Applied Analytical Systems

HPLC System Care and Troubleshooting Like a Boss (PART 2)

What’s Wrong with my HPLC?!?!? … Troubleshooting the Chromatogram

No Signal

Injector Failure – Sample is not introduced to the flow path. Check injector. Clean if it’s clogged. Repair if malfunctioning. If you’re using an autosampler, check to see if it’s aspirating sample successfully – bypass the autosampler and manually inject sample to see if it’s the problem.

Pump Failure – Mobile phase not flowing. Check/repair pump.

Detector Failure – Check/adjust detector settings (gain, sensitivity). Check/replace the lamp.

Column Issues – Sample is binding to the column and not eluting – change separation column and/or guard column type.

Solvent Issues – Sample is incompatible with mobile phase, or mobile phase is not compatible with column – change mobile phase solvent.

Drift

Solvent Issues (evaporation/concentration changing, atmospheric gas absorption/pH changing, leaks) – De-gas and sparge solvent with an inert gas. Seal solvent containers. Check for and repair leaks (tubing, fittings).

Gradient Issues (one solvent absorbs/retains more of the sample than other) – Change one or both mobile phase solvents. Employ baseline subtraction.

Contaminated Column – Flush column with solvent. Change column type to avoid build up and slow release.

Pressure Issues – Filter mobile phase and samples. Reduce sample or solvent viscosity. Use larger tubing.

Temperature Issues – Look for changing temperature over the run. Control temperature in the detector flow cell.

Cyclic Variation

Temperature Issues – Stabilize environmental control. Check for effects from the HVAC system or other nearby equipment that throws off heat or cooling. Control temperature in the detector flow cell.

Bubbles / Mixing Issues (in the pump) – Flush and prime pump. De-gas mobile phase. Increase system volume.

Clogs (partial) – Remove clog. Replace tubing. Flush with solvent.

Pump Issues – Repair or replace the pump.

Electrical Noise – Look for and remove sources of interference with a cyclical pattern. Change circuit. Install line conditioner.

Baseline Noise

Bubbles (in the mobile phase, column, or detector) – Check for and repair leaks (tubing, fittings). De-gas mobile phase. Flush or backpressure detector cell or column to removed trapped bubbles.

Contaminated Column – Flush column with solvent. Change the column.

Electrical Noise – Check connections. Look for and remove sources of interference. Clean contacts. Shield cables. Change circuit. Install line conditioner.

Detector Issues – Flush detector cell using manufacturer’s cleaning protocol. Adjust sensitivity/gain. Replace the lamp.

Spikes

Bubbles – Check for and repair leaks (tubing, fittings). De-gas mobile phase.

Electrical Wiring Issues – Check wiring, leads, and connectors. Repair or replace broken wiring or connectors. Clean connectors and leads.

Electrical Noise – Check connections. Look for and remove sources of interference with a sharp, intermittent pattern (valves, compressors, lighting). Clean contacts. Shield cables. Change the power circuit. Install line conditioner.

Detector Issues – Check and replace the lamp.

Synchronous Noise

Bubbles / Mixing Issues (in the pump) – Flush and prime pump. De-gas mobile phase. Increase system volume.

Pump Issues – Repair or replace the check valve. Repair or replace the pump plunger or seals. Check for and repair leaks.

Electrical Noise – Look for and remove sources of interference with a cyclical pattern. Change circuit. Install line conditioner.

Asynchronous Noise

Bubbles / Mixing Issues – Check for and repair leaks (tubing, fittings). De-gas mobile phase. Flush or backpressure detector cell or column to removed trapped bubbles. Increase system volume.

Clogs (partial) – Remove clog. Replace tubing. Flush with solvent.

Electrical Noise – Check connections. Look for and remove sources of interference with an intermittent pattern. Clean contacts. Shield cables. Change circuit. Install line conditioner.

Positive and Negative Peaks or Only Negative Peaks

No Issues – Negative peaks may be normal if you’re using a refractive index detector – no changes.

Solvent Issues – Filter solvent. Change to different solvent.

Bubbles – De-gas mobile phase.

Only Negative Peaks – Change detector polarity.

Injectors

Injectors transfer your sample from the source vial or plate into the LC system flow path. Once introduced, the sample flows through the column to the detector(s) and out to the waste. Once again, improper priming can be the main cause of issues relating to the injector. Any air present in the system can affect the injection volume and cleanliness. Priming the system properly removes air bubbles from the sample and wash syringes. This will improve the system reproducibility and reduce sample carryover.

Columns

Maintaining your separation column is critical for your LC system. This is the heart of the system. This component enables the separation of constituents in your sample. Problems with your column will translate directly to your results. Refer to your column’s documentation for appropriate wash and storage solvents to use. Always flush the column after runs with a compatible organic solvent. Failure to clean your column will impact column life and performance. This is especially important if you plan to store the column for an extended period. Replace your column if the packing material has become contaminated or ineffective. Run a test standard through the system regularly to track changing column performance.

Detectors

There are many detector types and models available for LC systems. But, detector maintenance is a simple procedure that’s common across types. You need to know the back pressure your detector flow cell generates at a known flow rate for a solvent. This will create a baseline for you when troubleshooting the system. You can use this value to gauge system readiness.

To do this:

Record the pressure while flushing solvent through the detector (D).

Remove the inlet line to the detector and record the pressure again while flushing (C).

Subtract C from D.

This is important because the most common issue with detectors is a clogged flow cell. If the flow cell is partially plugged, the pressure will be much greater at the detector inlet. The flow cell can rupture if completely plugged, resulting in an expensive repair. To maintain the flow cell, always flush with a clean solvent that is at least 20% organic. This will help maintain the flow cell and keep it clean and free of blockages.

Fittings & Tubing

Improper connection of tubing in fittings can also have an impact on results. A common error is to leave a void at the end of the tubing inside of the fitting. It’s important to seat the tubing flush to the end of the fitting.

LC tubing size can have a tremendous impact on chromatographic results. When replacing tubing, make sure to use the correct sizes. If the tubing internal diameter (ID) is too big it will cause peak shape issues. Peaks will become wider (and shorter with larger tubing). If the tubing is too long or too narrow, it will generate higher pressures and can also cause peak issues.

Mobile Phase (The Lifeblood of the System)

The mobile phase is the solvent that carries your sample through the HPLC system. This can be water, an aqueous buffer solution, or an organic solvent or mixture. You can use an isocratic mobile phase – a single solvent at a constant concentration. Or you can use a gradient mobile phase – two or more solvents at changing ratios over time. The majority of HPLC issues originate from mobile phase problems.

To fix these issues, follow these suggestions:

Start with fresh HPLC grade solvents. If using water, get it from an HPLC-grade purification system.

Filter the mobile phase using a 0.2-micron filter.

De-gas the solvents well before use.

Never add more solvent to open bottles; replace them when empty.

Bacteria can grow in pure water; be careful to use fresh, filtered water.

Use a 10-20% organic solvent/water mixture to inhibit bacteria growth for storage.

Maintaining Your Equipment

Of all of the types of lab equipment, HPLC systems tend to be the most DIY-friendly for maintenance – and their owners tend to be pretty skilled as well. It comes with the territory. Not every problem is easy to solve and regular preventative maintenance service is important for continued performance. Applied Analytical Systems Ltd. would be happy to work with you to help with your routine maintenance. Also, if you are buying a new equipment, or you want to replace an old one, we are just within your reach. Training, repairs, calibration and consultation are part of the services we render to our clients.

Please feel free to check out what we have to offer:

https://aasnig.com/

https://aasnig.com/services/

https://aasnig.com/products/

https://aasnig.com/contact/

Reference:

https://www.atlanticlabequipment.com/blog/hplc-system-care-and-troubleshooting-like-a-boss/

Posted by Opeyemi Akinbuli

[email protected], www.aasnig.com

 

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