Case Study Poor Peak Area Precision Encountered During HPLC System Calibration
Two real-world examples are used to illustrate how to resolve this common problem encountered during the calibration of an autosampler Chapter 9 . In Table 10.1, the precision of peak area was found to be gt 1 RSD, which was above the acceptance criterion of 0.5 RSD. Repeating the experiment showed similar precision results. Since a worn sampling syringe of the autosampler is the most common cause for poor precision, it was replaced. The peak area precision was found to be 0.3 RSD after this...
Refractive Index Detector RID
A refractive index detector measures the refractive index change between the sample cell containing the eluting analyte and the reference cell purged with pure eluent see Figure 4.11b . It has lower sensitivity 0.01-0.1 j.g and is prone to temperature and flow changes.1'4 It offers universal detection and is used commonly for analytes of low chromophoric activities such as sugars, triglycerides, organic acids, pharmaceutical excipients, and polymers. It is the standard detector in GPC.15 Modern...
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Figure 9.8. HPLC chromatogram of a system suitability solution SSS for a challenging impurity testing method for a drug product containing two APIs. This SSS contains both APIs and several key degradants and impurities at their expected concentrations as retention time markers . One of the component DGA1 present at 0.10 level also serves as a sensitivity check and must meet the acceptance criterion of having S N gt 10. Figure 9.8. HPLC chromatogram of a system suitability solution SSS for a...
Hplc System Maintenance
Common HPLC maintenance procedures that can be performed by the user are described, including procedures such as replacing check valves, filters, pump seals, detector lamps, flow cells, autosampler sampling syringes, and injector rotor seals. Other more elaborate maintenance tasks such as replacing pump pistons or high-pressure needle seals in some integrated-loop autosamplers are typically handled by the service specialists or internal metrology staff. Most laboratories that work in a...
Guard Columns
A guard column is a small column placed before the analytical column to protect it from particles or contaminants from the samples.26 Ideally, guard columns are packed with the same materials as analytical columns and should not cause a significant increase in pressure or performance degradation Figure 3.24 . Guard columns are commonly used in applications involving dirty samples environmental or bioanalytical samples where more extensive sample clean-up might not be feasible. Some users might...
Proteomics Systems Capillary LC and MultiDimensional LC
Proteomics research is currently driving HPLC technologies towards high sensitivity, speed, and peak capacities.8 Capillary LC sometime termed micro LC or nano LC 1819 is used to enhance mass sensitivity needed to analyze minute sample amounts. System requirements for handling extremely low flow rates and small peak volumes mandate the use of specialized HPLC system such as the one shown in Figure 4.14a, which has a micro-pump, a micro x-y-z autosam-pler, and a PDA detector with a low-volume...
MultiDimensional LC
The maximum theoretical peak capacity n of a single high-resolution column with 20,000 plates using gradient elution is about 400 a level insufficient for the analysis of many complex samples. One traditional approach to increase separation power is to use two-dimensional chromatography where the sample is fractionated by the first column and each eluted fraction is subsequently analyzed by a second, orthogonal column. This process can be automated in a 2-D system using switching valves, thus...
Natural Food Components
7.2.1.1 Sugars Common carbohydrates are monosaccharides glucose, fructose , disaccharides sucrose, maltose, lactose , trisaccharides raffinose , and polysaccharides starch . HPLC offers a direct, quantitative method for simple sugars, which requires a specialty cationic resin-based column and refractive index or evaporative light scattering detection.7,8 UV detection at low wavelengths 195nm can be used but is more prone to interferences.8 Different resin columns Ca , Pb , H , Ag , etc. are...
Pesticides Analysis
Pesticide analysis methods developed by manufacturers, in support of registration petitions to the regulatory agencies, are usually company proprietary. These are mostly HPLC methods since most pesticides are thermally labile and might not survive the GC process. These methods are used to generate assay, stability, residue, and metabolism data. In the United States, regulations require pesticide manufacturers to notify the EPA using a premanufacture notice PMN , describing structure,...
Operating Principles
Figure 4.8b shows the schematic of a PDA detector where the entire spectrum of the deuterium lamp passes through the flow cell onto a diode array element Orthogonal spray orientation and high-capacity drying gas system improve instrument perfomance and decrease maintenance requirements.
Mobile Phase Premixing
Premix mobile phase by measuring the volume of each solvent separately and combining them in the solvent reservoir.12 This is particularly important when mixing organic solvents with water because of the negative AV of mixing. For example, prepare 1L of methanol water 50 50 by measuring 500 mL of methanol and 500mL of water separately in a measuring cylinder and combine them together. Do not pour 500mL of methanol into a 1-L volumetric flask and fill it to volume with water as more than 500 mL...
Summary Of Hplc Operation
The following is a summary of important procedures, a sequence of events, for HPLC operation Filter and degas mobile phase. Prime pump, rinse column with strong solvent, and equilibrate column. Purge injector and make sure there are no bubbles in the sampling syringe. Perform system suitability test for regulatory testing, see Chapter 9 . Rinse column and shut down pump and lamp.
Case Study Poor Assay Accuracy Data an OutofSpecification Investigation
Table 10.3 shows a set of data generated during the assay of five pharmaceutical suspension samples in duplicate preparation prepared for clinical testing. Data showed at least two suspected out-of-specification OOS results because they were beyond the specification range of 90-110 of label claim. Data review indicated that the HPLC system was functioning properly and passed system suitability testing. Subsequent laboratory investigation indicated that incomplete extraction of drug substance...
Info Rtm
Figure 9.9. HPLC chromatogram of a drug substance assay method showing separation of the API, impurity 1, precursor, and other trace impurities. The inset is the chromatogram at full scale showing a peak height lt 1.0 AU for the API. Method validation results of this method are shown in Table 9.9. Figure 9.10. a Peak purity assessment using the photodiode array detector and Waters Empower purity algorithm by calculating the purity angle and comparing them to the purity threshold. The peak is...
Info Bfz
Figure 5.14. a Interference peak stemming from contamination of mobile phase A with the pH calibration buffer preservative during pH adjustment procedure by dipping the pH electrode into mobile phase A the weaker mobile phase . b Note that this ghost peak is substantial and elutes close to the active pharmaceutical ingredient API in a composite pharmaceutical assay combining assay and impurity testing. This ghost peak is eliminated by not dipping the pH electrode into the mobile phase A during...
Column Selection Guides
Column selection during method development often reflects personal preferences or prior experience.1'4'6 Nevertheless, some general guidelines can be suggested based on consensus of experienced chromatographers. Note that these recommendations focus on RPLC. Select columns packed with 3- or 5- m high-purity silica-bonded phases from a reputable manufacturer. 50-100mm x 4.6mm for simple samples e.g., assays and dissolution 50-150mm x 3.0-4.6mm for purity testing or more complex samples Waters...
Electrochemical Detector ECD
An electrochemical detector ECD measures the electrical current generated by electroactive analytes in the HPLC eluent between electrodes in the flow cell.1'4 It offers sensitive detection picogram levels for catecholamines, neu-rotransmitters, reducing sugars, glycoproteins, and compounds with phenolic, hydroxyl, amino, diazo, or nitro functional groups. ECD can be amperometric, pulsed-amperometric, or coulometric type and common electrodes materials are carbon, silver, gold, or platinum,...
What Is Hetp In Hplc
Figure 2.18. Van Deemter curve showing the relationship of HETP H vs. average linear velocity. The van Deemter curve has a classical shape and is a composite plot of A, B V, and CV terms plotted below to show their contributions . Hmin minimum plate height, Vopt optimum velocity. Figure 2.18. Van Deemter curve showing the relationship of HETP H vs. average linear velocity. The van Deemter curve has a classical shape and is a composite plot of A, B V, and CV terms plotted below to show their...
System Dwell Volume
Also known as gradient delay volume, system dwell volume is the liquid holdup volume of the HPLC system from the point of solvent mixing to the head of the column. This includes the additive volumes of the injector, the sample loop, all fluidic connection tubing, and any internal pump volumes of a low-pressure mixing system. The typical dwell volume of a modern HPLC is 0.5-2mL, but can be as high as 5-7mL in older systems. High-pressure mixing systems have lower dwell volumes because the point...
Trends
Modern pumps have more features and better reliability and performance than earlier models because of better designs in seals, pistons, and check valves as well as innovations such as dual-piston in-series and piston seal wash.2 Performance at low rates can be improved by variable stroke mechanism, micro pistons, or active check valves. The fluidic components in more inert pumps for bio-purification or ion-chromatography are often constructed from titanium or polyetheretherketone PEEK ....
Trends In Hplc Equipment
Table 4.4 summarizes some of the trends of HPLC equipment. HPLC is a mature analytical technique based on well-developed instrumentation with a definitive trend toward higher performance, better reliability, and easier maintenance. Demands in life sciences and drug discovery are driving HPLC technologies toward higher resolution, sensitivity, and throughput. Recent innovations in micro LC, ultra-high-pressure chromatography, multidimensional chromatography, parallel analysis, and...
I Efs
Table 5.1. Data System Method Types and their Primary Functions Controls and documents parameters of pump and detector Pump flow, gradient conditions, degasser, oven Detector wavelength, bandwidth, filter response, sampling rate Controls and documents parameters of autosampler and sample standard information Injection sequence vial , inject volume, injections, run time Functions inject samples, equilibrate, calibrate, quantitate Standard sample info name, amount, sample weight, label claim,...
Impurity Testing
The monitoring of process impurities and degradants is important to assure drug purity and stability. The Food and Drug Administration FDA has published updated guidelines, which outline the need for stability testing on drugs and biologics for use in humans.19 In addition, the International Conference on Harmonization ICH has recently revised guidelines for the validation of analytical methods to measure and to report impurities and d gradants in new chemical entities.20'21 For sample...
The Resolution Equation
The degree of separation or resolution Rs between two solutes is dependent on both thermodynamic factors retention, k, and selectivity, a and kinetic factors peak width and column efficiency, n .1-36-712 Resolution is controlled by three somewhat independent factors retention, selectivity, and efficiency as expressed quantitatively in the resolution equation To maximize Rs, k should be relatively large, though any k values gt 10 will drive the retention term of k k 1 to approach unity. No...
Chemiluminescence Nitrogen Detector CLND
Chemiluminescence nitrogen detector CLND is a nitrogen-specific detector based on pyro-chemiluminescence technology where N-containing compounds are oxidized to nitric oxide. The nitric oxide then reacts with ozone and emits light of a specific wavelength. It is highly specific and sensitive lt 0.1 ng of nitrogen and yields equimolar response to the nitrogen in the compound. It is particularly useful for the determination of relative response factors of unknown impurities and degradants in...
HighPressure and LowPressure Mixing Designs in Multisolvent Pumps
Multisolvent pumping systems are classified by how solvent blending is achieved, as shown in Figure 4a and 4b. In low-pressure mixing designs typically found in quaternary pumps, a single pump draws mobile phases from a four-port proportioning valve Figure 4.4b . The pump microprocessor controls the solvent composition of each intake piston stroke by the timed opening of each port. Note that solvent blending occurs inside the pump at low pressure. Effective degassing is mandatory in this design...
REFERENCES Qhe
1. S. Ahuja and M.W. Dong, eds., Handbook of Pharmaceutical Analysis by HPLC, Elsevier, Amsterdam, 2005. 2. J.M. Miller and J.B. Crowther, Analytical Chemistry in a GMP Environment A Practical Guide, Jossey-Bass, New York, 2000. 3. S. Ahuja and S. Scypinski, eds., Handbook of Pharmaceutical Analysis, Academic Press, New York, 2001. 4. S. Ahuja, Impurities Evaluation of Pharmaceuticals, Marcel Dekker, New York, 1998. 5. G. Lunn and N.R. Schmuff, HPLC Methods for Pharmaceutical Analysis,...
REFERENCES Qom
1. L. Nollet, ed., Food Analysis by HPLC, 2nd Edition, Marcel Dekker, New York, 2000. 2. R. McCrae, ed., HPLC in Food Analysis, Academic Press, London, 1988. 3. A. Grasfeld-Husgen and R. Schuster, HPLC for Food Analysis A Primer, Hewlett-Packard Agilent Technologies , Waldbronn, Germany, 1996, Publication number 12-5965-5124E. 4. R. Helrich, ed., Official Methods of Analysis of the Association of Official Analytical Chemists AOAC , 15th edition, Arlington, VA, 1990. 5. S. Chang, et al. Food...
Performance Qualification PQ
Performance qualification PQ is the process of demonstrating that an instrument can consistently perform an intended application within some predefined acceptance criteria. In practice, PQ testing is synonymous with system suitability testing conducted with specified columns, mobile phases, and test compounds. PQ is performed during initial system qualification or after the system Table 9.1. An Example of a Design Qualification DQ of an HPLC System for Method Development Table 9.1. An Example...
Radiometric Detector
A radiometric detector, also called a radio-flow detector, is used to measure radioactivity of radioactive analytes in the HPLC eluent passing through a flow cell. Most are based on liquid scintillation technology to detect phosphors caused by the radioactive nuclides. A liquid scintillator can be added post-column with a pump or a permanent solid-state scintillator can be used around the flow cell. This detector is specific only to radioactive compounds and can be extremely sensitive. This...
Summary of Method Development Steps
Table 8.4 summarizes the factors and suggested guidelines in method development. Although each method development scientist might favor his or her own individual approach, the adjustment factors and sequence of steps are likely to be similar. Table 8.4. Summary of Factors Optimized During RPLC Method Development Sequential isocratic steps or scouting gradients Modern columns packed with C8 or C18 phases of 3- or 5- im high-purity-silica support Use PDA to evaluate Xmax of all analytes. Select...
Mobile Phase Parameters B Buffer pH Solvent Type
In HPLC, the mobile phase controls the retention and selectivity of the separation and can be conveniently and continuously adjusted during method improvement. Lowering the solvent strength B of the strong solvent increases retention and typically increases resolution see Figure 2.16 . In isocratic separations, the analyte peaks are typically kept in the k range of 2-20 to maintain sufficient sensitivity of the late eluting peaks. Lowering solvent strength might result in peak shape distortion...
Isocratic Vs Gradient Analysis
Most HPLC separations are performed under isocratic conditions in which the same mobile phase is used throughout the elution of the entire sample. Although isocratic analysis is good for simple mixtures, gradient analysis, in which the strength of the mobile phase is increased with time during sample elution, is preferred for more complex samples containing analytes of diverse polarities.1-5 Advantages of gradient analysis are Better suited for complex samples and applications that require...