!
The HP CE instrument consists of a PC and a base unit consisting of
detection and separation subunits. Methods are developed on the PC
and downloaded to the base unit for independent execution. The control
electronics and firmware of the separation subunit takes care of tray and
vial movement, capillary voltage, current, and power control, capillary
temperature control, diagnostics, and related data capture.
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voltage trace acquired during the analysis to calculate the
apparent mobility of the species.
The temperature of the capillary in the cassette has to be
stabilized because it influences the electrophoretic results.
One can compare this with the thermostabilization of an
HPLC column. Electromobilities depend on solvent viscosity,
which changes at a rate of about 2%/°C for typical buffer
solvents. Chemical retention, if it occurs, is also influenced
by temperature. In general, cooling is required because the
current through the capillary heats the solvent and would
boil the liquid with the sample in it. The temperature must
be stable within at least ±0.1°C and perhaps as low as 10°C
below ambient.
For cassette cooling a Peltier element is used instead of a
chiller. This element is made of semiconductor material, and
with electrical current applied, one end is heated while the
other end is cooled. The direction of current flow defines
which end is cooled. Thus the unit can be used for both
•
heating and cooling by reversing the excitation voltage.
•
During automated sequences the samples to be analyzed are •
kept in the liquid handling tray. If temperatureĆsensitive
•
samples like biosamples are being analyzed, the sample storĆ •
age area has to be cooled to as low as 5°C to ensure that the
samples are not spoiled while they are stored in the instruĆ
ment. While capillary cooling is always required, the tray
cooling is an optional feature. The user can connect a chiller
•
to control the tray temperature and the HP CE instrument
•
will measure and display the tray's temperature.
•
ForcedĆair cooling is used. The forcedĆair circulation has to
be fast enough to ensure that water condenses only on the
surface of the cooling heat exchanger. This is important beĆ
cause a wetted surface is able to conduct electrical current.
With 30 kV in a sample bottle this can lead to unexpected
high voltages in other areas of the instrument.
An analogĆtoĆdigital converter (ADC) resolution of 12 bits is
sufficient for all of the signals that need to be converted. The
highest resolution is required for temperature control of the
capillary and for raw data generation. A 12Ćbit ADC allows a
resolution of 0.075 A in current and 0.025°C in temperature
measurements.
One ADC is multiplexed to measure the analog signals for
voltage, current, return current, pressures, and temperature.
Multiplexing requires more firmware support. It is a load to
the interrupt level of the system processor and is therefore
timeĆsensitive, but it saves hardware cost.
Fig. 1 shows the functional blocks of the separation subunit,
which are:
Operating system
Vial and liquid handling
HighĆvoltage control and safety functions
Thermal control
Replenishment and injection.
These functions are separated onto different printed circuit
boards. The separation subunit contains the following
boards (see Fig. 2):
Main processor board (CMP)
PC interface board (CRB)
Electromechanical interface board (EMI)
Injection and
Replenishment
Control
Operating
System
Injection
Transducer
DAC
To
ChemStation
HP-IB
Valves
Control
High-Voltage and
Safety Functions
Liquid Handling
Temperature Control
Lift
DAC
DAC
DAC
– HV
Lift
+ HV
Lift
Fan
Temperature
Sensor
Fan
Door
Temperature
Sensor
Functional block diagram of the separation subunit.
June 1995 HewlettĆPackard Journal
Temperature
Sensor
Peltier
Cooler
Control
Replenishment
Transducer
Valves
CMP
HV
Supply
Power
Control
HP-IB
CRB
Data
EMI
HVT
PDV
PDV
PDV
PDV
TDR
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16-Bit Control Data (14 Bits Used)
Calculated Every
Three Seconds
8 Bits MS Byte
6 Bits LS Byte
64 Cycles = 800 ms
n Cycles, n = LS Byte, Cycle = 12.5 ms
f = 30 kHz
H
L
PWȀ = MS Byte + 1
PW = MS Byte
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To guarantee user safety, the HP CE instrument implements
a number of safety features. Tray rotation is stopped after a
maximum of 400 ms when the tray door is opened. Any
movement that was in progress is finished after the door is
closed again. The highĆvoltage power supply is brought to
0V and switched off when the top cover is opened or when
a leak is detected. The cassette fan is switched off when the
top cover is opened to avoid finger injuries. Pressure on the
vials is released immediately when the top cover is opened;
otherwise, the vial contents would be sprayed inside the
detector compartment. Pressure is removed from the buffer
bottle if a leak is detected.
When a concentration dependent detector signal is inteĆ
grated over time the peak area is a measure of the amount
of separated sample compound. The sample area and the
amount are related by the speed of the absorbing particles
as they move through the detector cell.
In HPLC separations the speed of the sample is normally
known. It is the flow rate of the liquid. In electrophoresis,
the separating power is the difference in the mobility of the
compounds, which means that the compounds have differĆ
ent specific speeds while moving through the detector.
To correct for speed differences in later evaluation steps,
information about the moving force is needed. In a simple
analysis the voltage is constant during the complete run, but
temperature changes have an influence on viscosity and
mobility, and a timeĆprogrammed voltage or fraction collecĆ
tion can have a tremendous influence on absoluteĆareaĆtoĆ
amount calculations.
To support future evaluation developments the HP CE inĆ
strument supports five raw data channels in addition to the
detector signals:
• Voltage, current, and power of the highĆvoltage source
• Pressure across the separation capillary
• Temperature of the air from the cassette.
Traditionally, diagnostic routines built into instruments are
based on the perception that the failure modes are already
known during the development phase. For these failure
mechanisms special tests are implemented (RAM, ROM, comĆ
munication, display, etc.) which generate a pass/fail indicaĆ
tion or a value. Sometimes the knowledge of a specific failĆ
ure mode in the early development phase leads to changes
of the product that reduce or even eliminate the failure
mode. However, there is no possibility of avoiding the learnĆ
ing curve. Unexpected behavior and dependencies always
appear after the product is released, and may increase the
list of diagnostic functions needed. Thus, the diagnostics
implemented in the early phase might be of little use while
those most needed are not supported. The disadvantage of
the traditional concept is that the diagnosis of failure modes
discovered after the development phase is not possible withĆ
out product changes.
The HP CE instrument implements traditional diagnostic
functions as outlined above, but the firmware also impleĆ
ments basic functions for diagnosis, such as read sensors, or
write to hardware address. Macros on the PC can use these
basic functions to construct complex diagnosis functions to
address specific problems. Now if a new failure mode is
discovered, we just add a diagnosis macro for it and create
an updated version of the diagnostics disc to distribute it to
the manufacturing and service organizations. This leads to a
continuous improvement of the diagnostics for the instruĆ
ment, and we spend the time to create diagnosis functions
only for failures that have actually occurred in the field. A
valuable side effect is that most module test routines are
implemented within the product, so manufacturing does not
need a large amount of special equipment. The service perĆ
son at the customer's site has all the equipment needed to
perform the same tests as originally used in the factory.
The instrument normally collects equally spaced data for
voltage, current, power, vial pressure, and temperature in a
buffer memory in the instrument RAM. The diagnosis firmĆ
ware makes it possible to redirect other data like servo
speed, servo position, servo current, sensor data, valve states,
and so on to this buffer. Thus, we can store up to five conĆ
figurable measured values, with a configurable data rate of
up to 20 Hz. The PC software reads this buffer and generates
reports and graphics.
This tool greatly improved our ability to find bugs during the
development phase because we got the history of servo
positions, pressure values, and other measured values when
an error occurred. It also gave us much more insight (like a
builtĆin oscilloscope for electromechanics) into what really
happened in the unit than we would have obtained from
single measurements.
An advantage of this firmware feature is that service personĆ
nel can use it to find problems in the field and can send the
printout to the factory for a simple form of remote diagnosis.
An example is the macro for checking the pressure system.
It takes about five minutes and checks the power of the air
pump and the tightness of the pressure and vacuum system,
the injection system, and the replenish system and stores the
data from all of the pressure sensors. A graph of these quanĆ
tities characterizes most of the pressure system and helps
diagnose the problem if there are deviations from the normal
behavior.
The success of this product is a result of the exceptionally
good teamwork of all the people who worked on it. We
especially want to thank Gerda Biselli for her patience in
writing the online help and the manual, Arno Graf for the
discussions about serviceability and diagnostics, and Claudia
Maschke for designing the temperature controller board.
The great support of the production and marketing groups
during development helped us to find and realize some of
the ideas that make the instrument as good as it is.
1. A. Wiese, et al, A New Generation of LC Absorbance Detectors,"
HewlettĆPackard Journal, Vol. 41, no. 2, April 1990, pp. 36Ć43.
2. C. Büttner, et al, Firmware Development for a Modular Liquid
Chromatography System," ibid, pp. 44Ć50.
June 1995 HewlettĆPackard Journal