I am currently running an HPLC to quantify the activity of my enzyme. I incubate it with my substrate which shows a peak corresponding to its product and also the substrate peak which wasnt converted as well. Since i use the substrate i also do HPLC runs with the substrate alone as a control so i add no enzyme to it and all the buffer conditions, pH etc. are the same. I use the exact same methode (flow rate, injection volume, temperature) as well. But every run shows a substrate peak at a RT of 7.3min for the substrate alone without any enzyme. The peak corresponding to my substrate in the enzyme reaction however has a RT of 7.1min so a shift of 12 sec even though it is the same compound. How is it possible?
The retention time (RT) of a compound in HPLC analysis depends on various factors such as the nature of the column, flow rate, temperature, and the composition of the mobile phase. Even small changes in these parameters can cause significant differences in the RT of a compound.
In your case, it is possible that the presence of the enzyme in the reaction mixture has affected the properties of the mobile phase or the column, leading to a change in the RT of the substrate peak. This could be due to several reasons, such as the formation of a complex between the enzyme and the substrate, changes in the pH or ionic strength of the buffer, or alterations in the temperature or pressure of the system.
However, it is important to note that a shift of 12 seconds in the RT of a compound is generally considered to be within the acceptable range of variation for HPLC analysis. Therefore, it is possible that the observed shift in RT is not significant and may not affect the accuracy or reproducibility of your results.
To confirm whether the shift in RT is significant or not, you could perform a statistical analysis of your data, such as a t-test or ANOVA, to compare the peak areas or heights of the substrate peaks in the enzyme reaction and the control runs. This would help you determine whether the differences between the two sets of data are statistically significant or not.
How reproducible are your retention times normally? A small difference between runs could indicate that there is a leak in the HPLC system, or a problem with pump priming.
Thank you all for the answers. Adam B Shapiro i run the hplc for an activity assay of several mutants with the same substrate. All (3 different enzymes, only differ in a point mutation) showed the non converted substrate peak at the same RT of 7.1min. I did 2 hplc runs for each construct and it was always reproducible. Sourav Roy
Besides the Peak corresponding to my substrate i also observe the peak corresponding to the product. The product peak however has the same RT (in all constructs)in comparison to the standard (i have the product with 99% purity as well as a controle). Maybe an important note: the substrate i use is futalosine which is a tautomere and it has only a purity of around 60% at best. Lamia Fatima Thank you for the answer as well, during the runs the flow rate, temperature etc seamed very stable...so it might be the case that the enzyme has this big effect on the RT of the substrate. I will take a look into the statistical analysis to have an explanation for this shift as well.- Badges
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