Last year the Nobel Prize for Chemistry was granted to scientists, who were modeling physico-chemical interactions in biochemistry. Although we are modeling since 1985, the recognition of the importance of modeling for the whole Life Science is a great motivation for the Molnar-Institute to continue in finding the best column and the best separation in HPLC, by modeling a large number of possible chromatograms.
It is 40 years ago, that Csaba Horváth discovered in 1975 the reason for the success of Reversed Phase Chromatography at Yale – changes in the structure of water as part of the eluent. Complex calculations were required, but computers were just starting to emerge. He used a PDP11 “cabin” to describe, how acids dissociation might be modeled by equations.
Today we are modeling separation choices in a multi-variable Design Space to the most robust method for industrial drug production. With a computer generated separation model at hand, one can calculate the influence of various experimental parameters on the separation and, therefore, also model the robustness of a separation. This is extremely useful if a method should be used for a long time where one has to take into account day-to-day variations in method conditions.
The talk will demonstrate on case studies, how modeling software can work together with modern instruments to produce the best separation instead of what used to take months, in as fast as a few hours.
1 Restek Corporation, weerhaan 9, 4336KT, Middelburg, The Netherlands
For several reasons, there is interest to replace helium for a different carrier gas in gas chromatography. Hydrogen is the obvious choice, but there is a concern on safety and reactivity. It is also possible to use nitrogen, but this is often not considered because it has a low optimal flow and velocity.
By using method translation and chromatogram modeling it became clear that the loss of efficiency using nitrogen could be perfectly compensated by using a smaller ID capillary of a shorter length. By replacing a 30m x 0.25 for a 20m x 0.15mm, it is demonstrated that separations under nitrogen are exactly the same as obtained with Helium, in the same analysis time, while using exact the same conditions for oven programming. Even the inlet pressures are very close.
The only price that has to be paid, is a loss in loadability, which means that this concept will not work for every application, but for many, it will. Besides the guaranteed availability, using nitrogen offers a big advantage in the cost and consumption volume of carrier gas, meaning cost per analysis will also benefit significantly.
Antonella Cavazza, Bignardi Chiara, Claudio Corradini, Carmen Laganà, Paola Salvadeo
Food products may come in contact with polymeric materials during processing by kitchen equipment and tableware, and storage and/or heating in plastic containers. This contact can be responsible for the migration of several compounds depending on food and plastic chemical properties, temperature and time of contact.
Polycarbonate is one of the most studied food contact material; migration of its monomer bisphenol A, and additives such as UV-absorbers or antioxidants, employed to prevent polymer degradation, has been reported to occur in food simulants (1). Ageing and light exposure are responsible for polymer degradation; besides, mechanical or chemical abrasion occurring during use and/or washing can damage the surface leading to release of oligomers and genesis of degradation products belonging to the so-called non-intentionally added substances (NIAS).
In this context, the development of suitable analytical methods to identify and to determine even low concentrations of possible contaminants is essential for safety assessment. High resolution and accurate mass spectrometry has been demonstrated to be a very promising technique in this field (2). The present study was aimed at developing and validating a method based on UHPLC coupled to Orbitrap technology to be employed for the identification and the monitoring of several compounds through a both targeted and untargeted approach. The investigation was focused on tableware of different age. Qualitative and quantitative analysis of bisphenol A and common additives employed in plastic production was carried out. Besides, the identification of colouring agents and polymer products of degradation was performed by means of the high mass accuracy. Statistical analysis permitted to correlate age, degree of surface damage, and bisphenol A migration, and allowed to discriminate new and old samples by evaluating the occurrence of a different pattern of degradation products.
Gao Y, Gu Y, Wei Y (2011) J Agric Food Chem 59, 12982.
Bignardi C, Cavazza A, Corradini C, Salvadeo P (2014) J Chromatogr A 1372, 133.
Nowadays, protein and peptide analysis is on top of the research agenda of molecular biochemists, neurologists, geneticists, etc. At the same time, the enantioseparation of D/L amino acid pairs is on the one hand still a challenge and on the other, it is widely neglected. Consequently, a considerable confusion exists in various different areas of life sciences related to such issues, as homochirality of mammalian and human amino acids, geochronology making use of the amino acid clock, etc. Amino acid analysis with use of GC needs pre-column derivatization of amino acids to enhance the volatility thereof. Depending on pre-column derivatization type performed, the GC enantioseparation of amino acid pairs can be done with use of commercially available non-chiral or chiral capillary columns (i.e., in the so-called indirect or direct approach). The HPLC enantioseparation of amino acids is done mostly with use of regular non-chiral chromatographic columns (e.g., packed with C18, C8, etc.), yet upon pre-column amino acid derivatization with the Marfey’s reagent (or its analogues), to obtain respective diastereomers. For direct enantioseparation of underivatized amino acids, an absolutely limited spectrum of commercially available chiral HPLC columns is available from the market.
TLC is a flexible analytical tool which permits direct enantioseparation of amino acids, without a need to derivatize them prior to the analysis proper. Apart from commercial stationary phases used for direct enantioseparation of amino acids, a possibility exists of an in-home preparation of different chiral stationary phases and of specifically modified mobile phases which facilitate such enantioseparation. In most cases, molecular mechanism of direct amino acid enantioseparations by means of TLC is based on complexation of transition metals. Special attention will be given in this talk to selected examples of successful amino acid enantioseparations with use of TLC. In conclusion, on certain occasions direct enantioseparation of amino acids performed with aid of TLC can provide unequivocal results in shorter time and at a lower cost than the fully instrumental GC and HPLC technique.
We have succeeded for the first time to quantify biological compounds in human plasma using pillar array columns, to be specific, a fast and quantitative method for the determination of branched-chain amino acids (BCAAs) in human plasma using pressure-driven liquid chromatography (LC) on a chip was developed. BCAAs (valine (Val), isoleucine (Ile), and leucine (Leu)) have been found to be closely related to some diseases, such as obesity, diabetes, and chronic liver disease. Hence, development of a fast and quantitative analysis method for BCAAs should be significant. We performed fast separation of amino acids by using a long pillar array column with low-dispersion turns. However, a pillar array column has not been applied for any quantitative determination in real samples. Therefore, in this study, the pillar array column was utilized for the determination of BCAAs in human plasma sample. BCAAs were firstly derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and then analysis of NBD-BCAAs was optimized to be finished in 100 s. With the help of an internal standard (IS), the concentrations of BCAAs in human plasma samples were quantitatively determined. The results showed good agreements with the value with a conventional LC. The developed method should be a useful tool for fast diagnosis in clinical field.