In HPLC, the major aim of method optimization is to define the suitable conditions for robust, precise and reproducible analysis. A computer-assisted method development can be a useful tool to save resources. To characterize carbonyl content in air samples, usually a standard mixture of 13 aldehyde and ketone DNPH derivatives is used.
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A good separation of all 13 components has to be attained for precise analysis. In this experiment, the chromatography modeling software DryLab® with 3D Cube option was applied for the optimization of the analysis of the cabonyl standard mixture.
The examination of the combined influence of temperature, gradient time, and ternary eluent composition on critical resolution allowed for the development of robust method conditions. Moreover, the robustness space was studied in silico and experimentally verified with a high degree of agreement.
Figure 1. Chromatogram of 13 carbonyls, measured according to ISO DIN 16000-3 method with the DNPH column.
Figure 2. DryLab 3D Cube with 12 red pointed measurement conditions.
The analysis of the separation of 13 carbonyls was carried out in accordance with the method explained in DIN ISO 16000-3 . The chromatogram obtained from this experiment resulted in 11 peaks (Figure 1). The peaks that represented acetone-DNPH, acroleine-DNPH, 2-butanone-DNPH, methacroleine-DNPH, and butyraldehyde- DNPH were not separated.
To optimize method parameters in silico, DryLab needs measurements under 12 conditions (Figure 2). The measurements were performed as explained below. The obtained chromatograms were fed into the DryLab software, leading to the method operation design region (MODR). The red regions in the cube stand for the optimal chromatographic conditions (Figure 3). The selection of the best parameters from the predicted data pull is derived from high resolution values.
The optimal separation method was established with the solvent composition water and acetonitrile, with a gradient time of 14 minutes and a column temperature at 22 °C. From the results shown in Figure 4, it can be seen that the baseline separation of acetone-DNPH and acroleine-DNPH was reached with the resolution value of 2.69 (see Table 1). The lowest resolutions were realized between peak pairs 2-butanone-DNPH, methacroleine-DNPH (1.27) and methacroleine-DNPH, n-butylaldehyde (1.29).
Figure 3. MODR Method Operation Design Region.
Figure 4. Chromatogram of 13 carbonyls, measured according to DryLab® predicted method with the DNPH column.
Materials and Method
The HPLC system consists of the detector AZURA® DAD 6.1L, pump AZURA® P 6.1L HPG, column thermostat AZURA® CT 2.1 and autosampler AZURA® AS 6.1L. The method separation (as explained in DIN ISO 16000-3 ) and following method optimization was conducted on DNPH-column (150 x 3 mm).
The standard with 13 aldehyde and ketone derivatives, dissolved in acetonitrile, was collected from Sigma Aldrich and was diluted to a concentration of 1 μg/mL in acetonitrile. The DryLab® (Version 4) modeling software (Molnár-Institute, Berlin) was employed for method optimization.
The optimal separation conditions were predicted based on 12 chromatograms. The measurements were carried out by three different mobile phase compositions (100% MeOH, 50:50 MeOH:Acetonitrile, 100% Acetonitrile). Each composition was used for measurements at two different temperatures (20 °C and 40 °C) and two different gradient times (30 and 90 minutes). OpenLab chromatographic software was used to analyze the chromatograms.
Dwell volume of the system, initial gradient conditions, and column parameters were programmed in the DryLab® software for the method optimization. The chromatographic data files were changed into AIA (*.CDF) format and uploaded in the DryLab® software for the calculation.
The DryLab® software is a key part in the HPLC method optimization. As can be seen from the results, the software enables defining optimal separation conditions without carrying out several unwanted measurements. It helps to reduce the consumption of materials, conduct ecological `green`HPLC, and save time.
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 DIN ISO 16000-3; Indoor air – Part 3: Determination of formaldehyde and other carbonyl compounds in indoor air and test chamber air – Active sampling method (ISO 16000-3:2011).
This information has been sourced, reviewed and adapted from materials provided by KNAUER Wissenschaftliche Geräte GmbH.
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