High-throughput Method Development for Aldehydes and Ketones Using an Agilent 1290 Infinity LC-system

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The Application Notebook

The Application Notebook, The Application Notebook-07-02-2010, Volume 0, Issue 0
Pages: 28

This application note deals with the development of a fast method for the determination of 13 aldehyde and ketone derivates with the Agilent 1290 Infinity LC-system. The method, which used acetone as organic co-solvent, separates the analytes within 3.5 minutes.


Aldehydes and ketones are important compounds in the chemical industry. The majority of analytical methods for their determination makes use of the derivatization with 2,4-dinitrophenylhydrazine yielding the corresponding 2,4-dinitrophenylhydrazone. After that, an HPLC separation with UV detection is performed.



For method development, an Agilent 1290 Infinity LC-system was used. The system consists of: 1290 Infinity binary pump with integrated degasser, 1290 Infinity high performance autosampler, TCC SL thermal controlled column compartment, 1290 Infinity diode array detector

Analyte mixture

The mixture of aldehyde-2,4-dinitrophenylhydrazones and ketone-2,4-dinitrophenyl-hydrazones is a certified reference material from Sigma-Aldrich (catalogue no. 47651-U) diluted in acetonitrile. In the mixture, each analyte has a concentration of 30 µg/mL of carbon of aldehydes and ketones, respectively.

Results and Discussion

Figure 1 shows the separation of 13 aldehyde-2,4-dinitrophenylhydrazones and ketone-2,4-dinitrophenyl-hydrazones within 3.5 minutes. Acetone was used as organic co-solvent. All peaks are baseline separated with a critical resolution of 1.6 between peak pair 7/8. Moreover, Figure 1 also shows a comparison of programmed and effective solvent gradient. Due to the very small dwell volume of 125 µL of the Agilent 1290 Infinity LC system, there is only a minor difference between the programmed and effective solvent gradient when compared to a conventional HPLC-system, which exhibits a dwell volume of approximately 1000 µL. This means that at a flow rate of 1.2 mL/min, the programmed solvent gradient for a conventional HPLC system reaches the column inlet with a delay of about 0.83 minutes, so that the elution of the early eluting analytes occurs under isocratic conditions. In other words, the elution of the early eluting analytes cannot be affected by the solvent gradient. Using the Agilent 1290 Infinity LC-system with a dwell volume of 125 µL at a flow rate of 1.2 mL/min, the programmed solvent gradient reaches the column inlet after 6.25 seconds and enables fast separations within a few minutes.

Due to the applied flow rate of 1.2 mL/min and the 1.8 µm particle packed column, a pressure drop of 1100 bar during the solvent gradient can be observed. To constitute the robustness and reproducibility of the developed method 10 consecutive chromatograms were compared. There were virtually no differences between the 10 chromatograms (not shown). This conclusion is also confirmed by the relative standard deviation (RSD) of retention times of the analytes, which is in a range between 0.03 and 0.09%.


It could be shown that the Agilent 1290 Infinity LC-system is very suitable to develop fast HPLC methods. The separation of 13 aldehyde and ketone derivatives succeeded in around 3.5 minutes by using acetone as organic modifier in the mobile phase. Furthermore, the presented method highlights that fast HPLC separations are only possible using HPLC-systems with a very small dwell volume.

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