Basic Guide on How to Use the HPLC

Summary

This video provides an in-depth guide to using the Agilent 1260 Infinity II HPLC system, focusing on the essential components, such as the injector, column, pump, and detector. It explains the step-by-step process of setting up the HPLC method, preparing samples, injecting them into the system, and analyzing data through the instrument's software. The video also covers the importance of filtration, proper sample handling, and cleaning the system post-run to ensure optimal results. Whether you're collecting fractions or refining chromatographic data, this guide helps ensure successful operation of the HPLC system.

Takeaways

• 😀 HPLC (High-Performance Liquid Chromatography) is a technique for separating and purifying compounds based on polarity.
• 😀 The Agilent 1260 Infinity 2 is a reverse phase HPLC instrument that utilizes hydrophobic interactions for separation.
• 😀 Key components of an HPLC system include the solvent, fraction collector, detector, column, pump, and injector.
• 😀 The injector is used to introduce the sample into the system, and the sample volume is managed by the loop size.
• 😀 Solvents like water with 0.1% TFA and acetonitrile with 0.1% TFA are commonly used to prevent microbial growth.
• 😀 An HPLC method is created by specifying solvent volumes, column settings, and detection parameters in the software.
• 😀 Before using solvents, it is crucial to filter them to ensure they are free of debris.
• 😀 Sample data should be named and saved in a designated folder to ensure proper organization and storage.
• 😀 Before injection, rinse the syringe with the solvent used in the sample to avoid contamination and ensure a clean sample injection.
• 😀 During the HPLC run, peaks on the graph represent the compounds or analytes exiting the column and can be collected for further analysis.
• 😀 After completing an HPLC run, it's important to rinse the column to prevent contamination and ensure accurate results in future runs.

Q & A

What is the purpose of the Agilent 1260 Infinity 2 HPLC system?

The purpose of the Agilent 1260 Infinity 2 HPLC system is to purify and separate compounds based on polarity, utilizing reverse phase high-performance liquid chromatography (HPLC) techniques.

What are the key components of an HPLC system?

The key components of an HPLC system include solvents, the fraction collector, the detector, the column, the pump, and the injector.

How does the injector function in an HPLC system?

The injector in an HPLC system is where you introduce your sample. It is connected to a loop, which determines the amount of sample the system can take, and is sized according to the column's capacity.

Why is the volume of the loop important in an HPLC system?

The loop volume is crucial because it ensures that the sample injected is appropriate for the size of the column. A too-large or too-small volume can result in inaccurate peaks on the chromatogram.

What is the role of the mobile phase in an HPLC run?

The mobile phase, which consists of solvents like water with 0.1% TFA and acetonitrile with 0.1% TFA, helps carry the sample through the column and ensures proper separation based on polarity.

How can you customize the method for an HPLC run?

You can customize the HPLC method by selecting the instrument, adjusting the solvent volumes, setting the correct wavelength, and configuring the fraction collector. These adjustments are made in the software interface.

Why is it important to filter solvents before refilling the HPLC bottles?

It is important to filter solvents before refilling the HPLC bottles to ensure that they are free of debris, which could clog the system and affect the analysis.

What are the steps involved in preparing a sample for HPLC injection?

The steps for preparing a sample for injection include ensuring the syringe is correctly sized, rinsing it with the same solvent used for the sample, avoiding air bubbles in the syringe, and correctly positioning the injector lever to load the sample.

How do you collect fractions during an HPLC run?

To collect fractions, you can either manually collect by clicking the 'collect fraction' button or set the system to automatically collect fractions based on pre-set criteria. Ensure that vials are in place to avoid wasting samples.

Why is it necessary to rinse the HPLC column after completing a run?

Rinsing the HPLC column is necessary to prevent any residual material from clogging the column or contaminating future runs. Running the same method without the sample helps to clean the system effectively.