Automating Method Development with an HPLC System Optimized for Scouting of Columns and Eluents Marco Karsten, Bas, Dolman, Giovanni Maio, Frank Steiner, Holger Franz, Frank Arnold and Remco Swart LC Packings, AA, Amsterdam, The Netherlands, Dionex Softron GmbH, D- Germering, Germany INTRODUCTION HPLC method development is still considered one of the crucial bottlenecks impeding productivity in analytical laboratories. Usually, due to the variety of available columns, the proper selection of the stationary phase represents the greatest challenge. Despite all efforts in the field of phase characterization and phase property indexing, an unambiguous selection approach is still missing. With the introduction of alkaline stable silica-based stationary phases, a large palette of mobile phase additives and ph values are applicable in reversed phase chromatography. This results in a vast number of parameters which may have to be tackled in method development. In this paper, we present a new integrated system that allows automatic and intuitive scouting of columns and eluents. The system includes quaternary gradient capabilities, an autosampler compatible with well plates and standard sample formats, and a powerful diode array detector. Switching valves in the column compartment are used to select between columns. The application of this system to automatically screen possible combinations of different columns, different organic solvents and different buffers for the separation of diuretics is presented. Compounds are identified via UV/VIS spectra comparison. A spreadsheet-based reporting tool provides a peak resolution chart. The most promising combinations of eluents, stationary phase, and temperature are automatically depicted. UltiMate for Automated Method Development The complete system is controlled using Chromeleon Chromatography Management software. The instrument setup is shown in Figure. A. Ammonium Acetate ph B. Ammonium Bicarbonate ph C. Methanol D. Acetonitrile TCC- LPG-A WPS-TSL Acclaim C Acclaim Polar Advantage II PDA- Hypersil Phenyl Hypersil C Figure. Instrument setup for automated column and eluent scouting. The quaternary pump allows the use of two buffers and two organic modifiers for eluent scouting, while two -position, -port valves were used to select between four different columns. In a later variant of the TCC-, two -position, -port valves will be available and, thus, markedly improve column scouting abilities. Chromeleon Tools For Automated Column Selection and Equilibration Optimization of LC methods utilizing column scouting requires automated column change and equilibration. Chromeleon fully supports these procedures in sequences as well as in manual control of the instrument. The two switching valves are controlled directly from Chromeleon software allowing: The UltiMate for automated column and eluent scouting consists of: LPG-A quaternary pump SR- solvent rack TCC- thermostatted column compartment with two integrated switching valves WPS-TSL cooled well-plate autosampler PDA- photodiode array detector Direct valve control without the need to program contact closures Automated selection of columns Automated column flushing and equilibration procedures Figure shows the Chromeleon interface for the manual selection of columns. By changing from one to the next column, the equilibration procedure is started automatically. The script file takes the valve position into account, starts the flow of the pump, and pops up with the message when the procedure is finished. PITTCON Presentation PITTCON Presentation
In addition to the compound retention time, UV spectral information can increase the reliability of peak assignment. However, the applicability is limited to compounds that have different UV spectra and do not completely coelute. An example of peak tracking tools in Chromeleon is shown in Figure. Figure. -D UV contour plot and peak spectral comparison with library. Figure. Chromeleon interface for direct column selection and column equilibration. Script file for automated equilibration can be created easily. The experimental LC conditions applied in this study are listed in Table. Table. LC Conditions for Automated Column and Eluent Scouting Columns. Dionex Acclaim C, µm. mm i.d.. Dionex Acclaim PolarAdvantage II -µm particles Temperature C. Thermo Hypersil Phenyl. Thermo Hypersil C Buffers Ammonium formate buffer, mm, ph Organic modifiers Flow Rate Gradient Sample Ammonium bicarbonate buffer, mm, ph Methanol Acetonitrile. ml/min % organic modifier in min Equilibration min Diuretics,. µg/µl ) Amiloride ) Furosemide ) Ethacrynic acid ) Chlorthalidone ) Benzthiazide ) Probenecid ) Triamterene ) Bumetanide ) Canrenoic acid Injection Split loop, µl Detection UV Spectra nm and nm, µl flow cell nm to nm Optimization of the RP-HPLC Separation of Diuretics Many compounds of pharmaceutical or biological importance are ionizable. For these compounds, the ph of the elution buffer can be used to adjust selectivity of the separation. In the present study, diuretics were chosen as test compounds consisting of acids, bases, and zwitterions. The compounds were separated on reversed-phase columns applying different organic modifiers and different buffers. In Table an overview of the examined LC methods is given. Table. Overview of LC Columns and Eluent Scouting Methods Method Column ph Organic modifier A MeCN B MeCN C MeCN D MeCN E MeOH F MeOH G MeOH H MeOH I MeOH J MeOH K MeOH L MeOH M MeCN N MeCN O MeCN P MeCN Peak Tracking A photodiode array detector was used for compound identification. UV spectra were acquired from separately injected compounds and stored in a library. In the method optimization study, mixtures were separated and the acquired spectra were compared to those contained in the library. A matching factor provides the degree of correlation. Automating Method Development with an HPLC System Optimized for Scouting of Columns and Eluents
The selectivity between different LC methods was compared by calculation of the squared correlation coefficient (r ) between two sets of retention times. The resolution between two adjacent peaks was calculated and functioned as the main selection criterion. Influence of ph The influence of the ph of the buffer on retention of diuretics on a C column is illustrated in Figure. In changing the ph of the buffer from ph to ph, a drastic change in the selectivity was obtained. For most of the compounds the retention was higher at ph. This could be explained by the fact that most compounds have acidic groups in their structure that are protonated at low ph, thereby increasing the hydrophobicity. The opposite was observed for triamterene, a compound with several amine groups, that carries a positive charge at ph. In general, the correlation plots between ph and showed a large scatter of the retention times, indicating the usefulness of ph as a parameter to influence the separation selectivity. -, -..... Minutes Retention time (min) - ph Influence of ph on retention R =. Retention time (min) - ph Figure. A) Separation of diuretics on Acclaim C column using ammonium bicarbonate buffer ph (upper chromatogram) and ammonium formate buffer, ph (lower chromatogram) with acetonitrile as an organic modifier. B) Correlation plot of retention times between the two LC methods. Influence of Stationary Phase The influence of the type of stationary phase on retention and selectivity was less pronounced compared to the influence of the ph of the buffer. For many column-to-column comparisons, the squared correlation coefficient was close to. In Figure, the separation of diuretics on Acclaim C and Hypersil C is compared. Despite the highly similar selectivity for these columns, as indicated by the value of r =., the C stationary phase has the advantage that all compounds are baseline separated, in contrast to the C stationary phase. - Retention time (min) - Hypersil C, - Minutes Influence of stationary phase on retention R =. Retention time (min) - Acclaim C Figure. A) Separation of diuretics on Acclaim C (upper) and Hypersil C (lower) columns using methanol as organic modifier and ammonium bicarbonate buffer, ph. B) Correlation plot of retention times between the two LC methods. Influence of Organic Modifier The type of solvent used in the reversed-phase separation of diuretics was found to have a strong influence on the retention and selectivity. In Figure the separation of diuretics on Acclaim C is compared for methanol and acetonitrile as solvent. The higher solvent strength of acetonitrile resulted in a significantly faster separation while maintaining good overall resolution. The separation selectivity was also affected by changing the solvent from methanol (protic solvent) to acetonitrile (aprotic solvent), demonstrated by the large scatter of retention times in the correlation plot. PITTCON Presentation
... Minutes... Retention time (min) - Acetonitril Influence of organic modifier on retention R =. Retention time (min) - Methanol Figure. A) Separation of diuretics on Acclaim C with methanol (upper chromatogram) and acetonitrile (lower chromatogram) as an organic modifier using ammonium bicarbonate buffer ph. B) Correlation plot of retention times between the two LC methods. Resolution (average) Resolution of Chlorthalidone................ A B C D E F G H I J K L M N O P Method A B C D E F G H I J K L M N O P Method Figure. Resolution chart for all investigated LC methods. A) Average resolution of all compounds. B) Resolution for Chlorthalidone between previous (black) and next (blue) eluting compounds. Method Selection The most important selection criterion in LC method development is chromatographic resolution. Depending on the analytical information required, the resolution of one or more compounds is decisive. Chromeleon software provides useful tools to aid in decision making based on chromatographic information. After finishing a sequence, in the present study consisting of different LC methods, charts showing resolution for each method can automatically be generated. A few examples of such plots are shown in Figure. The average resolution is maximum for methods A and E. The methods run on the Acclaim C column with a low ph buffer, combine baseline separation of all compounds in a reasonable time. Method M combines the fastest analysis time with baseline separation of all compounds (see Figure ). If quantification of a particular peak is of more interest, the resolution for that compound with adjacent eluting peaks can be plotted as well. Conclusion UltiMate LC systems can be easily configured for automated column and eluent scouting The thermostatted column compartment has two integrated switching valves allowing for automated column selection Chromeleon software features graphical user interfaces with dedicated tools for column and eluent scouting Chromeleon post processing tools assist in decision making with respect to optimal LC methods The ph of the mobile phase and the type of organic solvent are powerful parameters to influencing the separation selectivity of acids and bases in RP-HPLC The PDA- photodiode array detector is a useful tool for peak tracking during method development. Automating Method Development with an HPLC System Optimized for Scouting of Columns and Eluents
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