Using Mass Spec and TLC for Intelligent Flash Purification

The whole process of synthesizing, purifying and confirming compounds can pose challenges. From ensuring successful product synthesis, preparing a flash purification method, and eventually confirming ID of fractions, this long work flow is often repetitive and time consuming.

This article explores a new seamless work flow that can rapidly and easily drive the whole process from reaction monitoring through to flash purification and fraction ID.

This new process is essentially broken down into three steps. These are summarized below and explained in more detail throughout the article.

Using Mass Spec and TLC for Intelligent Flash Purification

Image Credit: Advion

Step 1: A Suzuki Reaction is performed, followed by push-button TLC/CMS analysis using the Advion Plate Express® TLC Plate Reader and expression® Compact Mass Spectrometer (CMS).

Step 2: TLC to Flash takes place, and the compound is purified using the Interchim puriFlash® XS520 Flash Chromatography Genius® system, utilizing a method auto-developed from a cell phone image of the TLC plate.

Step 3: Fraction ID is conducted in <30 seconds using the Advion Atmospheric Solids Analysis Probe (ASAP®) working alongside the expression CMS.

Method

Experimental Setup

Table.  Source: Advion

. .
Mass Spectrometer & Sampling Techniques Advion expression CMS Plate Express TLC Plate Reader ASAP
Flash System Interchim puriFlash XS 520
Sample Mixture See Figure 1
Solvents Cyclohexane, 10%
Dichloromethane, 90%
Quantity to Purify 400 mg

 

Step 1: Reaction Monitoring Using TLC

The reaction of Phenylboronic acid and Bromoaniline in ethanol using a palladium catalyst (Figure 1) takes places at room temperature and over a 2-hour period. After 2 hours have elapsed, an aliquot is taken and spotted on a silica TLC plate before being developed in Cyclohexane and Dichloromethane solvents.

The sample mixture including molecular weight, structure and reaction amounts.

Figure 1. The sample mixture including molecular weight, structure and reaction amounts. Image Credit: Advion

The reaction is monitored via TLC/CMS, with the Plate Express connected to the expression CMS, enabling push-button MS analysis of the TLC plate (Figure 2). The system can accurately identify spots by collecting a mass spectrum from the TLC plate directly (Figure 3).

(Left) With the Plate Express TLC plate reader, place the developed TLC and align your spot of interest. At the push of a button, the head lowers and extracts the sample with a solvent to send directly to the mass spectrometer.

Figure 2. With the Plate Express TLC plate reader, place the developed TLC and align your spot of interest. At the push of a button, the head lowers and extracts the sample with a solvent to send directly to the mass spectrometer. Image Credit: Advion

Spot 1. The TLC data clearly shows the typical isotopic doublet at an m/z of 172 and 173, the M+H of Bromoaniline, the unreacted starting material. Spot 2. A MS signal at m/z 170 indicating the desired reaction productp-NH2- Biphenyl.

Figure 3. Spot 1. The TLC data clearly shows the typical isotopic doublet at an m/z of 172 and 173, the M+H of Bromoaniline, the unreacted starting material. Spot 2. A MS signal at m/z 170 indicating the desired reaction productp-NH2- Biphenyl. Image Credit: Advion

Step 2: TLC to Flash: Purifying the Compound

TLC separation information is employed to intelligently program the Interchim puriFlash system, using the TLC plate image. This ensures peak separation for fraction collection. Once the product is confirmed via the Advion Plate Express, it must then be prepared for purification with the Interchim puriFlash XS520.

The process starts with an app - the TLC to Flash Converter (Figure 4) – allowing users to purify compounds using a tablet, cell phone or similar device.

With the TLC to Flash app, take a picture of the TLC plate. The compounds will be detected automatically. Select the ones of interest with a tap. The application calculates Rf and ?CV (= ?K). Finally, indicate the solvents, their proportions and your comments in the dedicated areas and send to the puriFlash.

Figure 4. With the TLC to Flash app, take a picture of the TLC plate. The compounds will be detected automatically. Select the ones of interest with a tap. The application calculates Rf and ΔCV (= ΔK). Finally, indicate the solvents, their proportions and your comments in the dedicated areas and send to the puriFlash. Image Credit: Advion

A photograph is taken of the TLC plate, and a title or other identification details are entered, such as the product of interest and solvent front, the amount requiring purification (400 mg) and the solvents used (Dichloromethane, 90% and Cyclohexane, 10%).

Once acquired, this data is transferred wirelessly to the puriFlash system, allowing the Genius software to help construct the rest of the method (Figure 5). The screen displays the total UV profile after the run has taken place (Figure 6), with the touchscreen functionality illustrating where within the fractions the peaks are located, enabling fraction confirmation analysis in the following step.

The TLC data appears on the Genius software and the system suggests the best gradient purification method for you based on your TLC data.

Figure 5. The TLC data appears on the Genius software and the system suggests the best gradient purification method for you based on your TLC data. Image Credit: Advion

The TLC plate and total UV profile after the completed run

Figure 6. The TLC plate and total UV profile after the completed run. Image Credit: Advion

Step 3: Fraction ID In <30 Seconds with ASAP/CMS

Flash fractions are located by examining their UV profile. These must be confirmed in order to ensure they have eluted as anticipated, so the central fraction for each compound is analyzed using ASAP/CMS (Figure 7).

The ASAP is dipped in to one of the identified fraction tubes and inserted in to the mass spectrometer, where is it instantly analyzed by the APCI ion source.

Figure 7. The ASAP is dipped in to one of the identified fraction tubes and inserted in to the mass spectrometer, where is it instantly analyzed by the APCI ion source. Image Credit: Advion

The fraction displayed in Figure 8 has been confirmed using ASAP, with the signal at m/z 170 indicating the desired reaction of productp-NH2-Biphenyl, as anticipated.

The data from the run quickly and easily identifies 170 m/z, which is exactly what was expected.

Figure 8. The data from the run quickly and easily identifies 170 m/z, which is exactly what was expected. Image Credit: Advion

Finally, the ASAP sampling technique for the expression CMS is used to confirm fractions. This can be completed in just seconds by dipping the probe in the preidentified tube, then directly inserting this into the mass spectrometer.

Summary

This article explored the use of the Advion expression CMS, the Plate Express and ASAP sample techniques, as well as the Interchim puriFlash flash chromatography system. This novel combination of instruments offers advanced detection technology, user-friendly sampling techniques, and intuitive software interfaces.

Utilizing this streamlined workflow enables users to confidently and easily synthesize, characterize and purify a range of compounds.

The intuitive, seamless workflow ensures that the right TLC spot is identified for purification via the Plate Express and expression CMS. A highly optimized Flash method is then proposed and programmed via the Interchim puriFlash XS520 and Genius software before flash fractions can be identified using the ASAP dipping probe and expression CMS.

This information has been sourced, reviewed and adapted from materials provided by Advion.

For more information on this source, please visit Advion.

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