ChromSword in action.

Computer-assisted optimization of HPLC separations Examples for users 1

ChromSword Optimization

Aromatic amines

Gradient profile optimization

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

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

Dear chromatographer,

This example illustrates 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 in this example, you will gain valuable experience working with ChromSword
in particular and in computer-assisted method development in general.

Objective: to optimize separation of mixture containing 21 compounds

Compounds: aromatic amines

1. 2,4-diaminotoluene
2. 2,4-diaminoanisole
3. o-anisidine
4. o-toluidine
5. p-cresidine
6. 4,4´-diaminodiphenylmethane
7. 2,4,5-trimethylaniline
8. 3,3´-dimethoxybenzidine
9. 2-naphthylamine
10. 4,4'-diamino-3,3'-dimethyldipenylmethane
11. 3,4-dichloraniline
12. 4-aminobiphenyl
13. 4-aminoazobenzene
14. 3,3´-dichlorbenzidine
15. 4,4´-diamino-3,3’-dichlorodiphenylmethane
16. o-aminoazotoluene
17. 4,4´-oxydianiline
18. 4,4´-thiodianiline
19. Benzidine
20. N,N-dimethylaniline
21. 4,4'-tetramethyldiaminodiphenylmethane

In this example the mobile phase gradient is optimized. To select the optimum
methanol gradient profile in a mobile phase:

1. Two gradients with different methanol concentration slopes are run (Step 1).
2. Dwell time, zero time, retention, efficiency and peak area data are entered (Step
2).

3. ChromSword finds the optimum gradient profile (Steps 3 and 4).

STEP 1: Separation using two linear gradients

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

1. The two linear gradients are run under the following conditions under two
different gradient slopes.

 Run 1

Column: LiChroCART Purospher RP18e (250 x 4mm)
Organic Modifier: methanol
Water component: 30mM phosphate buffer, pH = 5.0
Starting acetonitrile concentration: 5%
Final acetonitrile concentration: 95%
Gradient time: 45 min
Temperature: 30°C

 Run 2

Column: LiChroCART Purospher RP18e (250 x 4 mm)
Organic Modifier: methanol
Water component: 30mM phosphate buffer, pH = 5.0
Starting acetonitrile concentration: 5%
Final acetonitrile concentration: 95%
Gradient time: 45 min
Temperature: 30°C

2. The retention order for the 21 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 separations 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 ARAM21B.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, select the
ARAM21B.smp file and click the OK button to open the sample file (Fig. 2).

Fig. 2 The Compounds page with the ARAM21B.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 separations 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 concentration of methanol (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 of 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 separations 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). You are now back on the Runs page.
- To browse the data of the second run, set the Current Run # to 2 in the
Current Run Scroll box.

STEP 3: Gradient profile optimization (I)

1. Open the Gradient page by clicking the Gradient button on the upper 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 separations Examples for users 7

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

2. Click the Linear button to display the Linear Gradient Map (Fig. 6). The
Linear Gradient Map shows values of the optimization function (Fopt) for
different gradient times. The lower the Fopt value, the closer a gradient profile
corresponds to the desired results. The red cross on the map indicates the
minimum value of Fopt.

The best time for

linear gradient

Fig. 6 The Linear gradient map

3. Click the OK button to display the optimum linear Gradient Profile (Fig. 7).

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

Fig. 7 The Gradient profile page showing the optimum linear gradient profile

4. Open the Chromatogram page to simulate the corresponding chromatogram

(Fig. 8).

Fig. 8 The Chromatogram page of the Gradient page showing the simulated
chromatogram using the optimum linear gradient profile

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

 Conclusions:

- The analysis time is acceptable.

- Overlapping peaks: benzidine/o-anisidine; p-cresidine/3,3’-
methoxybenzidine; 2-naphthylamine/4,4’-diamino-3,3’-
dimethylphenylmethane; 3,3´-dichlorobenzidine/4,4´diamino-
3,3’dichlorodiphenylmethane/4-aminoazobenzene.
- Gradient profile optimization is needed.

STEP 4: Gradient Optimization (II)

1. Return to the Input page. Click the Separation button to display the Desired
Separation Results dialog box (Fig. 9).

Fig. 9 The Parameters page of the Desired Separation Results dialog box

2. Enter the desired values for the difference in retention between two neighbouring
peaks (Topt = 1), the retention times of the first (Tmin = 4 min) and the last
peaks (Tmax = 60 min) and click the OK button.
3. Click the Process button to open the Search process parameters dialog box
(Fig. 10). If you enter some values for the starting and the final concentration
ChromSword will search for the optimum gradient profile only between these
limits.

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

Fig. 10 The Search Process Parameters box

4. Click the OK button to set the parameters and to start the optimization process.
5. Open the Optimize page (Fig. 11) by clicking the Optimize button on the lower
button bar to start automatic optimization of the gradient profile.

Fig. 11 The Optimize page

The Optimize page contains several windows. These 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 best profile using the Monte-Carlo

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method. The program automatically optimizes both the number of nodes

(breakpoints) and their position on the Concentration-Time surface.

6. Wait several minutes until the gradient profile will contain 6 nodes (breakpoints).
7. Open the Input page to stop optimization process. Click the OK button in the
Confirm box to confirm interruption of the optimization. The Select Gradient
dialog box with the Optimization Map is displayed. (Fig. 12).

The Optimization Map shows the relationship between the optimization function
value and the number of nodes in the gradient profile for each type of gradient profile
(containing 1,2,3... and more nodes). The red cross indicates the gradient profile
with the minimum value of the optimization function. Each point corresponds to the
optimum gradient profile the program has been found during optimization.

Click here

Fig. 12 The Select Gradient box

8. Click the OK button to open Select Gradient profile in the Input page. To load
the gradient profile just found, click the Load button and select the
ARAM21.grl. sample file (Fig. 13).

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Fig. 13 The Input windows with the recommended optimum Gradient profile

9. Click the Profile button to display the Gradient Profile table (Fig. 14). Click the
OK button to close it.

Fig. 14 The Gradient profile dialog box

10. Open the Chromatogram page (Fig. 15) to simulate the chromatogram using
this gradient profile and analyse it.

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

Fig. 15 The simulated chromatogram in the Chromatogram page

11. 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 following steps:

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

- Move the cursor to the left (right) desired 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 (a part
of the chromatogram will be displayed in the limits set).

12. To determine the peak identity and the peak retention time for a particular peak,
move the cursor to the peak and click it. Alternatively, you can click the Table
button to display the Table with the predicted results (Fig. 16).

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ChromSword in action. Computer-assisted optimization of HPLC separations Examples for users 14

Fig. 16 The Retention Prognosis table

13. If you want to find the peak of a particular compound, click the Find button to
display the Find dialog box (Fig. 17).

Fig. 17 The Find dialog box

Conclusion: The simulated chromatogram is acceptable.

FINAL CONDITIONS SELECTED

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ChromSword in action. Computer-assisted optimization of HPLC separations Examples for users 15


Column: LiChroCART Purospher RP18e (250 x 4 mm)
Organic modifier: methanol
Water Component: 30mM phosphate buffer, pH = 3.0
Gradient elution

Gradient Profile:

N t C
1 0.0 8.0
2 11.5 14.0
3 18.3 24.0
4 33.5 30.0
5 38.0 44.0

Temperature: 30°C
Flow rate: 0.7 ml/min
Analysis time: 45 - 48min

Fig. 18 Experimental chromatogram

Final conclusion

The mixture has been well separated. There are no substantial differences between
the predicted and experimental chromatograms. (compare Figs. 15 and 18).

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ChromSword in action. Computer-assisted optimization of HPLC separations Examples for users 16

Dear colleague!

We have guided you through this optimization procedure. In time, when you
gain more experience in rapid 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