Separation methods in the CE require a large number of parameters which strongly influence the separation of the involved components. It takes a lot of time and work to consider all those parameters in the separation method. The simulation software CESAR enables the CE user to simulate applications and separation methods at the PC without using the CE device and thus receiving important clues about the planning of experiments. This means that it is no longer necessary to carry out costly and time-consuming optimization experiments for separating new samples. CESAR simulates changes in parameters and their effects on sample separation in real time and thus shows when a real experiment is useful and necessary.
You have the possibility to enter data which were defined before in an experiment into the simulation program to compare the simulation results with the real data. This means that CESAR optimizes applications for special tasks.
The program simulates the changes of parameters such as pH, voltage and temperature in real time. The effects can be followed immediately on the screen. CESAR can also be applied for training and teaching persons who are not yet experienced in carrying out capillary electrophoresis experiments.
Features
With the help of CESAR, you can combine all components which are contained in the database and simulate CE runs. As CESAR provides detailed evaluations you receive the most important information about the reaction of the sample during the CE.
Features of CESAR are:
l Simulation of sample separationComponent Databasel Experiment values in correlation to simulated data
l Component database including all samples and their parameters
l Database with standard EOF (Electro-Osmotic Flow) conditions which were defined in real experiments
l CE-applications database for the managing experiments and documenting them in detail (according to GLP Good Laboratory Practice)
l Definition of the best parameters for the separation by using numerous diagrams such as resolution plots and selectivity plots
The component database contains all components and their characteristics which are available to CESAR. You can always add data to the database and edit the components. The data which are most important for a simulation are the molecular weight and all dissociation constants (pK). In addition, you can enter the ionic equivalent conductance and the mobility of a sample at a certain pH and temperature.
EOF Database
The EOF database contains the standard conditions of the experiment which are necessary for calculating the EOF. They include:
l Concentration of the puffer solution [mol/l]As well as in the component database, you can edit and add values or delete values entered before.l Viscosity of the puffer solution [Pa·s]
l Total length of the capillary [cm]
l Capillary length to the detector [cm]
l Capillary diameter [µm]
l Electro-osmotic mobility at different pH values (captured in an experiment)
Experiments
Generally speaking, you can simulate each experiment as long as the components required are defined in the database. Simply flag the components and EOF conditions which are to be simulated. The most important parameters, such as the pH of the solution, the voltage or the temperature, can be changed in the diagram so that you can immediately see the effects of the alteration.
You can also add real experiments to the simulation program which makes a direct comparison of the real experiment to the simulation possible.
Diagrams
The elution diagram shows the migration times of the single components during the later stage of the CE experiment. Experiments which were already carried out can be added to CESAR and are shown in the elution diagram. In addition to the elution diagram, CESAR shows a couple of other diagrams which are also meaningful for method optimizations.
l Dependency of the elution time on the temperatureDocumentationl Dependency of the elution time on the pH
l Dependency of the diffusion coefficient on the temperature
l Dependency of the resolution on the pH
l Dependency of the EOF mobility on the pH
l Dependency of the separation on the pH; „Separation Plot" (shows the separation of all components referring to one selected component)
l Dependency of the component mobility on the pH
l Dependency of the component mobility on the temperature
l Dependency of the total mobility on the pH
l Dependency of the selectivity on the pH (the selectivity is calculated out of the mobility differences of the components which are nearest to each other. E.g., if two components out of a total of 10 are not separated, the result will be a selectivity of 0)
l Elution profile
l Dependency of the voltage on the pH
All diagrams and simulation reports can be printed out to be documented in the lab book. Thus, CESAR is a great aid for the work routine in the laboratory.
Comparison of the Simulation (CESAR) to the Experiment
Extract of Bernhard Ausperger’s Dissertation, FH Reutlingen (University for Applied Science), June 1997.
The following comparison is to demonstrate the efficiency of CESAR. It also shows how the simulation results can be transferred to real experiments which is achieved because of the great precision the program operates with.
Material and Methods:
| Device: | Hewlett Packard HP3DCE-System with the software | |
| HP Chemstation HP3D G1601A, Rev A.01.02 | ||
| Configuration (Presettings) | Voltage | 15 kV |
| Polarity | positive | |
| Capillary | Untreated Quartz Capillary | |
| Total Length LGes 48.5 cm | ||
| Length to Detector LD 40 cm | ||
| Capillary Diameter DI 75 µm | ||
| Detection | DAD Detector | |
| Measurement at 200 nm | ||
| Temperature | 20 °C | |
| Injection Pressure | (2 sec at 50 mbar) | |
| Puffer System | 25 mM Phosphate Puffer | |
The puffer system (25 mM PO4-Puffer) was produced with the following
chemicals:
| phosphoric acid 85% | |
| sodiumdihydrogenphosphate | Fluka, # 71500 |
| disodiumhydrogenphosphate | Riedl-De-Haèn, # 30412 |
| Benzyl alcohole 99% | Janssen, Lot: 44847/1 |
| NaOH 0.1 N | Fluka, # 72079 |
| NaOH 1 N | Fluka, # 72082 |
| Cytochrom c from horse heart | Fluka, # 30400 |
| Trypsin | Fluka, # 93611 |
| Tris(hydroxymethyl)-aminomethan e | Merck. # 8382 |
| Ammoniumacetate | Fluka, # 09692 |
| Calciumchlodrid Hexahydrate | Fluka, # 21110 |
| HCl 32 % | Merck |
| Tryptic digest of the protein Cytochrom c |
| Basic substance: Cytochrom c from horse heart |
Figure 1: Simulated elution profile of the Cytochrom c Fragments after 24 Stunden tryptic digest for a puffer solution with pH 4.
Table 1: Assignment of the experimentally
defined migration times of the single peaks to the peaks calculated in
the simulation at pH 4 [t in min].
| 1. Run | 2. Run | Simulation | Fragment | |
| 1. Peak | 6,719 | 6,668 | 12,03 | GITWK |
| 2. Peak | 7,071 | 7,025 | 15,25 | TGQAPGFTYTDANK |
| 3. Peak | 9,709 | 9,596 | 17,49 | TGPNLHGLFGR |
| 4. Peak | 10,205 | 10,099 | 22,43 | GGK |
| 5. Peak | 10,373 | 10,240 | YIPGTK | |
| 6. Peak | 11,973 | 11,871 | 33,56 | ATNE |
| 7. Peak | 13,045 | 13,181 | 40,85 | HK |
| 8. Peak | 13,369 | 13,702 | 41,07 | MIFAGIK |
| 9. Peak | 13,883 | 14,518 | 41,80 | NK |
| 10. Peak | 14,094 | 14,880 | 43,91 | GK |
| 11. Peak | 14,197 | 14,967 | 44,69 | TER |
| 12. Peak | 16,465 | 19,231 | 45,54 | EETLMEYLENPK |
| 13. Peak | 20,175 | 23,202 | 57,19 | EDLIAYLK |
| 14. Peak | 25,044 | 28,495 | 59,19 | IFVQK IFVQK |
| 15. Peak | 27,222 | 31,240 | 92,68 | GDVEK |
| 16. Peak | 30,808 | 35,842 | 114,79 | CAQCHTVEK |
Diagram 1: Graphical comparison of the simulated migration times with the experimentally defined migration times of Cytochrom c fragments at pH 4
Diagram 1 shows in graphics which was already indicated in Fig.1: The resolution as well as the order of the fragments agree very well.
Discussion of the Cytochrom c Fragmentation
The comparison of the cytochrom c fragments at pH 4 shows a perfect agreement of simulation and experiment. There are differences in the migration times which are higher in the simulation than in the experiment. However, the migration times can be optimized in any case by adapting the correction factor. Apart from that, all data and results of the simulation can be totally undertaken to the experiment.
Requirements
The simulation program CESAR can be operated under Windows 3.x, Windows95
or WindowsNT. The prerequisites are:
| l IBM Compatible 486 or more |
| l 4 Mbytes RAM |
| l 3 Mbytes Hard Disk Space |
| l VGA Graphics |
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