How to start HPLC/ HPLC Setup
Hplc is a one of the most used tool in analytical chemistry used to separate, identify and quantify different components present in the complex substance mixture. There are many instruments that use different setup systems based on their software and hardware. In this guide we will go through some of the important points and a step by step guide for you to get started with your first HPLC run. Whether you’re a student or a new lab analyst, starting up an HPLC system correctly is crucial to ensure accurate results, prevent contamination, and extend the life of your instrument.
1.Prepare Mobile Phase Bottles
Depending on type and amount of solvent required for your Hplc method prepare the solvent as required, during preparation always use HPLC-grade solvents only. While using buffer always use freshly prepared and filter through a 0.22 µm or 0.45 µm filter and avoid using the salts that precipitate in organic solvents. After preparation of required amount of the mobile phase solvent transfer into a mobile phase bottle each labelled clearly with required information like(Solvent name and ratio, pH and Date of preparation.)
Inset the solvent inlet filters into each of the bottle depending on the solvent lines(A,B,C,D)
2.Empty the Waste Bottle
Before and during the running of hplc it is necessary to check the amount of solution present in the waste bottle used to collect the hplc running waste solution .
Make sure to connect the detector outlet tubing and purge waste line securely into the waste container.
Label the container “HPLC Waste”(check the procedure for any specific labeling)
3.Turn On the HPLC System
Ensure all the electrical connection and then turn on main console with components like Pump module, Autosampler/injector module, detector module,column oven and start the software in the computer connected to the instrument .
For different instruments the starting sequence may vary and may also have safety checks, always check the instruction module of the instrument before starting the instrument.
4.Hplc Priming
Priming of an Hplc is done to remove the air bubbles, filling the pump and solvent lines with mobile phase is done to ensure the consistent flow and reliable chromatographic results.
This process is carried out
– During initial setup or when solvents are changed.
– When there is a case of air bubbles entering the lines or if there is an unstable pressure.
During this process depending on the model you use, opening of the purge valve may be required, By opening the purge valve the solvent from the mobile phase solvent is bypassed from entering into the column to the waste bottle . this step is necessary as if the air enters into the column there is a chance of the column getting damaged or producing unreliable result. Opening the valve reduces backpressure during priming and allows faster flow, close the purge valve after use.
Some of steps include are
1. Dry Priming
2. Wet Priming
3. Needle Wash
4. Seal Wash
5.Connect and Prepare the Column
Choose the correct HPLC column according to the procedure used and hplc preformance, before using column inspect for any damage and ensure that column is compatible with the analysis
Install the column in the correct flow direction for this check for the arrow mark or any sign which indicates the flow of solvent through the column.
Connect the inlet fitting and outlet fittings
Set a low flow rate (e.g., 0.2 mL/min) and start flushing the column with your mobile phase.
Increase the flow slowly according to method flow rate
Allow the column to equilibrate as required and check for any pressure fluctuations.
6.Temperature set up
Set Column Oven Temperature
Set the column temperature using software or on the instrument and allow it to reach the required temperature before starting the test. Always check the temperature capability of column before setting the temperature
Set Autosampler Temperature
Depending on the instrument and procedure set the autosampler temperature, presenting the autosampler before use is important for some temperature sensitivity analysis.
7.Check System Baseline
After turning the detector ON, select the lamp from the software, set a wavelength and wait for 10 -15 minutes to monitor the baseline signal. This signal should be stable (minimal noise or drift ).
For advanced detection methods check the user manual before turning the detector