5 causes and solution for changing retention time.

-




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 will result in change in retention time of the eluting peak.

5) Temperature: If temperature is not constant of column oven or room temperature is continuously fluctuating, then it will impact the elution pattern of the peak. It is hence always recommended to keep the uniform temperature where HPLC systems are kept.

Comments

Post a Comment

Popular posts from this blog

Case study of Incident observed during HPLC analysis

-
Image
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 some hel
Read more

Drift in the analyte Retention time in HPLC analysis

-
Image
Drift in Retention time of Analyte peak during HPLC analysis: Causes:  There are several reasons of observing drift in Analyte peak in HPLC analysis  like: a. There is slight leakage in pump due to which RT shift is observed in the chromatogram b. Insufficient  saturation of HPLC column as every column behaves in different manner and take different time to get saturated c. Insufficient sonication of Mobile phase which involves solvents like Acetonitrile which requires minimum 15 to 20 mins. of sonication to remove entrapped air bubbles d. Incorrect selection of column for analysis. Remedies :  a. Any type of leakage which might at pump or detector, must be immediately fixed. It might be possible fer rules are not tightened properly which must properly tightened  b. Column saturation  should be done to get stable baseline. Once it is free from any types of noise, standard injection to be done. c. Mobile sonication should be done for minimum 20 minutes to avoid any bubbles entrapment in
Read more

Sample and Standard peak area found in the ratio of 1:6 ratio during HPLC analysis

-
Image
 Sample peak at RT about 4.5 min. with height of about 90 Standard peak at RT about 4.5 min. with height of about 630 Investigation : When such phenomenon was observed then analyst was interviewed and detailed discussion done about level of dilutions done during sample and standard solution preparation. As per the Standard testing procedure, std was to be taken as 50 mg and sample was to taken as Equivalent to 50 mg active substance. Now, the analyst weighed correctly 50.2 mg of standard but during sample weighing he took the similar weight as 50.5 mg. Here the point is weight of tablet under analysis is 300 mg. Hence, the actual weight is to be taken as 300 mg as each tablet contains 50 mg of active content. This resulted in Sample peak height to Standard peak height in the ratio of 1:6. Conclusion : This is the common mistake in pharma lab which is sometime done by analyst. They should clearly distinguish between the word Equivalent and Equal . Though this seems silly mistake but m
Read more