What causes peak tailing HPLC?
The primary cause of peak tailing is the occurrence of more than one mechanism of analyte retention. Secondary analyte interactions, with ionised silanols on the silica surface, give rise to peak tailing. These interactions need to be minimised to achieve acceptable peak shapes.
What causes tailing?
The most common cause of peak tailing for nonactive compounds is column contamination. These contaminants are relatively nonvolatile, and they accumulate in the column over time. These types of contaminants usually originate in the sample and are species such as polymeric materials, salts, and proteins.
How do I fix peak broadening in HPLC?
In both cases the solution is quite simple: Dilute the sample or reduce the injection volume. The pH of the mobile phase can also have a strong influence on the peak width. Depending on the chemical property of a sample, it can be ionized in different ways, i.e., completely protonated, deprotonated or neutral.
What is tailing factor in HPLC?
Tailing Factor (Tf) is the USP coefficient of the peak symmetry.
How can I improve my peak shape?
Steps that can be taken to improve early eluting peak shape:
- Use a split injection. This limits the amount of solvent that gets onto the column and reduces how much analyte dissolves in pooled solvent.
- Decrease the injection volume.
- Use a pressure pulsed injection.
- Use a guard column.
- Increase the column film thickness.
What causes RSD failure in HPLC?
Column temperature fluctuations (especially evident in ion exchange systems). Column overloading. (Retention times usually decrease as mass of solute injected on column exceeds column capacity.) Sample solvent incompatible with mobile phase.
What causes peak shouldering?
Split Peaks / Shoulder Peaks This phenomenon also occurs when the column is poorly packed and that the packing bed settles under the system pressure or that the mobile phase pH is too high and dissolves the silica thus creating a void at the column inlet.
Why are there no peaks in HPLC?
Make sure that you only inject samples that you’ve filtered through at least a 0.45um filter before injecting into your HPLC. When you observe no peaks, first check to insure that your needle valve seat and injection needle are not plugged.
What causes peak broadening?
Peak broadening or splitting in capillary gas chromatography may be due to condensed solvent flooding the inlet of the column. Solvent introduced by cold on-column injections or recondensed after a splitless injection (solvent effect) travels into the column as a liquid.
What is a good tailing factor?
Acceptable Tailing A new column is considered acceptable if the As value is 0.9 – 1.2 (0.9 indicates slight fronting). In practical terms, an As value below 1.5 is usually OK to work with, and up to As = 2.0 may be acceptable depending on the separation and resolution of the peaks.
What is the resolution formula in HPLC?
HPLC resolution R Resolution is an important HPLC performance indicator usually assessed by how quickly and how completely target components in a sample separate as they pass through a column. Resolution is measured by dividing the difference in peak retention times by the average peak width.
Why is peak broadening bad?
Aiming to be in peak shape Poor peak shape can cause integration and resolution errors in your analysis — which ultimately means poor analysis and wasted time and money.
When does tailing come and go on a HPLC?
Interfering coelution- If you have tailing only for one or two of your peaks, there may be an interfering contaminant that coelutes. If that is the case, you might see the tailing come and go, and a shoulder may appear at times.
How to troubleshoot peak shape problems in HPLC?
Troubleshooting – HPLC Peak Shape Problem & Ghost Peak 1 Small Peak. Using different mobile phase and HPLC columns to separate and compare samples, and select suitable separation conditions. 2 Tailing Peak & Leading Peak. Interference peak. 3 Double Peak Phenomenon. 4 Ghost Peak
What causes peak tailing in an eluting compound?
Cause 4: Sometimes, the presence of excessive extra column dead volume can result in peak tailing. This problem usually effects the peak shape of the early eluting peaks. Later eluting compounds will be more influenced by the packing material itself as these reside longer in the material.
What does the tail of a HPLC peak look like?
The trailing edge (tail) of the peak slowly drops off towards the baseline and is non-Gaussian in shape. For those with GC experience it appears similar to a peak that “bleeds” and continues to interact with the column for an extended period of time. Flow path Diffusion (from extra-delay volume).