Tips for Troubleshooting in Chromatography

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

There are multiple reasons of %RSD failure during HPLC analysis but four are the major factors impacting %RSD: 1) Degassing of mobile phase: There are some solvents which requires proper degassing. So after preparation of mobile phase, it should be properly sonicated. If it is not properly degassed it will definelty result in variation of peak area which will finally give RSD failures. 2) Standard stability: There are certain molecules which are very sensitive to temperature. So if run time is long then till they reach up to 5th or 6th replicate, there area drops drastically which results in RSD failure. Hence method should be developed in such a manner that such molecules should be taken care of. 3) Pressure drop: Due to pressure drop in chromatograms it will impact the area of standard. Hence again this can be attributed to improper degassing of mobile phase which results in %RSD failures. 4) Improper priming of HPLC system : Before analysis, all solvent lines should be prime pro

Definitions: ODS column: Full form of ODS column is Octadecasilane because it contains Octadecasilane chain in it. This column is mainly used in reverse phase chromatography and has free -OH group. Also, as a trade name it is also called as Inertsil column. Using this column usually gives less tailing in peak.  Pros : The main advantage of these columns is it's low cost and sharpness of peaks. Con's: The main disadvantage of using these column is they take too long for column saturation. Elution of peak is fast and so it becomes difficult to separate certain components. BDS Column: The full form of BDS is Base deactivated silica. This column is mainly used in reverse phase chromatography but has blocked -OH group. These column is also called End capped columns. Also, its trade name is Hypersil column. Using this column gives too high tailing in peak. Pros: The main advantage of this column is that it is used for analysis of basic compounds.   Con's: The main disadvantag

 %Carryover in HPLC system is the residue which interferes during routine analysis on HPLC which impacts the actual results. This is one of the most common problem which every analyst encounters during HPLC analysis. Though as a part of system limitations there are certain acceptance criteria for % Carryover i.e. it should not be more than 0.1% of the principle peak, as a common practice. It is very easy to get rid of such %carryover by following three (3) methods: 1) Injection pattern: During analysis injection pattern matters a lot. If high concentration solution is injected first followed by low concentration silution then %carryover is more. So if any high concentration solution is injected it should be followed by blank injection to avoid any %carryover. This is acceptable even by the auditors if more blanks are added. This can also avoid creation of any type of lab events or incidences. 2) Frit cleaning: There is a part called frit after which solution pass into the detector in

There are 5 major causes for too much noise during HPLC analysis? Noise is the basic problem which every analyst faces during day to day HPLC analysis. Below are the causes which create noise and if properly handled, will definitely solve this problem. 1) Sampling rate: If too much noise is observed then very first check the sampling rate in HPLC method. It should be 1.0 instead of 10 (preferably in case of waters HPLC systems). 2) Pressure graph: Pressure graph to be monitored. If pressure fluctuations are more then it will give rose to noise creations during analysis. 3) Degassing: Degassing of mobile phase. If mobile phase is not degassed properly then it will take time to stabilize the column which will again generate the noise. 4) Detector: If room temperature is fluctuating too much then it will impact on detector which will disturb the baseline and will result in disturbed baseline.  5) Column saturation: Some mobile phase requires some long time to saturate during HPLC analy