Automated HPLC Separation Using LC-Mate: An Integrated Repetitive Autosampler and Fraction Collector for Microscale Purification

Our research focuses on designing an instrument that plugs right into HPLC setups to automate all those repetitive manual steps. Manual sample injection and the fraction collection are very tedious. LC-Mate offers a cross-platform automated solution for HPLC sample purification.

To begin, verify the solvent plumbing connections between the HPLC system, the LC-Mate, and the chromatographic column to ensure proper flow paths. To check the connection of the signal transmission line between the UV detector and LC-Mate, connect the black wire to the red terminal and the white wire to the black terminal to match the polarity of the analog signal output. Observe the LC-Mate display to confirm that the analog signal waveform appears.

If no waveform is visible, reverse the wire connections by attaching the black wire to the black terminal and the white wire to the red terminal. To check the trigger connection between the ultraviolet detector and the LC-Mate, plug the single end of the data cable into the output port of the ultraviolet detector. Connect the black and white wires from the other end of the cable to the black and blue trigger terminals on the LC-Mate respectively Tighten the terminals to ensure a firm trigger connection.

Next, ensure the proper startup of the HPLC system and LC-Mate. Click on the LC Mate icon on the interface to automatically execute the system self-test program and initialize key hardware modules. Wait for the brief prompt tone and the disappearance of the self-testing message to confirm that initialization is complete.

Configure the HPLC separation conditions and begin elution to equilibrate the column. In the test interface, input the injection volume, elution time, and repetition times, then press the Start icon to initiate the auto-sampler, fraction collector, and HPLC operation. Identify feasible separation conditions, including resolution, running time, retention time, injection volume, and ultraviolet wavelength.

Select the Threshold function module on the left side of the interface to open the parameter setting screen. Enter the injection volume, repetition times, threshold, and elution time, and press Start to begin automated operation. Monitor the status indicators.

Observe injection in progress to confirm sample introduction. Check the Separation in progress indicator to verify that the chromatographic separation is going on. Note syringe emptying indicator to identify when the syringe is purging residual liquid.

To perform fraction collection based on time, select the timing collection module from the left side of the interface to open the parameter menu. Input injection volume, repetition times, elution time, and the required collection times. Press the Start button to initiate the repeated automated sequence.

Next, select the Collect All function module on the left side of the interface. Set a collection threshold value to collect all chromatographic peaks separately. For pipeline cleansing, insert the injection pipeline of the autos-sampler into a small bottle containing clean solvent.

Select the Cleansing module from the left side of the interface to open the parameter menu. Then set the injection volume between zero and 1000, and the repetition times for injector pipeline cleansing. Click on Cleansing below Cleansing Completed to start injector pipeline cleansing.

Monitor the cycle count to track completed wash cycles. Set the cleansing time for the collection pipeline cleansing. Click on Cleansing below Current Pipeline to start collection pipeline cleansing.

Monitor the current pipeline display to confirm sequential cleaning from pipeline 0 to pipeline 10. Enable automatic saving to allow the system to save chromatograms after each elution cycle. Alternatively, click on the Save button after elution to manually save data.

After entering the metadata in the dialogue box, choose the storage location from local storage, an SD card, or an external universal serial bus drive. Semi-preparative HPLC separation with LC-Mate achieved continuous and stable sample injection and fraction collection during purification of 2"O-galloyl-hyperoside. Minor impurities were effectively removed to obtain purified 2"O-galloyl-hyperoside using the LC-Mate system.

Continuous and stable sample injection and fraction collection were also successfully achieved during the purification of phloridzin and trilobatin. Demonstrating that the LC-Mate system significantly improves operational efficiency by greatly reducing manual intervention. The relative standard deviation of the full ring injection peak area was 0.813%indicating excellent stability and reproducibility of LC-Mate injections.

This system facilitates automated respective purification and the precise fraction collection for efficient macro-scale HPLC workflows. Correct signal variant, proper mode selection, and the parameter setting are critical for successful application of the LC-Mate instrument. We are planning to boost channel capacity and integrate new detectors for better cross-platform compatibility.