Multi-format Sample Introduction
Automated Sample Introduction from 96-well plates, 2 mL autosampler vials, PCR* vials and 0.5 mL tubes can be accommodated. Automation is certainly a means by which many laboratory bottlenecks may be reduced. One of the major bottlenecks is in sample handling. The use of 96-well plates as a sampling vessel not only increases system capacity but allows compatibility with automated sample preparation devices which utilize the 96-well plate format. Additionally, 2 mL vials (which are autosampler industry standards) may be used; or sampling can be achieved directly from PCR* vials and microfuge tubes
Multiple Separation Modes
Modes of separation include: Voltage, Current, Power, Pressure and Vacuum. All electrophoretic separations allow the programming of both step and linear gradients along with the simultaneous application of pressure or vacuum on both ends of the capillary. Voltage gradient programming is beneficial for gel-sieving techniques, improving separations over a wide range of fragment sizes. This is especially critical for the separation of nucleic acids. The application of simultaneous voltage and pressure is beneficial for the high efficiency mobilization of proteins during iso-electric focusing as well as detecting material not migrated off the capillary. Also, several CEC methods have specified the use of simultaneous pressure applied to both inlet and outlet reservoirs. This system is compatible with those methods.
High Sensitivity UV Detector
The Modular High Sensitivity Selectable Wavelength UV Detector utilizes up to seven filters in an assembly, which allows wavelength changes during a run. Detector sensitivity in Capillary Electrophoresis is critical since small mass loads are introduced and small detection path lengths are used (governed by the capillary dimensions, i.e. 25 - 200 um). Fixed wavelength detection in a filter wheel assembly maximizes detector sensitivity yet still allows wavelength changes during a run. In a Q.C. or regulated environment it is beneficial to "lock-down" a method once it is developed in order to retain the integrity of the data. The PA 800 high sensitivity UV detector may be configured with a "single" wavelength from a filter traceable to NIST making both the instrument certification and method validation much simpler. Additionally the system employs complete fiber-optic technology bypassing the necessity of an optical bench. This lowers the noise on the detection system (by removing reflective surfaces where energy loss can occur) and simplifies the system making it more rugged for routine use.
GLP/CGMP Compliance Features
32 Karat™ Software includes the following GLP/CGMP compliance features: 1) Original method is stored with sample data. 2) Raw data cannot be overwritten. 3) The instrument logs all major events. 4) Software security includes password protection with multiple levels of operator access. Many laboratories are mandated by law to follow current good manufacturing practices (CGMP) and good laboratory practices (GLP; the 32 Karat™ Software has many features which are geared towards the maintenance of data integrity. With the GLP flag turned on, sample data and methods parameters cannot be overwritten or overridden, ensuring the data that was generated was as described. To track instrument operation, all events including errors are logged onto the system. To provide security of operation users may be coded to different security levels with selected access to: system configuration, methods programming, data reprocessing, specific instrument operation and data comparison. All of these features can be used to "lock down" the system for regulated use or logged in as a "toggled off" state in laboratories where flexibility of operation is the critical issue.
Specialized CE Calculations
Capillary Electrophoresis requires specialized data handling. 32 Karat™ Software provides specialized CE calculations including: Apparent Mobility, Mobility, Corrected Peak Area, Molecular weight and Iso-electric Point. The software also includes system suitability which will use these calculations in actuating suitability decisions. The suitability algorithms are capable of triggering re-injections, methods pausing, methods aborting or running shutdown methodology upon failure. Mobility calculations establish the identity of a compound independent from changes in electroosmotic flow, voltage, temperature and column dimensions. This can be very useful in compound identification and method validation especially when comparing data between different laboratories where analytical conditions may vary slightly. However, to calculate this value one must utilize actual voltage values, not simply programmed values. Since CE detection is "on-column," it is important that peak area be normalized to a compounds velocity. When characterizing a protein, it is essential to be able to calculate its molecular weight and iso-electric point.
Sample Tray Temperature Control
Temperature regulation controls samples independently from the electrophoresis buffers. Sample temperature maintained from 4º to 60º C. Sample temperature control is beneficial in minimizing the degradation of temperature-labile compounds and controlling reaction rates when enzyme kinetics. However, it is essential (when regulating the sample temperature) to ensure that the temperature of the electrophoresis buffers remains unaffected. Lowering the temperature of buffers, detergents, high salts or chiral additives will result in precipitation to occur. Furthermore, temperature gradients due to cooled buffers may negatively impact separation efficiency, peak symmetry and even reproducibility.
Electrokinetic, Pressure and Vacuum Sample Introduction
Sample introduction is accomplished using one of the three injection modes: Electrokinetic, Pressure and Vacuum. Each mode allows variable control of all introduction parameters and is equally accessible from either end of the capillary. All sample injection modes are equally accessible from either end of the capillary. An instrument designed to operate with all commonly used sample introduction modes and the capability to dial in the parameters (i.e. injection pressure or vacuum), allows greater ease in transferring methods developed on other instrument brands. Being able to inject from both sides of the capillary, provides a rapid low resolution screen and a high resolution analysis all from the same capillary.
Diode-array provides high resolution spectral scanning detection for Capillary Electrophoresis. Capillary Electrophoresis generates peak efficiencies much greater than HPLC, making scanning with traditional "fast-scanning" detectors impractical. Diode-array detection provides high quality spectral information without compromising resolution. The exceptional spectral resolution provided, allow library search algorithms to more accurately identify a compound even with large analyte concentration differences. With 32 Karat™ Software, four channels of data can be viewed, collected and analyzed in real-time. Any wavelength in the scan range may be viewed during a run and selected for extraction and analysis after the run. This allows an operator to make methods development decisions in real time as all data may be accessed.
These systems use an internal mercury lamp to calibrate the Diode-array detector providing excellent wavelength accuracy (± 1 nm) and precision. This ultimately improves the ruggedness and reproducibility of the system.
CAESAR Integration Algorithms
Lower limits of quantitation can be obtained with CAESAR Integration Algorithms. CE, by its nature, separates components based on differences in their inherent mobilities and as such produces peaks, which are typically non-gausian in distribution. This a function of the analytes mobility with respect to the mobility of the separation electrolyte. CE baselines also tend to demonstrate more perturbations and drift as lower detection wavelengths are commonly used challenging traditional integration algorithms. The CAESAR algorithm has been optimized for CE data reduction, using moving median data filtering to lower the limits of quantitation. Up to five fold improvements in LOQ's have been documented by using this means of data reduction which is optimized for CE analysis.
Automatic Standards Co-Injection
Automatic co-injection from multiple standards in combination with the sample injection is offered. Sample injections can be automatically spiked with reference standards for positioning or for positive identification. This is useful to help minimize errors resulting from sample matrix effects. Additionally, with high salt samples, it may be necessary to do multiple injections within the same run in order to employ an electrokinetic salt elution step. Practical examples of this include the electrokinetic injection of PCR* samples directly after amplification. The interfering salts and metals would need to be be removed through a sample handling step if one were not able to perform an electrokinetic elution from ddH20 prior to sample introduction. Additionally, once a sample table has begun, the data that is generated may indicate the necessity to run new sample combinations. On-the-fly sample table editing may be accomplished with the 32 Karat™ Software allowing the addition of new samples to a running sequence.
Dual Wavelength LIF Option
High sensitivity laser-induced fluorescence detection extends the capabilities of Capillary Electrophoresis for the analysis of carbohydrates and nucleic acids and other compounds which either naturally fluoresce or that can be made to fluoresce through sample derivitization. LIF provides both selectivity and sensitivity to any analysis and typically yields 500 x better sensitivity than UV for compounds which fluoresce. The LIF detector is modular and will allow simultaneous excitation using two laser sources; 488 nm and 635 nm. Two different wavelength emissions can be collected simultaneously. This is important where you wish to run your sample and standard together in the same run, using two "colors" to discriminate between the two species.
Guards against system obsolescence and lowers cost by allowing the use of multiple detectors on a single instrument platform. For example, a methods development platform may be rapidly reconfigured to a routine use platform by changing the detector module and locking down the software methods.
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