Tips for Troubleshooting in Chromatography

  In an reputed pharma company, in IC system, XZ drive failure was observed.  Root cause analysis:  The system was shutdown for about 8 months. So initially it was not known that why such thing happened. Just as instrument was started and XZ drive failure took place. During investigation it was found that earthing observed as 2.4 ohms which is more than the requirement of NMT 1 ohms. Actually at the time of installation it was 1.4 ohms two years back. Due to increase in load, earthing increased upto 2.4 ohms which resulted in failure of XZ drive of IC instrument.  Cost of new drive is about $ 9000. Precaution: It is highly advisable to keep close check on earthing during operation of sophisticated instruments like IC system otherwise it can result in high costly affair.

Case study of Incident observed during HPLC analysis on Waters auto sampler:   Recently during HPLC analysis on Waters make HPLC, a very unique incident happened. During Related substance analysis, after 3 standard injection, in fourth injection missing vial was observed though injection was taken from same single vial. Root cause analysis: Further on investigation it was identified that at the time of vial labelling, analyst used small sticker. So at the time of vial picking by robot in autosampler, small piece of sticker on inside wall of vial carousol. Due to this robot tried to pick the vial but due to resistance from vial, robot was not able to pick the vial. This resulted in above incident and missing vial message was obseeved. On prima facie it was difficult to find the root cause but due to so small sticker, such sticker can cause the incident. Remedy :  Never apply sticker labels on vials and always use marker pen to assign the identification on vial. Hope you may get...

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...

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.

There are many factors impacting Retention time during HPLC analysis. Among them 5 most impacting parameters and there solutions are as below: 1) Contamination build up: This is related to some contaminants which gets retained in the column. This can be solved by continuous flushing using strong solvents. This will flush out the residue and constant retention time will be achieved. 2) Equilibration time: If equilibration time for gradient is not sufficient then it will give variation in retention time. The equilibration time should be sufficient so that constant retention time is achieved. 3) Online degassing: If online degassing is not proper then it will affect the retention time of the peak. Online mobile phase degassing is done by the in build degassed on HPLC system which should be checked from time to time. 4) Evaporation : If reservoir bottles of mobile phase and solvents are not covered properly, slow evaporation of solvent will cause change in mobile phase composition. This ...