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Solutions for organic synthetic chemistry

Synthetic chemistry not only synthesizes new compounds but also designs and prepares new compounds that can result in numerous applications in materials detection, pharmaceuticals and life sciences. Nonetheless, effectively developing a synthetic route for organic compounds is never easy as synthetic chemists are always facing a range of analytical challenges such as impurity isolation, preparation and purification; molecular weight measurement; accurate yield acquisition; TLC (spot) and method development. To ensure fast and economical organic compound synthesis, it is vital for manufacturers to up their game in the aspects of profiling and identification of impurities as well as verification of synthesis.

Profiling and identification of impurities as well as verification of synthesis

Fig.1 Systemic solutions for the profiling and identification of impurities

To develop a synthetic route for organic compounds, the verification of impurity profiling as well as examination of synthetic compounds is required. To predict the structure of impurities, the sample needs to be analyzed using mass spectrometry by direct sample introduction and/or a Nuclear Magnetic Resonance (NMR) or Fourier Transform Infrared Spectroscopy (FTIR) measurement after concentration of the sample by preparative-LC (Prep-LC). Impurity structure may also be predicted based on the molecular weight information from LC/MS and GC/MS as well as on the structure predicted from a synthetic route. Moreover, a time-of-flight mass spectrometer (Tof-MS), which analyzes the components based on their accurate mass, is available to improve the accuracy of prediction. Waters provides a variety of solutions, including the Atmospheric Pressure Solids Analysis Probe (ASAP) — which is available to directly analyze impurity spots appearing on the TLC — along with UPLC technology optimal for the separation of impurities, the peak-purity test using Empower software, a PDA detector that enables the reduction of the overlooking of impurities, and UPLC H-Class systemic solutions, which increase the efficiency of method development.

Examples of the identification of impurities and reaction monitoring using the ASAP/QTof MS


ASAP (Atmospheric Pressure Solids Analysis Probe)

The probe is exchangeable within a couple of minutes from ASAP for the direct introduction of solid samples to an ESCi probe for LC/MS.

In the ASAP, the sample is put on a glass capillary at the tip of the detachable introduction probe before being introduced to the vacuum system. In contrast to the direct introduction probe for the GC/MS, a procedure for separation from a vacuum system is no longer required because ionization is carried out under atmospheric pressure. In addition, any contamination in the vacuum system that may negatively affect the sensitivity can be avoided because the sample is not introduced directly into the vacuum system.

Any synthetic solutions and powders, as well as any intermediate solutions, can also be analyzed through adherence to the tip of the glass capillary. Whether or not the target compounds are successfully produced can also be confirmed in a timely manner. Therefore, this method can be applied to reaction monitoring. The analysis of major component and impurity, and structure prediction by MS/MS are also available using the QTof MS. Figure 2 shows an example of the results of analysis.

Fig.2 Analysis of impurities from Octahydroacridin using the ASAP/MS

Preparative column technology in the Prep-LC

Have you ever experienced the following problems during a scale-up from an analytical condition using HPLC to a Prep-LC system?

1. Decrease in separation capability
2. Lack of reproducibility
3. Too many times injections needed to obtain the volume required for an NMR

Most of these issues occurring during a scale-up to the preparative scale result from the lower bed density of the preparative column. The bed density depends on the ratio of the column length (L) to the inner dimension (D) (L/D). Therefore, the bed density is lower in traditional high-pressure slurry packing preparative columns with a larger inner diameter and a smaller L/D than the analytical columns.

Fig.3 Effect of bed density on L/D (column length/inner diameter)

OBD (Optimum Bed Density) technology
The Optimum Bed Density (OBD) preparative column*, which applies innovative hardware and packing methods, is a breakthrough preparative column that provides not only a consistent bed density independent of the ratio of L (column length) / D (inner diameter), but also a uniform density gradient from the entrance of the column to the exit.
* U.S. Patent No. 7,399,410/U.K. Patent No.GB 2 408 469


Fig.4 Chromatogram of theoretical scale-up by OBD technology

Theoretically predicted scale-up

The theoretically predicted scale-up is realized from the analytical column (XBridge C18 4.6 x 50 mm) to the OBD preparative column (XBridge C18 19 x 50 mm), which adopted the same materials as the analytical column. For the preparative columns of XBridge, HSS and XSelect, we provide product lines of UPLC columns (BEH, CSH, and HSS) that have adopted the same materials. Therefore, the theoretically predicted scale-up from a UPLC column to an OBD preparative column can be performed.

Solution for precise yield measurement

Yield measurement system (with a peak-purity test function)
UPLC H-Class/PDA

Challenges in acquiring accurate yield:

  • Different impurity profiles in the synthetic routes development
  • Inefficiency when optimizing an LC condition for each synthetic route
  • Difficulty in ensuring the purity of the peak of the major components

Workflow of accurate yield measurement in synthetic route development

Fig.5 Workflow of accurate yield measurement in synthetic route development

Fig.6 Peak-purity test by PDA spectrum in Empower3 software

Peak-purity test arrows to visually understand the presence of impurities in the sample

Peak-purity test using Empower 3
The combination of the UPLC H-Class/PDA system and Empower 3 software enables the ability to solve analytical challenges that may occur while developing a synthetic route. This combination facilitates response monitoring and impurity isolation through ultra-high-speed separation. Additionally, a more accurate yield can be obtained with a peak-purity test to confirm that no peaks from impurities are overlapped in the peak of the major component.

Solution for the development of analytical methods

System for assay development
UPLC H-Class/PDA with a column manager

For the prompt development of a separation/analysis condition:

  • Prompt and more cost-effective optimization of the mobile phase by Auto Blend Plus
  • The developed assay condition is also able to be applied for analysis using the HPLC columns
  • Comprehensive study of separation condition, utilizing the advantage in speed of the UPLC
  • Study of separation condition using templates that enable the ability to develop a condition without experience
  • Automatic switching of columns using the column manager (optional equipment)

Examples of prompt optimization of the mobile phase
Generally, acidic organic solvent-based and water-based mobile phases are needed to be prepared for optimization of the acidity in a whole gradient run time at the same ratio. Therefore, many of the mobile phases prepared are disposed of for optimization. The Auto Blend Plus function incorporated into the UPLC H-Class allows the ability to set the mixing-ratio arbitrarily for each mobile phase as shown below, as well as enable the ability to optimize the acidity and analytical conditions without preparing every mobile phase for each condition. This is efficient for the optimization of the mixing-ratio of the organic solvents, such as THF and ACN, which are frequently used as organic materials.

Fig.7 Example of optimizing mobile phase by Quaternary Solvent Manager.

Fig.8 Effect of additive concentration in mobile phase on separation.

These charts suggest that the separation was clearly influenced by TFA concentration. The waveforms within the red frame indicate that the separation was improved by increasing TFA concentration, and the order of elution peaks within the green frame was changed. Six types of solvent mixture are needed to be prepared for a study of the mobile phase with these three conditions shown at left using a binary system.

If the Auto Blend Plus function is used, a set of results will be provided by changing the mixing-ratio of solvents on the control software without preparing solvent mixtures of different acidity.

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