Firma:              Beckman

Modell:            P/ACE 5000

Kommentar:   Dokumente engl.

Principles of Operating

Utilizing the electrokinetic principles of electrophoresis and

electroosmosis, the P/ACE 5000 Series instrument separates sample components within a fused-silica capillary. This type of separation is known as high performance capillary electrophoresis (HPCE). In the P/ACE System, sample is injected using either pressure or voltage. Under the influence of high voltage, sample components migrate differentially through the capillary. As these components pass a window in the capillary, a wavelength-selectable UV detector monitors them and transmits a signal (a voltage that is proportional to sample absorbance) to a recorder, integrator, or computer. The signal is then plotted graphically in the form of an electropherogram.

The P/ACE instrument can be used to separate many different kinds of samples, including amino acids, peptides, proteins, DNA fragments, and nucleic acids. Precious and biologically active materials can be separated using this system because efficient separation can be achieved using very small sample amounts (5 to 30 microliters, with the actual injection volume being typically between 10 and 50 nanoliters). Capillary electrophoresis also requires very little time when compared with traditional forms of electrophoresis. Depending upon the length of the capillary used, the applied potential, and the mobility of the sample components, baseline separations can be achieved within a few minutes; typical run times are between 10 and 30 minutes.

P/ACE SYSTEM DESCRIPTION

In the P/ACE instrument, a fused-silica capillary, which is contained within a temperature-controlled cartridge, bridges two electrolyte vials located on an autosampler. The autosampler trays rotate until vials containing the necessary fluids for specific operations are in position beneath the ends of the capillary. The vials are then pneumatically raised so that the ends of the capillary are immersed in the fluid in the vials. Also immersed in one of these vials is a positive electrode (anode) that can deliver up to 30 kV; a negative electrode (cathode) is immersed in the other. *

Sample is injected into the capillary using either pressure or voltage. Pressure injection may be used with free-zone capillaries to avoid sample discrimination based on electrophoretic mobility. Voltage injection is used with gel-filled capillaries that do not permit bulk fluid flow. Continued application of voltage during separation causes sample components to migrate through the capillary at different velocities. Separated sample component zones thus pass by a detector window in the capillary at different times, until all components have passed the detector.

The detector is wavelength-selectable and is located near the outlet of the capillary. It measures the absorbance of each component as it passes and transmits this information to a data system. The data system produces what is known as an electropherogram, which is similar to an HPLC chromatogram in that absorbance values are plotted as peaks, and sample component may be identified by its migration time and may be quantitated by the area under the corresponding peak in the electropherogram.

OPERATION

You can operate the instrument by initiating processes individually or by programming a sequence of processes (methods). The same process values can be run again by simply reinitiating the process or method; alternatively, one or more values can be changed (and retained in memory) before the process or method is run again.

The controller requires that values for various process parameters be entered into memory to perform a process effectively. Required parameters generally include time duration, inlet and outlet vial number, and voltage or current limits. In addition to these process parameters, other types of information can also be entered into the controller memory and some settings can be controlled using certain front panel keys. These other settings include the wavelength to be used during separation, whether high or low pressure will be used during a rinse, the cartridge coolant temperature, the rise time used to reduce noise in the detector signal, the point at which the instrument will automatically calibrate (zero) the detector during a method run, and whether or not multiple repetitions of the same method will be performed automatically.

During a run, all displays show real-time values. The data shown in the DETECTOR OUTPUT display during a separation is filtered to compensate for the eyes" inability to read rapidly changing numeric data. The display can still change quickly, showing changes in absorbance as sample components pass the detector, but not so fast that our eyes cannot perceive the data. The output from the back panel to the data system is not filtered beyond that determined by the selected rise time.

The main components of the P/ACE 5000 Series instrument include an autosampler (two rotating trays) that holds vials of sample and other solutions, a cartridge holder and Interface, a high-voltage power supply and electrodes, an optics module and detector, temperature control hardware, a sample injection mechanism, and a controller with front panel displays and function switches (see Figure 3-1).

SPECIFICATIONS

Dimensions and clearances           see Figure 1-1

Weight (uncrated)                            100lb (45.4 kg)

Power requirements                        50/60 Hz; voltage selectable

                                                         Voltage 90–130 VAC

                                                         or

                                                         190–254 VAC

Fuse requirements

100-120V                                         P,vo ULJCSA T4/250V: IEC T3.15A/250V

220-240V                                         'livo UUCSA T2A/250V: IEC 1.6A/250V

Compressed nitrogen requirements

Pressure                                          80-85 psig (552-586 kPa)

Purity                                                99% (prepurified)

Max. dew point                                 0°C (32°F)

Maximum heat dissipation to the room         300 W (1024 Btu/h)

Ambient operating temperature range          4-40°C (39-104°F)

Recommended range for best performance            15-30°C (59-86°F)

Humidity restrictions                        < 95% (non-condensing)

Pre-rinse time                                  0 to 99.9 min. (resolution: 0.1 min.)

Post-rinse time                                0 to 99.9 min. (resolution: 0.1 min.)

Inject time                                        1 to 99.9 sec. (resolution: 0.1 sec.)

Separate time                                  0.1 to 99.9 min. (resolution: 0.1 min.)

Temperature                                    15 to 50°C* (resolution: 0.1°C)

Inject Voltage                                   1 to 10 kV

Separate Voltage                             1 to 30 kV (resolution

                                                         programming — 0.1 kV;

                                                         operation — 0.01 kV)

Current                                             1 to 250 µA (resolution 0.1 µA

Inlet vial number                               11 through 34

Outlet vial number                           1 through 10

UV Detector

Wavelength range                           190 to 380 nm

Filter selection                                 200-, 214-, 254-, and 280-nm standard

                                                         three open positions for additional

                                                         wavelength selections

                                                         Filter diameter 1/2-in. (12.7 mm)

UV source                                        deuterium lamp, 30 W

Wavelength accuracy                      ± 2 nm

Output range (programmable)        ± 0.005 to +0.200 AUFS

High Voltage Power Supply

Voltage range                                   1.0 to 30 kV

Selectable voltage increments        100 V

Display resolution                            10 V

Absolute accuracy                           ± 3%

Repeatability                                    within 0.3%

Current range                                  1 to 250 µA

Selectable current increments        0.1 µA

Display resolution                            0.1 µA

Absolute accuracy                           2%

Repeatability                                    within 0.2%

Shipping Test Results

The packaged P/ACE instrument has been subjected to the following tests without degradation to the performance or appearance of the instrument

Drop                                                 Free drop 18 inches on all sides

Vibration                                           15 minutes duration composite "Thick-Air" spectrum at 1.2 g's

RMS with the package oriented on the base and left side; and 15 minutes at the resonant frequency of the package (15 Hz) with the package oriented on the base and back at 1.2 g's RMS.