The path C:\ProgramData\PurityChrom6 is recommended and used per default as a working directory. Therefore, system configurations should be saved there. However, this path is per default not visible under Windows. To see the folder ProgramData in the data explorer, the option “hidden items” have to be activated in the tab ”View”.

Run the application PrepConConfiguration.exe that is saved under the installation directory (per default C:\Program Files\PurityChrom6). This opens the installation setup. In the step „authentication“, the login mode „user name and password“ has to be chosen to activate the user administration. The user can choose between the connection modes SQLite, MySQL and MongoDB to save the user administration database locally on the computer or a server, respectively.

In the next step of the setup, the location of the local database (in case of SQLite option) or the server connection settings has to be defined.

Then, close the setup via the „Configure“ button. The default account settings (user: admin, password: 0000) will be shown in the report window of the setup. After the first login with this initial administrator account, the user is forced to create a new password.

PurityChrom 6 is the new generation of Knauer´s preparative software PurityChrom, which you will easily recognize at first glance. The whole software was developed further so that an upgrade to PurityChrom 6 will offer you new beneficial features for your purification tasks as the following ones:

• Animated flow path
• Easy method writing via clicks into the system visualization
• Configuration of multiple systems
• User-friendly, modern software interface
• Extended functions of the user administration (detailed permission definition, grouping of users, Windows SID login, …)
• GAMP 5 and 21CFR Part 11 compliance can be supported
• …

To upgrade your existing PurityChrom 5 system to PurityChrom 6, article A2687 or in case of required software qualification A2689 has to be ordered. Your PurityChrom 5 dongle will be also valid for PurityChrom 6, thus please state your existing dongle serial number in your order.

The old software files cannot be transferred automatically to the new software. Therefore, we offer to configure your existing system in the new software. This service is included in A2687 and A2689. You only have to deliver the following PurityChrom 5 files of your existing system to your local sales contact:

• PurityChrom.ini (C:\Windows),
• PurityChrom.cfg and PurityChrom.lic (C:\PurityChrom),
• Visualisation Background (C:\PurityChrom\Visualisation\Visualisation Backgrounds),
• Visualisation File (C:\PurityChrom\Visualisation\Visualisation Files)

 Please note, that the user administration cannot be transferred from PurityChrom 5 to PurityChrom 6.

1. Methods are called “Time Control Files”.

2. You have the possibility to stop your pumps if you pause your run with the “Hold” button. Therefore check “Stop Pumps at Time Control Hold” in “Options”.

3. Start your method at time/volume point 0 with the function “Composition Major Pump” or “Composition Minor Pump” and with the Function “Flowrate”.

4. If you are using both pumps during the run, choose the composition and flow rate for both pumps at time point 0. You can choose a flowrate of 0 ml/min for the pump you want to start later.

5. Choose the default position of all your used valves at time point 0

6. Choose your wavelength (function “Wavelength”) and your required channels with the function “Start Chromatogram” from time point 0.02 (not 0.0).

7. Don’t forget to program an autozero.

8. To program a gradient, you need to include all the angles of this gradient as compositions. A linear gradient will be calculated between two different compositions at two different time points. Program a small time difference between the compositions to create a step in your gradient. Using a P2.1L the smallest time difference should be 0.06 minutes.

9. To start fractionation using a fraction collector, change the position of your fraction collector valve from “Waste” to “Fraction”. The function “Collector” enables you to move the collector arm to the wished position (“Step”: The arm will perform a step to the next position). Don’t program the function “Collector” at time point 0. To set a volume use the function “Fraction Limiter”. The current position of the fraction collector arm will be remembered until the program is switched off and on again.

10. To start fractionation using a fraction valve, change the position of your fraction valve from “Waste” to “Fraction”. You can choose a distinct position or the command “Next Step” to go automatically to the next position. To set a volume use the function “Fraction Limit”. The current position of the fraction valve will not be remembered by the software.

11. Program the function “Stop All” at the end of your method. The function “Stop Chromatogram” is only needed to if you want to restart your method automatically (function “Restart Time Control File”), if you want to link another method to your method (function “Load new File”) or if you are using your method in a sequence table.

PurityChrom gives you the possibility to fractionate peaks via thresholds. This is an optimal fractionation method for well baseline separated and defined peaks. A threshold is set at a specific UV Level as indicated in the example by the blue line. If a signal is overstepping this value, fractionation will be automatically started. If the signal falls below the threshold, fractionation will be stopped, and the valve will switch back to waste position. Both levels can also differ. Be aware that if a peak also consists of a shoulder, which is above the threshold, this shoulder will be collected in the same fraction.

You can also combine the limiter with a threshold. Every fraction, that is recognized by the threshold settings, will be divided into smaller sub-fractions by the limiter function and thus, fractions will not be bigger than the defined limiter value. If you adjust the limiter volume according to your working flowrate and regular fraction sizes, you will minimize contaminations by peak shoulders.

The fractionation via threshold can be defined in a method (time control file) using the threshold tab. Here you can specify at which start and end point the threshold function should be active and which level of signal is triggering the threshold actions. It is important to define the actions that should be carried out upon exceeding or undershooting the signal. This you can do by clicking on the red dots indicated in the screenshots. After defining all parameters for the threshold function, don´t forget to insert them into your time control file by using the button “insert”.

If you want to have different UV values for start and end point of your fraction collection, simply add two thresholds with different UV signal intensities and then define for one only the “Over Event actions” and for the other only the “Under Event actions”. When you would like to combine the limiter and the threshold, simply add the “fraction limiter” command to your method.

The error message "Please wait until UV Detector is ready" indicates that the lamp of the UV detector is off and thus, not ready for analysis. If you don´t want to receive this message, simply deactivate the checking of lamp status in the ini file. For that, open the file "PurityChrom.ini", which you will find under C:\Windows. In this file, please change under [Detector] the command "CheckLampStatus=0". Now you should be able to open methods, although the UV lamp is not ready for running this method.

In PurityChrom a user-defined channel can be created, which will be calculated based on the chosen data channels. 

Settings for a user-defined channel have to be done:

1. In the setup menu of PurityChrom: Open the setup menu. Go to the tab „user-defined channel“ and choose the data channels and the mathematical operation, which should be used for the calculation of the new channel.

2. In the chromatogram window: Open the chromatogram window and activate the user-defined channel by clicking on the „U“ button. Open now the setup menu of the chromatogram window and rename the channel and its dimension.

Using external pressure sensors (one before and one after the column), the user-defined channel can display for example the pressure difference. Or monitor the purity of your sample by defining the user-defined channel as a ratio of UV signals. 

You can fractionate peaks using the automated peak sampling in PurityChrom. In this mode the peaks of your run are analyzed by several factors like slope and baseline and a customizable fractionation is carried out while the peak situation (e.g. peak start, peak valley, peak maximum, peak shoulder and peak end) is determined

In the time control file, you can find the tab “sampling”. Here one defines the time range in which the automatic fractionation should be active. Also, you can define which data source is used for the peak sampling and how many data points are averaged over (“filter factor”). The value for “slope sensitivity” is the most important point, because here a new peak can be identified by its steepness. The “maximum baseline level” is the value, which determines whether a peak valley or a peak end is identified. You must put in a value higher than the usual baseline of the chromatogram you want to fractionate. The settings under “maximum fractions” and “excessive fractions to” are used with specialized valves but do not apply to KNAUER hardware.

To define which actions are taken at which peak situation you click on the dot of the peak situation and then define the actions, that should be carried out by the software. To get a rough idea of the values “maximum baseline level”, “filter factor” and “slope sensitivity”, which are compatible with the peak situation of your chromatogram you can use the integration function in PurityChrom.

Fractionation via a limiter is often used in standard FPLC runs. It means, that upon setting the fraction collector into fractionation mode, it will collect the same size fractions as long as it is in this mode. An example of the fractionation behavior you can see pictured. You see here that the same volume is collected for each fraction unless you interfere with the fractionation manually. This is convenient especially for runs with less defined peak shapes, low UV intensities and to make sure, that no material is lost during the run. However, the possibility of contaminating the product of interest is likely because no active surveillance of UV signal is involved in this fractionation type, as you can see in the exemplary run for fraction 8.

To implement the limiter in a method, you add the command “Fraction Limiter” and define the volume which is repeatedly collected during the run. In this example, the tube is changed after a volume of 1 ml is collected. The fractionation is started by switching the fraction collector valve into the fractionation position at the point of your choice.

The variables functions allow you to freely adjust certain parameters in a method flexibly upon starting the method or when creating sequences. Hence you can run different applications with just one method or screen quickly the most important method parameters.

Variables have to be defined in the Time Control File in the tab „Variables“. Here you should decide which parameters you would like to hold flexible and give them a short name as well as a default value. Don´t forget to push the button „insert“ to save your settings.

To add a variable to your method, go to the tab „Functions“ in the Time Control File and choose the function related to your variable. With a long-term left mouse click in the parameter field, a shortcut will be opened with the short name of your defined variable. After clicking on the short name and inserting the function in your time control file, the variable will be shown in the timetable of the method.

If you start a method including variables as a single run, a window will be opened before the method is executed, in which you can change the values for your defined variables.

Suppose you add a method including variables in a sequence table. In that case, new columns will be automatically added to the sequence table, in which you can change the settings for your defined variables. This option enables you a quick screening of method parameters and thus a fast optimization of your method.

The following parameters could be used as variables in PurityChrom:

Solvent Composition Major / Minor

Flowrate Major / Minor

Wavelength (UVD/DAD)

Mass (MS)

Channel No.

Threshold value

Valve position

Limiter value

Collector position

Temperature

Function Time

Time Shift

Also check our tutorials about variables in PurityChrom 5! 

If the administrator account of PurityChrom 5 is locked because of too many wrong login attempts, deliver the following information to support@knauer.net:

• Dongle serial number
• Date at which the new account should be valid

The KNAUER customer support will send you a number that can be used as user name and password to enter PurityChrom. However, this account is only valid for one day only on the defined date. Please make sure, that time and date are correctly set on your system computer.