What is reference wavelength in HPLC?

What is reference wavelength in HPLC?

The reference window is typically chosen to be wide (100 nm) with the reference wavelength around 50 nm above the wavelength at which the analyte spectrum falls below 0.1 mAU.

How do you troubleshoot an HPLC?

  1. Start pump.
  2. Check mobile phase level in reservoir(s). Check flow throughout system. Examine sample loop for obstruction or air lock.
  3. Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises.
  4. Disconnect tubing at guard column (if present) or analytical column inlet. Check for flow.

What are the reasons for getting negative peaks in HPLC?

Negative peaks are most often caused by difference in refractive index between the sample solvent, sample and mobile phase. They are also caused after routine maintenance when the system has not been reconfigured correctly.

How do you avoid 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.

Why do we use reference wavelength?

Hi Shinsmon, for colorimetric assays, the purpose of using reference wavelength is to correct/normalize any changes that is not from the measuring wavelength of your analyte, such as well to well variation in ELISA plate etc .

What is a reference wavelength?

Based on the displacement effect between solvent and solute molecules in a solution, the signal at the reference wavelength is used as an internal reference to correct the spectrum of the sample under test.

What is tailing factor in HPLC?

Tailing Factor (Tf) is the USP coefficient of the peak symmetry.

How do I fix tailing in HPLC?

There are a few methods that can be used to avoid peak tailing:

  1. Operate at a lower pH.
  2. Use a highly deactivated column.
  3. Consider the possibility of mass overload.
  4. Consider the possibility of column bed deformation.
  5. Work at high pH when analyzing basic compounds.
  6. Use a sample clean-up procedure.

What is Unretained peak in HPLC?

When solvent is injected into a high-performance liquid chromatograph (HPLC), it will elute at the unretained peak position. The first item to check is if the peak coming before the void volume is resulting from a previous injection. This can be determined by varying the amount of time between injections.

What does a negative peak mean in HPLC?

less absorbance
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.

What is unretained peak time?

Therefore, the term void volume is more correctly referred to as the volume of an unretained peak, sometimes called the time of the unretained peak (t0 ). When solvent is injected into a high-performance liquid chromatograph (HPLC), it will elute at the unretained peak position.

How do you select wavelength in HPLC?

Use UV-Vis spectrophotometry to determine the wavelength at which the target compound shows maximum absorbance and also check the solvent effect on the absorbance maxima of the target compounds. It is helpful while choosing the mobile phase i.

What are the advantages of reference wavelength HPLC?

The benefit of this method is that the signals are all acquired using the same time base (unlike Method #1). Using the concept of Method # 2 described above, many HPLC manufactures added a software feature known as a the ‘Reference Wavelength’ to their systems.

How do I solve the problem of negative peaks in HPLC?

Using the problem of negative peaks described above, the HPLC Troubleshooting Guide finds a solution as follows: • From the app’s home screen, select “Start Troubleshooting” (Figure 2a). • Because the issue is related to peaks, select “Peaks” (Figure 2b). • Choose “Negative peaks” (Figure 2c).

What is the best HPLC troubleshooting guide for mobile devices?

An alternative for people with smart phones is the Thermo Scientific™ HPLC Troubleshooting Guide – an app for iPhone, Android, and Windows platforms. This mobile application incorporates all the information from the HPLC troubleshooting poster, and expands it to include electrochemical and nano LC solutions.

How do I use the reference wavelength feature on a dad?

If you want to use the “Reference Wavelength” feature on a DAD: We recommend setting up two different signals in the settings box; Signal #1 & Signal #2. If you want to experiment with a Reference signal to improve the baseline of your chromatogram, then analyze the sample using…