double Jeopardy: What does ambient temperature mean?
Retention time shift of the analytes. Besides shorter run times – usually a good thing – an increase in temperature can have other beneficial effects. As the temperature increases, the viscosity of the mobile phase will decrease, resulting in lower pressures. Lower mobile-phase viscosity also improves diffusion in the chromatographic system, giving narrower peaks. Shorter retention times will generate narrower peaks. Narrower peaks mean taller peaks, assuming the same mass of sample on the column, and thus lower detection limits. Ambient temperature can be frustrating if we try to operate at room temperature in a laboratory that doesn’t have very good climate control. Someone in Antarctica might suggest that 17ºC was ambient, someone in Bellefonte may suggest 30ºC was ambient. Who is right? Bottom line- use your oven temperature.
M
e
n
u