What are troubleshooting problems in HPLC?

What are troubleshooting problems in HPLC?

HPLC troubleshooting

• No pressure reading, no flow. Possible cause.
• Now pressure reading, flow is normal. Possible cause.
• High back pressure. Possible cause.
• Low back pressure. Possible cause.
• Fluctuating pressure. See section High back pressure.
• Pressure dropping to zero.
• Pressure dropping, but not to zero.
• Pressure cycling.

What causes low pressure in HPLC?

Low backpressure is generally caused by a flow rate that is set too low. Using an improper HPLC column, having the column temperature set too high, a system leak, and controller malfunction can also cause low backpressure.

Why does RSD failure in HPLC?

Re: RSD failing HPLC OR use continuous high-purity Helium sparging to degass the two liquids. Sonication and vacuum filtering of mobile phase are of no use here as they only temporarily remove gas from the liquid resulting in poor RSD.

How do you resolve negative peaks in HPLC?

Solution: Adjust or change sample solvent. Dilute sample in mobile phase whenever possible. d) Mobile phase more absorptive than sample components to UV wavelength (vacancy peaks). Solution: Change UV wavelength or use mobile phase that does not adsorb chosen wavelength.

What is Ghost peak in HPLC?

These peaks are due to large particles either present in your sample or bleeding from your HPLC system. For the latter ones, they are called system peaks or “ghost” peaks since they are not real sample peaks.

How do you remove air bubbles from HPLC column?

Place a thumb over the sealed column top and invert the column until the bubble is in the exit tip. 4. With your thumb, apply gentle pressure to the “diaphragm” created by the laboratory film until the trapped air is expelled from the tip.

What is maximum pressure in HPLC?

Check your programmed max pressure limit. Operating pressure limits for regular HPLC systems (not UHPLC) are often in the range of 5800 – 7000 psi (400 – 500 bar), which is usually also appropriate for HPLC column hardware and (fully porous particle) packing material.

What is a good RSD percent?

Accepted range of relative recovery? The recovery for accepted range in analytical chemistry is 70-120%.

What causes baseline noise in HPLC?

Baseline noise can be caused by contaminants washing off from a dirty column or from a column that has degraded due to phase dewetting. If it’s suspected that the column is the source of excessive baseline noise, it should be replaced with a union and the method run without the column while observing the baseline.

What is negative peak?

A negative peak means that there is less absorbance while the peak is passing through the detector than when the mobile phase is passing through. Two likely reasons for this are: 1) The mobile phase has more absorbance than the analyte at the monitored wavelength. Inject a sample of pure water.

Are there any good HPLC troubleshooting guides?

Although the web is full of HPLC troubleshooting guides, many of these are dozens of pages long.1,2,3 They can be helpful in teaching systematic approaches to troubleshooting, but when you need an answer fast, digging through these resources to find what you need can be slow and tedious. A free app that will save time and frustration

What are the most common HPLC pump problems?

HPLC pumps incorporate single or dual piston, syringe, or diaphragm pump designs. Pumping system problems are usually easy to spot and correct. Some of the more common symptoms are erratic retention times, noisy baselines, or spikes in the chromatogram. Leaks at pump fittings or seals will result in poor chromatography.

How do I choose the tubing for my HPLC system?

The internal diameter of tubing used in a HPLC system varies with the position in the instrument. Refer to your system maintenance manuals to determine the recommended tubing for a specific application. Note:Most suppliers of HPLC tubing now color code their products for ease of internal diameter identification.

How do I change the phase of my HPLC system?

Close the purge valve and pump the new phase around the HPLC system to flush out any remaining contaminated phase. Allow the system to equilibrate prior to use.