Tips for Troubleshooting in Chromatography

Though this is funny word 'Ghost' but after all this is also one of the major issue during HPLC analysis. There are many factors which gives rise to such unidentified peaks. Some of important factors are mentioned below: 1) Late eluting peaks: During analysis many times late eluting peaks are found in which they interfere in principle peak in subsequent injections. This can be solved by increasing the run time. 2) Contamination: It sometimes happens that due to some contamination ghost peak used to found during analysis. To handle such situations Ghost buster columns are available in market which can be used. It looks like shown in below image: 3) Water Quality: While preparing mobile phase water quality is not proper then it may give rise to ghost peaks. Such situation should be handled by using either Milli Q grade water or HPLC grade water. 

Many times peak splitting is observed during HPLC analysis. Majorly there are 3 main impacting factors due to which peak splitting is observed. 1) Contaminated guard column: Usually guard columns are not washed and this gives splitting of peaks when used for long time. This can be solved by reversing the guard column and flushing it with solvents. 2) Partially blocked frits:  This does not come to notice easily but is very important that frits should be timely changed or cleaned. Since coagulated frits will give rise to splitting of peaks. 3) Small uneven void at column inlet: Repack column with the particles or column should be flushed by reversing it. This will definitely give rid of peak splitting.

Many times it happens that no peak is found during HPLC analysis. There are many such factors which gives rise to such situation. Out of that we will talk about 4 major factors today: 1) Solvent issue:   Practically during analysis many times it happens that analyst uses wrong solvents for example instead of us in ACN they use Methanol. Due to this change in mobile phase, peak is not eluted. So very first it should be checked that correct solvent is used during HPLC analysis. 2) Detector Response:   Second important factor is detector response. If there is any issue in detector response peak will not elute. Ensure that at the time if peak is not eluting then check the detector for any type of error. 3) Injector issue:  Third major issue is with Injector. There can be any bubble entrapment which may give low response or no peak during HPLC analysis. Hence system should be purged properly to avoid such incidences. 4) Leak issue:   Leak is also one of the most impacting parameter for no-p

During HPLC analysis many times a issue is observed namely peak broadening. Due to this area increases abnormally giving rise to erroneous results.  Some of the reasons and solutions for peak broadening are given below : 1) Temperature: Ambient temperature or room temperature can impact the broadening of peaks. To control it, column temperature should be kept in equilibrium mode using column heater or column cooler. 2) Equilibrium of column: Column and detector should be stabilized properly. During gradient system, sufficient time should be allowed between analyses and re-equilibriation.  3) Incorrect column: Check that whether correct column is used during analyses with respect to dimensions as well as packing. If incorrect column is used it will give broad and improper peaks.