Enantioselective reaction monitoring utilizing two-dimensional heart-cut liquid chromatography on an integrated microfluidic chip †

Chip-integrated, two-dimensional high performance liquid chromatography is introduced to monitor enantioselective continuous micro-flow synthesis. The herein described development of the first two-dimensional HPLC-chip was realized by the integration of two different columns packed with reversed-phase and chiral stationary phase material on a microfluidic glass chip, coupled to mass spectrometry. Directed steering of the micro-flows at the joining transfer cross enabled a heart-cut operation mode to transfer the chiral compound of interest from the first to the second chromatographic dimension. This allows for an interferencefree determination of the enantiomeric excess by seamless hyphenation to electrospray mass spectrometry. The application for rapid reaction optimization at micro-flow conditions is exemplarily shown for the asymmetric organocatalytic continuous micro-flow synthesis of warfarin.


Additions to the instrumental setup and operation principle
The instrumental setup to realize two-dimensional HPLC-MS on a single glass chip is schematically shown in Figure 1 in the main publication.Special clamps described earlier 1 were used for fluidic chip to tube connections.At a flowrate of 1.5 µL•min -1 in the first dimension and 5 s transfer time the maximal transfer volume calculates to about 100 nL.Due to compression of the solvent in the closed off arms of the on-chip cross in transfer mode, the actual transfer volume will be somewhat smaller.
The transfer process can also be operated in a fully automated mode by triggering the 6-port valve when a threshold of the detected fluorescence intensity is exceeded.In injection mode, the pressure at the on-chip cross increases linearly, since both arms of the cross are closed off.This is monitored by the integrated pressure sensor and can be used to observe the successful injection and furthermore, to automatically end the injection when a certain pressure threshold is reached.The injection scheme for the second dimension is very reproducible.The exact amount of transferred analyte is only dependent on the reproducibility of the first dimensional chromatography run, if a runtime based switching mechanism is utilized.
For a successful operation the solvents in first and second dimension should, as in classical 2D-LC, match in solvent strength and miscibility.In the current study the solvents consisted of ACN/H 2 O (50/50 vol%) in the first and MeOH/H 2 O (70/30 vol%) in the second dimension.Both solvent compositions are fully miscible.The solvent in the second dimension has a higher elution strength than the solvent from the first dimension.This allows the formation of a defined injection plug on the column head of the second column and hence, a high quality separation.In the second configuration a fiber guided 325 nm laser was utilized in combination with a 420-480 nm emission filter.This setup was utilized to detect the components of the warfarin synthesis.The detection point is marked in Figure 1    In order to investigate the effect of the reaction temperature on the ee-value, the micro-flow reactor chip was placed in a thermostat.The effluent was analyzed with the developed 2D-HPLC-MS-chip system.The results are shown in Figure S-4, displaying the determined eevalues and the normalized peak area of (S)-wafarin as the main product.An increase in reaction temperature reduces the ee-values drastically, while the overall turnover increases.

Materials
The utilized materials and instruments are listed below.

Material Source
ProntoSIL

Figure S- 1
Figure S-1 Optical setup.Left: Picture of the utilized custom-built portable epi-fluorescence microscope.Right: Schematic illustration of the light path.A portable custom-built epi-fluorescence microscope allowed simultaneous optical and mass spectrometric detection.It was utilized for the optical detection of the first dimensional separation and to monitor the fluidic situation at the on-chip cross.Two different configurations were applied.The first configuration is shown in Figure S-1.It includes a 365 nm excitation LED, a 350/50 excitation filter, a 380 nm DCLP (dichroic mirror) and a 390 nm emission filter.It was utilized to detect the model analytes 7-amino-4-methylcoumarin, pirkle's alcohol and anthracene and to observe the on-chip transfer of sample from the first to the second column.

4 Figure S- 2
Figure S-2 Spectroscopic characteristics of the warfarin reaction components and the chip material.Dotted line: absorbance; solid line: emission at 325 nm excitation)

Figure S- 3 .
Figure S-3.Reaction time and flow rate dependent measurements of the enantiomeric excess for different solvents.To investigate whether the enantiomeric excess (ee) of the warfarin synthesis reaction is dependent on the reaction time, different reaction times between 5 and 57 min were employed by varying the reactant flow rates and the enantiomeric excess was determined.The resulting plots of the reaction time and the equivalent flow of the reaction solution versus the enantiomeric excess are shown in Figure S-3.

Figure S- 4 .
Figure S-4.On-chip temperature screening for the asymmetric warfarin synthesis.Solvent: 95% ACN/H2O.Reaction time: 11 min.Separation parameters as described in the caption of Figure 4 in the main publication.