Improving quantification using curtain flow chromatography columns in the analysis of labile compounds: A study on amino acids☆
Introduction
High speed, high resolution separations are an essential component of our modern analytical requirements. Separations coupled with mass spectral detection represent a powerful analytical technique, however, its limitations need to be taken into account. In particular, analysis through-put is limited by the solvent capacity of the MS. The drive towards smaller particles, and the developments in monolithic columns has meant that efficient separations can be undertaken at higher flow velocities. Hence the design strategy of column technology is at variance with the operational limitations of the mass spectrometer, since in MS detection solvent must be removed from the sample, and as the flow rate increases solvent removal becomes more difficult. Strategies that are employed by the analyst to overcome the solvent removal limitation of the MS are either, (1) the use of post-column flow stream splitting, which generally results in a reduction in sensitivity and separation efficiency, or most commonly nowadays, (2) the use of narrow bore columns, rather than analytical scale columns, albeit with perhaps a compromise made in the performance of the chromatographic separation [1], [2], [3].
Recently, a new column design has been developed that unites the benefits of both large and small internal diameter columns. This column technology has been referred to as active flow technology (AFT), and in this work we demonstrate the performance advantages of the curtain flow (CF) column, a member of the suite of AFT columns. The concept of the CF column has been discussed in detail in numerous publications [3], [4], [5], [6], [7], [8], but it is discussed again here briefly to provide context to the current study. Effectively, AFT columns, including the CF column, provide an environment that establishes a ‘virtual’ narrow diameter column inside a larger format column [4], [5], the flow properties of which emulate that of conventional narrow bore columns that have the same internal diameter as that of the virtual column internal diameter, but with improved performance. The advantage of the virtual column is that it is free from wall effects [4], [5]. The key design features of the curtain flow column are the end fittings (both inlet and outlet) and the frits that are housed in these fittings. The frit at both the inlet and the outlet is an annulus, containing a central porous region that is separated from an outer porous region by an impermeable barrier, which prevents cross flow between these two zones. The frit is housed inside a multi-port end fitting, such that the inner porous region of the frit aligns with a radial central port, and the outer porous region of the frit aligns with peripheral port(s). Sample and mobile phase enter the column via the radial central inlet port, and pure mobile phase (no sample) enters the column through the peripheral inlet port. This sample introduction process has the purpose of confining the sample to radial central region of the column, and dispersion to the wall is hindered by a curtain flow of mobile phase along the wall region. Mobile phase that flows along the radial central region of the column then exits via the radial central port, and flow along the wall region of the column exits via the peripheral port. The flow proportions at the column outlet can be varied by changing the relative pressure drop across the various exit ports. If for example, 21% of the flow from a 4.6 mm i.d. column exits the column via the radial central exit port, then a virtual 2.1 mm i.d. column is established. If that flow proportion is changed to 43%, then the diameter of the virtual column is 3 mm, and so forth [5]. The advantages of the curtain flow column design are; (1) increased column efficiency, since the most efficient portion of the chromatography column is employed [5], effectively eliminating the wall effect; (2) increased sensitivity, since the entire sample is confined to the radial central region of the column [3] and (3) the solvent flow volume of the virtual column is equal to that of the conventional column having the same internal volume. Hence, a 4.6 mm i.d. CF ‘virtual’ column can be employed at the same linear velocities and the same exit elution volumes as the narrow bore column that it emulates, making it just as compatible with MS-type detectors. More details of the virtual column, and curtain flow chromatography can be found in Refs. [4], [5].
In the present study, we demonstrate another advantage of these columns, greater assay reliability in the analysis of labile compounds. Such reliability arises from the ability of AFT separations with MS detection to be undertaken at higher volumetric flow rates, currently five times faster than conventional columns with the same physical diameter. Consequently, when labile samples are analysed, the overall duration of the assay is decreased and this leads to less variation within the sample during the total analysis time.
Section snippets
Chromatography columns
Columns were supplied by Thermo Fisher Scientific (Runcorn, Cheshire, United Kingdom). Two columns were standard Hypersil GOLD columns with internal diameters of either 2.1 or 4.6 mm. A third column, also supplied by Thermo Fisher Scientific was an AFT column fitted out in curtain flow mode [7]. This column had an internal diameter of 4.6 mm. All columns were 50 mm in length, packed with 5 μm particles.
Chemicals and reagents
All mobile phases were prepared from HPLC-grade solvents purchased from Thermo Fisher Scientific,
Results and discussion
The total analysis time required for undertaking the testing protocol described in Section 2.5 was 4.5 h for the conventional 2.1 mm i.d. column and the 4.6 mm i.d. curtain flow column, and 13 h for the conventional 4.6 mm i.d. column. Amino acids were chosen for this study because of their instability in an unregulated atmospheric environment. As such no effort was made to prolong their storage lifetime, since the purpose of this study was to highlight the importance of high speed separation
Conclusion
The separation speed afforded by curtain flow column technology whereby mobile phase can be separated between the peripheral outlet and central outlet ports of the column when coupled to MS detection can significantly increase analytical productivity. Sample through-put can be maximized, yielding a decrease in the required number of standards in order to maintain the appropriate levels of quantitative reliability. High speed analyses are especially important in the analysis of labile compounds,
Acknowledgement
D.K. acknowledges the receipt of a Australian Postgraduate Research Award.
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Cited by (4)
The parallel segmented flow column as an alternative front-end LC strategy for trace analyses
2019, Microchemical JournalCitation Excerpt :As such, these details are not discussed here further. The advantages of speed, sensitivity and resolution implementing AFT to decrease the volumetric flow to the MS has been well studied, with sensitivity gains of up to 66-fold for amino acid analyses [18–22,24] when curtain flow columns (CF) were used. In such experiments this required extra expertise and consumables to control the injection process in what is effectively an automated central point injection technique that concentrates the bulk of the sample in the radial central region of the column.
High through-put liquid chromatography - mass spectrometry requires new strategies for the management of fluid at the interface
2017, Journal of Chromatography AUsing curtain flow second-generation silica monoliths to improve separations at pressures less than 400 bar
2016, Microchemical JournalCitation Excerpt :Using AFT columns, ‘virtual’ columns [2] are established that utilize only the more homogeneous radial central section of the column bed and this greatly improves the separation performance. In addition to improving the efficiency of chromatography, columns other advantages are also apparent; AFT columns when coupled to the mass spectrometer for example, can be operated at high flow rates [11–14], but because the flow stream is radially split, only portion of the flow is directed to the MS, and in some instances the volume load to the detector can be dramatically reduced, enabling ultra-high through-put HPLC-MS [9]. In addition, AFT columns configured in the curtain flow (CF) mode of operation provided for a substantially higher level of sensitivity in detection [1,4,14–16] since the analyte injection process is confined in the central radial region of the column.
Using active flow technology columns for high through-put and efficient analyses: The drive towards ultra-high through-put high-performance liquid chromatography with mass spectral detection
2015, Journal of Chromatography ACitation Excerpt :Despite the advances in instrumentation that have enabled more efficient utilisation of the modern column, the general conclusions made by Guiochon are still relevant today, since the definition of ‘high-through-put’ has also changed, as will be addressed in the brief discussion on how AFT columns can be ultilised with MS detection. To date there are just three publications that detail the application of AFT columns in concert with MS detection, yet the results show substantial promise [13–15]. Primarily the greatest advantage lies in the fact that the flow to the detector is reduced in proportion to the segmentation ratio.
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Presented at the 41st International Symposium on High Performance Liquid Phase Separations – HPLC 2014, 10–15 May 2014, New Orleans, LA, USA.