ChromSword in action.

Computer-assisted optimization of HPLC separation Examples for users 1

ChromSword Optimization

Prohibited azo dyes

1. Gradient profile optimization

This example illustrates how to optimize the separation using data from the actual
chromatographic runs without taking into account chemical structures and column
properties

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 2

Dear chromatographer,

This example will illustrate how you can carry out a step-by-step optimization procedure of an
HPLC separation using your own PC. Acceptable separation conditions can be found after
only a few runs. Just follow the steps described below and you will see how easily you can solve

your separation problem using ChromSword . While carrying out the optimization

procedure described below, you will gain valuable experience working with ChromSword
in particular and in computer-assisted method development in general.

Objective: to optimize the separation of a mixture containing 17 compounds:

Compounds: prohibited azo dyes

1. 2,4-diaminotoluene
2. o-toluidine
3. p-cresidine
4. benzidine
5. 4,4-diaminodiphenylmethane
6. 2,4,5-trimethylaninline
7. 3,3’-dimethoxybenzidine
8. 2-naphthylamine
9. 4-chloro-o-toluidine
10. 4,4’-diamino-3,3’-dimethyldiphenylmethane
11. 4-aminobiphenyl
12. 3,3’-dichlorobenzidine
13. 4,4’-methylene-bis(2-chloroaniline)
14. p-chloroaniline
15. 4,4’-oxydianiline
16. 4,4’-thiodianiline
17. 3,3’-dimethylbenzidine

In this example, the 17 azo dyes are separated using a methanol/water gradient. To
find the optimum methanol gradient profile, the following steps are carried out:

1. Two gradients with different methanol concentration slopes are run (Step 1).
2. Dwell time, zero time, retention, efficiency and pea areas data are entered (Step
2).

3. ChromSword finds the optimum gradient profile (Step 3).

STEP 1: Separation using two linear gradients

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 3

1. The two runs are carried out using the following separation conditions at two
different gradient times:

Run 1


Column: LiChroCART Purospher RP18e (250 x 4 mm)
Organic Modifier: methanol
Water component: 20 mM phosphate buffer, pH = 7.0
Starting methanol concentration: 5%
Final methanol concentration: 95%
Gradient time: 63 min
Temperature: 55°C

Run 2


Column: LiChroCART Purospher RP18e (250 x 4 mm)
Organic Modifier: methanol
Water component: 20mM phosphate buffer, pH = 7.0
Starting methanol concentration: 5%
Final methanol concentration: 95%
Gradient time: 21 min
Temperature: 55°C

2. The retention order for the 17 compounds is determined for these two runs.

STEP 2: Sample file creation

Working Procedure:

1. Start ChromSword ( Fig. 1).

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 4

Click here

Fig. 1 ChromSword: The Sample module showing the blank Compounds page

2. Enter the names of the compounds and save the sample file as AZODYES.smp.
See on-line Help (Tutorial, problem 6, steps 1-5) on how to create a sample file
3. Click the Open button, to display the Open file dialog box, then select the
AZODYES.smp file and click the OK button to open the sample file (Fig. 2).

Fig. 2 The Compound page with the AZODYES.smp file opened

4. Click on the Gradient button on the upper button bar to open the Gradient
module, then click on the Runs button to open the Runs page (Fig. 3).

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 5

Fig. 3 The Runs page of the Gradient module

 Input 1:

- Experimental zero time value (retention of 1 µl of 0.01% KNO3) (see

the experimental value in the Zero Time edit field)
- Experimental dwell time (see the experimental value in the Dwell
Time edit field)
- Gradient time (see the experimental value in the Gradient Time edit
field)
- Starting concentration of methanol (see the experimental value in the
Conc. Starting edit field)
- Final methanol concentration (see the experimental value in the Conc.
Final edit field)

 Input 2:

- Retention time, efficiency and peak area data for each compound in the
mixture for Run 1 and Run 2
- Information about the column and the eluent being used

5. You can edit data of existing runs or add data for a new run. You can also
browse the data using the Current Run scroll box on the Runs page. To edit (or
only to browse the experimental values), the retention time, efficiency and
peak area data for the First Run use the following procedure:

- Set the Current Run # to 1 in the Current Run scroll box.

- Click the Edit button to open the Run Editor dialog box (Fig. 4). (To enter
data for a new run click the Add button).

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 6

Fig. 4 The Run Editor dialog box

- Enter (or only browse experimental values) the retention time, efficiency
and peak area data for Run # 1 and click the OK button. (If you are only
browsing, click the Cancel button).
- To enter (or browse) the data for the second run, set the Current Run # to 2
in the Current Run scroll box, enter the data and click the OK button. (If
you are only browsing, click the Cancel button).You are now back on the
Runs page.

STEP 3. Gradient profile optimization

1. Open the Gradient page by clicking the Gradient button bar in the Runs page.
The default linear gradient profile appears on the screen (Fig. 5).

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 7

Fig. 5 The Input page of the Gradient page showing the default gradient profile

2. Click the Separation button to display the Desired Separation Results dialog
box (Fig. 6).

Fig. 6 The Parameters page of the Desired Separation Results dialog box

3. Enter the desired value for the difference between the retention of two
neighbouring peaks (Topt = 1), the retention time of the first peak (Tmin = 4
min) and the last peak (Tmax = 40min), the weight value for optimization of the
retention time of the last peak (Wmax = 5, which indicates Tmax as being
extremely important) and then click the OK button.

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 8

4. Click the Process button on the Gradient page to open the Search process
Parameters dialog box (Fig. 7). If you enter values for the starting and the final
concentrations C0 and C1, ChromSword will search for the optimum gradient
profile between these two limits.

Fig. 7 The Search Process Parameters dialog box.

5. Click the OK button to select the parameters.

6. Open the Optimize page (Fig. 8) by clicking the Optimize button on the lower
button bar on the Gradient page (Fig. 5) to start the optimization of the gradient
profile.

Fig. 8. The Optimize page.

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 9

The optimize page contains several windows. These windows enable you to see and
to control the optimization process. During optimization, ChromSwordgenerates
many thousands of gradient profiles and simulates chromatograms for each gradient
profile, evaluates them and searches for the optimum profile using the Monte-Carlo
method.
The program automatically optimizes both the number of nodes (breakpoints) and
their position on the Concentration-Time surface.

7. Wait several minutes until the gradient profile will contain 6 nodes (breakpoints).
8. Open the Input page to stop the optimization process. Click the OK button in
the Confirm box to confirm interruption of the optimization process. The Select
Gradient dialog box with Optimization Map is now displayed. (Fig. 9).

Click here

Fig. 9 The Select gradient box

The Optimization Map shows the relationship between the optimization function
(Fopt) value and the number of nodes in the gradient profile for the each type of
gradient profile (containing 1,2,3... or more nodes). The lower the value for Fopt,
the more a simulated gradient profile corresponds to the ideal gradient. Each point
corresponds to the optimum gradient profile that the program has found for different
types of profiles. The red cross indicates the gradient profile with the minimal value
of the optimization function.

9. Click the OK button to load the Selected Gradient Profile into the Input page.
To load the optimum gradient profile just found, click the Load button and select
the AZODYES.grl. sample file (Fig. 10).

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 10

Fig. 10 The Input window with the recommended Gradient Profile

10. Click the Profile button to display the Gradient profile table. (Fig. 11). Click
the OK button to close it.

Fig. 11 The Gradient profile table

11. Open the Chromatogram page (Fig. 12) to simulate the chromatogram using
this gradient profile and analyse it.

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 11

Fig. 12 The simulated chromatogram in the Chromatogram page

12. If you want to see part of the chromatogram in detail you can set the Cursor
Detail mode of the cursor. To do this, carry out the following steps:

- Press the Space key (the cursor changes the colour).

- Move the cursor to the desired position (left or right) and press the Space
key to set the first limit (a vertical line appears indicating the first limit).
- Move the cursor to the right (left) position and press the Space key (part of
the chromatogram will now be displayed in the limits set).

13. To determine the peak identity and the peak retention time of a particular peak,
click on the peak. Alternatively, you can click the Table button to display the
Table with the predicted results (Fig. 13).

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 12

Fig. 13 The Retention prognosis table

14. If you want to find the peak of a particular compound, click the Find button to
display the Find dialog box (Fig. 14).

Fig. 14 The Find dialog box

15. Choose a compound of interest and click the OK button. The cursor will set the
position of the selected peak on the chromatogram.

FINAL EXPERIMENTAL CONDITIONS SELECTED

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ChromSword in action. Computer-assisted optimization of HPLC separation Examples for users 13


Column: LiChroCART Purospher RP18e (250 x 4 mm)
Organic modifier: methanol
Water Component: 20 mM phosphate buffer, pH = 7.0
Gradient elution

Gradient Profile suggested

N t C
1 0.0 28.0
2 19.9 28.0
3 20.0 60.0
4 29.8 60.0

Temperature: 55°C
Flow rate: 1.0 ml/min
Analysis time: 30 min

Fig. 15 Experimental chromatogram

Final conclusion

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The compounds have been separated. There are no substantial differences between
the predicted and experimental chromatograms (see Figs. 12 and 15).

Dear colleague,

We have guided you through this optimization procedure. In time, when you
gain more experience in computer assisted method development using
ChromSword you will find other ways and other separation conditions using
the experimental data for this example. The steps described here is not a
definitive procedure but rather a set of guidelines to get successful results
quickly, working with a stand-alone PC rather then using an HPLC data
system.

MERCK