EuroChrom 2000 for Windows Basic Edition version 2.05

EuroChrom 2000 for Windows
Basic Edition
version 2.05
Addition to Users’ Handbook v.1.2
1. Support of the KNAUER mini-instruments
The additional detectors are implemented in the Hardware Constructor’s Instrument Category List:
• the KNAUER UV Filter Photometers K200, K2000/1
• the KNAUER UV Spectral Photometer K2500
• the KNAUER RI detectors K2300/K2400/1.
Adjustment of all mini detectors (and pumps) is performed with the same Setup dialog box.
Digital (through RS232 serial COMx port) or analog (through integrating output and HPLC Interface
box) data transmission to a PC can be chosen.
Modification in the Hardware Configuration will result in the modification of the time table in the
method description. For example, if the Filter Photometer K2000/1 or the Spectral Photometer K2500/1
is included in the hardware configuration, a new column (wavelength control) will appear in the time
table of the method. In case of analog data transmission the HPLC Interface box produces an output
voltage on the Analog Output Nr.4 (Wavelength control) which corresponds to the wavelength. It is at
the responsibility of the user, to define the instrument which should be controlled by the box.
The KNAUER pumps K-500/1, K-120 and K-100 support serial communications and can be controlled
by the EuroChrom 2000 v.2.05 software directly via COM port. A separate COM port is required for
every instrument!
2. Support of the new KNAUER switching valves
Three types of new KNAUER switching valves are supported: the Injection valve (possible positions
are Inject/Load), the 6-port and the 16-port switching valves.
They can be adjusted in the same manner as mini-instruments and controlled by either the interface
box serially via RS232 port.
In case of valve control via the Interface box, the following connection will be necessary:
Knauer
GROUND
HOME
BACKW./INJ.
FORW./LOAD
OUT
HOME in the Valve
To GROUND Pin of the Interface Box
To a Pin, which is the next relatively to
one defined in the Hardware Constructor
To a Pin defined in the Hardware Constructor
In the case of digital control, it is necessary to setup the number of COM port.

Depending on the type of valve used a new column appears in the time table that allows the user to
define the position of a switching valve (I/L, 1-6 and 1-16 correspondingly). The EuroChrom 2000 for
Windows software analyses the value from the cell and produces a sequence of pulses or sends an
appropriate command to move the valve into the desired position.
3. Support of the Spark Autosamplers
When you work with the Spark autosamplers, please pay attention to the following:
In the hardware constructor, in the Basic Marathon SETUP choose the communication port and the
Autosampler ID (all other fields like Manufacturer, Type, Serial Number have no influence on control of
the instrument and used for documentation only). The port and ID MUST correspond to the actual
connection and settings.
If you chosen the serial mode for the Spark autosamplers, it is necessary to press F4 button for
Basic-Marathon to switch the autosampler to serial mode;
To setup the ID on the autosampler, press PROG/END button when you are in the serial mode.
This ID MUST CORRESPOND to the ID selected in the Hardware constructor.
The control of the Spark autosamplers Basic-Marathon and Triathlon in the EuroChrom 2000 for
Windows software is accessible from a macro table.
Autosampler
Settings
The software detects the autosampler described in the system and after selecting the EDIT
AUTOSAMPLER SETTINGS function a tool corresponding to the autosampler type will appear. In
principle, it is possible to use different methods in the macro table, although they must have similar
hardware configuration.
General settings for the Basic-Marathon autosampler can be adjusted in the MARATHON SETUP
dialog (Partial loopfilling option can be chosen for the Basic Marathon Plus)

and for the Triathlon autosampler in the TRIATHLON SETUP dialog:
The First Vial and Last Vial fields in a Macro table are dependent of the autosampler used and its
settings.
For the Basic Marathon autosampler the following values are acceptable:
• First Vial
from 1 to 96 samples without washing (1.5 ml vials);
from 1 to 92 samples with washing (1.5 ml vials);
from 1 to 48 samples for large sample vials (5 ml);
• Last Vial
Position of the last vial of the sample series in the tray;
Empty positions in the sample tray must be avoided
• Inj./Vial (Repeat)
Number of Injections of the same sample.
9 Inj./Vial
For the Triathlon autosampler the values depend on a tray configuration:

• First Vial
from 1 to 96 samples for 1.5 ml vials;
from 1 to 72 samples for 4.0 ml vials;
from 1 to 32 samples for 10 ml vials;
from 1 to 160 samples for micro vials (0.8 ml);
• Last Vial
Position of the last vial of the sample series in the tray must be in the range defined
above.
• Inj./Vial (Repeat)
9 Inj./Vial
Settings for a particular injection method are defined in every Macro line and stored in the INJECTION
METHOD cell. Double-click on the cell allows to edit the injection method.
The programming possibilities are dependent of the autosampler:
The Basic Marathon autosampler can perform full loop injections only. The injection volume is defined
by the loop volume and Flush Volume or Flush Time can be defined for the injection method. For the
Basic Marathon Plus you can specify Injection Volume.
• Injection Volume
the loop volume for the Basic Marathon
from 0 to 50 % of the programmed loopvolume with steps 10 ul (10, 20, 30, ....) for the Basic
Marathon Plus
• Flush Volume
flushing volume for needle and connecting tubing prior filling the loop;
from 30 (50 for Marathon Plus) to 999 µl
this field is enabled for Volume Base only.
• Flush Time (sec)
duration of headspace pressure for filling the loop.

from1 to 999 sec.
this field is enabled for Time Base only.
The Triathlon autosampler allows to select different injection methods. The maximum programmable
injection volumes are:
• Partial loopfill injections
50 % of the programmed loop volume
• µl pick-up injections
volume = (loop volume - 3 x needle volume)/2
• Flushed loop injections
not programmable, is equal to the loop volume but needs more sample to fill the loop
completely.
• Flush Volume
from 0 to 999 µl
Programming of time based methods is also supported for the Triathlon autosampler.
Please refer to a corresponding autosampler user manual for more details.
Tip: the serial mode is recommended for the Spark autosamplers;
it is necessary to press F4 button for Basic-Marathon and MENU / SERIAL for
Triathlon on the autosampler to switch the autosampler to serial mode;
PIN-Layout for the interface cable PC/Spark autosampler:
25 Pol Marathon male 9 Pol PC female
Pin 2 Pin 2
Pin 3 Pin 3
Pin 7 Pin 5
Pin 4/5 bridge Pin 1/6 bridge
Pin 6/8 bridge Pin 1/4 bridge
Pin 8/20 bridge Pin 7/8 bridge

4. Realisation of batch protocol printing
In the previous version it was possible to print protocol either from a graphic window or from a peak
report. Independently of how many chromatograms are displayed in the graphic window, only the
protocol for the current chromatogram could be printed.
The current version now allows printing of all displayed chromatograms after activation of the
FILE/PRINT PROTOCOL operation.
5. Setup the target directory for macro
In the previous version the files that were created after running a macro were saved in only one and
the same directory. It was not possible to choose between specific directories or sub-directories.
The possibility to select a target sub-directory for measured data is now supported. After activation of
MACRO/SET TARGET DIRECTORY the user selects the desired sub-directory and all the measured
and related data is saved in this directory.
6. Start macro from any position
To start a macro from any line is supported. The version 1.2 of the software allowed the start of a
macro from the last executed position (if the macro was interrupted) or to start it from the very
beginning. Sometimes it is necessary to run a macro for one or several predefined samples (i.e. to
change usual macro run).
7. Changing sorting mechanism for the tables
In the previous version a sorting mechanism was supported for a time table. After modification of time
the lines are reordered to keep consistent time order. Focus was defined by the current position of line,
i.e. it kept the same position after reordering the lines and currently edited line lost the focus. Now, the
focus is linked to the edited line, so after reordering the edited line keeps the focus.
8. Table column Auto-fill possibility
A possibility to fill all the cells in one column by the value taken from the current table cell (Menu
EDIT/FILL COLUMN) is supported.
9. Extension of a Header in Protocol printing
A new check-field appears in Print Protocol (Header structure) settings - Print Linked Objects. If this
field is checked, the measurement method name and analysis parameters name as well as their
creation time appear in a header of a chromatogram.
This certainly makes sense for the option of printing the protocol consisting of two objects:
chromatogram and peak report (which seems to be the most often used option).
Additionally, Creation Time as well as an Operator name are added for all objects which are printed in
the Protocol.
10. Calibration as an entry in the main menu
To speed up an access to a manual calibration utility, a new entry CALIBRATION appeared in the
EuroChrom 2000 main menu. It has exactly the same functionality as an option
MEASUREMENT/SERVICE/CALIBRATE from a measurement method window.
11. Support of various performance calculations
EuroChrom 2000 for Windows Basic Edition calculates the following values that can be used to assess
overall system performance:
• Relative Retention
• Theoretical Plates
• Capacity Factor
• Resolution
• Peak Asymmetry
The following diagram shows the parameters used to calculate these system performance values for
the separation of two chromatographic components.

Injection
Unretained Peak
t a
Time
t
1
t
2
W 1
Separation of Two Chromatographic Components
Note: To accurately calculate suitability values, the sampling frequency (set in Acquisition Setup) must be
set to provide at least 20 data (10 for the leading edge and 10 for the tailing edge of the peak) points
for the narrowest peak of interest.
Relative Retention (Selectivity)
t 2 - ta
α =
t 1 - ta
Where
α = Relative retention.
t2 = The retention time measured from point of injection
Capacity Factor (k’)
Where
t 2
k' = −1
ta
ta = The retention time of an inert component not retained by the column, taken
from "Unretained R(etention) T(ime)" in the Detection Parameters section.
t1 = The retention time from point of injection for reference peak defined in the
Detection Parameters section. If no reference peak is found, this value
becomes -1.
k' = Capacity Factor
t 2 = The retention time measured from point of injection
t a = The retention time of an inert component not retained by the column, taken
from "Unretained R(etention) T(ime)" in the Detection Parameters section.
USP (United States Pharmacopia) calculation method
Theoretical Plates
t
n = 16( )
W
2
Where
n = theoretical plates
t = The retention time of the component
W = The width of the base of the component peak using tangent method.
W 2

Peak Asymmetry (Tailing Factor)
.
T = W 0 05
2f
Where
T = Peak asymmetry, or tailing factor
W0.05 = The distance from the leading edge to the tailing edge of the peak, measured
at a point 5% of the peak height from the baseline
f = The distance from the peak maximum to the leading edge of the peak at the
position of 5% peak height
Peak Front Peak Tail
f
W 0.05
Peak Maximum
Asymmetric Peak
0.05 h
Resolution
2( t 2−t1) R =
W2 + W1
Where
R = Resolution between a peak of interest (peak 2) and the peak preceding it
(peak 1).
t2 = The retention time measured from point of injection of peak 2.
t 1 = The retention time measured from point of injection of peak 1.
W 2 = The width of the base of the component peak 2
W 1 = The width of the base of the component peak 1
EMG (Exponential Modified Gaussian) calculation method
Theoretical Plates
Where
N =
� t �
�
W
�
� 0.
1�
41.
7 ×
b0.
1
+ 1.
25
a0.
1
2
N= The number of theoretical plates
t= The retention time of the component
W0.1 = The width of the peak at the position of 10% peak height
a0.1 = The width of the first half (start to top) of peak at the position of 10% peak
height
b0.1 = The width of the second half (top to end) of peak at the position of 10% of
peak height
Peak Asymmetry (Tailing Factor)
.
T = W 0 05
2f
Where
h

Resolution
Where
T = Peak asymmetry, or tailing factor
W0.05 = The distance from the leading edge to the tailing edge of the peak, measured
at a point 5% of the peak height from the baseline
f = The distance from the peak maximum to the leading edge of the peak at the
position of 5% peak height
( t
R = 2.
15×
W
2 − 1)
0.
1
+
t
W
p0.
1
R = Resolution between a peak of interest (peak 2) and the peak preceding it
(peak 1).
t2 = The retention time measured from point of injection of peak 2.
t 1 = The retention time measured from point of injection of peak 1.
W 0.1 = The width of peak at the position of 10% peak height
W p0.1 = The width of previous peak at the position of 10% peak height
DAB (German Pharmacopia) calculation method
NOTE: This calculation equation is also called the BP (British Pharmacopia), EP (European Pharmacopia),
and ASTM.
Theoretical Plates
Where
N
=
5.
54
� t
×
�
�W
0.
5
�
�
�
2
N = Theoretical plates
t = The retention time of the component
W0.5 = Width of peak at the position of 50% peak height
Peak Asymmetry (Tailing Factor
T W005
.
=
2f
Where
T = Peak asymmetry, or tailing factor
W0.05 = The distance from the leading edge to the tailing edge of the peak, measured
at a point 5% of the peak height from the baseline
f = The distance from the peak maximum to the leading edge of the peak at the
position of 5% peak height
Resolution
( t
R = 1.
18×
W
Where
2 − 1)
0.
5
+
t
W
p 05
R = Resolution between a peak of interest (peak 2) and the peak preceding it
(peak 1).
t2 = The retention time measured from point of injection of peak 2.
t 1 = The retention time measured from point of injection of peak 1.

W 0.5 = The width of the component peak at 50 % peak height.
W p0.5 = The width of the previous component peak at 50 % peak height.
Japanese Pharmacopoeia (JP) calculation
Theoretical Plates
Where
N
=
5.
55
� t
×
�
�W
0.
5
�
�
�
2
N = Theoretical plates
t = The retention time of the component
W0.5 = Width of peak at the position of 50% peak height
Peak Asymmetry (Tailing Factor)
T W005
.
=
2f
Where
T = Peak asymmetry, or tailing factor
W0.05 = The distance from the leading edge to the tailing edge of the peak, measured
at a point 5% of the peak height from the baseline
f = The distance from the peak maximum to the leading edge of the peak at the
position of 5% peak height
Resolution
( t
R = 1.
18×
W
Where
2 − 1)
0.
5
+
t
W
p 05
R = Resolution between a peak of interest (peak 2) and the peak preceding it
(peak 1).
t2 = The retention time measured from point of injection of peak 2.
t 1 = The retention time measured from point of injection of peak 1.
W 0.5 = The width of the component peak at 50 % peak height.
W p0.5 = The width of the previous component peak at 50 % peak height.
12. Database viewer: new status information, „Put into directory“- and „Copy
with links“- options
The number of different objects stored in a current sub-directory of a database as well as the number
of selected objects is shown on a Status bar of a database viewer.
A possibility to copy a set of linked objects without breaking the links is implemented. There are two
modes: copy all linked objects (if someone copies one chromatogram which is linked to a method and
the method is linked to more chromatograms, all the chromatograms will be copied); stop at method
(other chromatograms linked to the method will not be copied).
Contrary to Copy/Move operations, the option PUT into DIRECTORY does not perform any physical
movements of the chosen data. It modifies a structure of internal informational links in a way that the
objects are visible in a selected sub-directory. The option is especially helpful for re-organization of
large customer databases.

13. „Locate“ function for every object: jump into a corresponding sub-directory
in a database
The function „Locate“ is available for every object and accessible from menu FILE/LOCATE of a
corresponding window. It allows to jump into the sub-directory of a database where the object is stored
even if the database is already closed. If there is no opened database viewers at the moment, the
function activates new one.
14. „Find object“ function:
Search for an object of all possible types by name with wildcards (‘*’ and/or ‘?’ symbols), case
sensitive/insensitive, pre-search by date, search in all registered databases, current database or
current directory, option „look in sub-directory“ is supported. The function is accessible from a
Database Viewer window menu BASE/FIND OBJECT.
15. Tracing LogBooks
The EuroChrom 2000 v.2.05 software supports multiple log books. Contrary to previous versions of the
software supporting only one HISTORY file, the current version creates a new history file (log book)
every time it starts a new working session. The log book is stored into a subdirectory HISTORY/MM-
DD-YY (the date of start of a working session) in the working directory of the EuroChrom software. The
possibilities to open one of the previous log books (the EuroChrom main menu OPTIONS/SHOW
LOGBOOK) or the current log book (OPTIONS/SHOW CURRENT HISTORY) are supported.
16. Validation utility with report printing possibility
A new feature - a possibility to validate all vitally important files with following report printing is
supported now. The following parameters are checked by the validation program: size, time, date,
check sum. A copy of the validation result is stored also in an ASCII data file in the EuroChrom working
directory.
17. Support of multiple HPLC systems under Windows NT
The EuroChrom 2000 v.2.05 software allows to run several HPLC systems from one PC. A special
ECW2000 Multiple utility is required to organize a PC environment for multi-System operation. Please
call KNAUER customer support for the details.
18. Installation of the Diode Array Detector (ISA and PCI Interface cards)
The EuroChrom 2000 v.2.05 software supports the K-2700 Diode Array Detector (PhotoDiode Array)
with two possible interface cards: for ISA bus and PCI bus. If you install the K-2700 Diode Array
Detector support you have to chose the appropriate interface card type:

NOTE: Do not check both PCI DAD and ISA DAD support simultaneously.
In case of PCI DAD you should install the PCI DAD driver.
To install the PCI DAD driver:
1. Insert the CDROM into your CD drive
2. Double-click on “My Computer”
3. Right-Mouse click on your CDROM
4. Select “Explore” from the right-mouse menu, go to Drivers\K2700
5. Double-click on Setup.exe
You should also select the appropriate Interface Board Type when creating the hardware configuration
from the EuroChrom hardware constructor