I'm doing determinations of amino acid concentrations (using OPA, Agilent Zorbax Eclipse C18 column & a variation/combination of an Agilent and a Dionex instrumentation methods). I get good peaks in some regions, but some blend together too closely. I don't want to go slower, I'm already running at 40 minutes per sample.
I currently have the HPLC running at 0.72mL/min, but in playing with the method I found decent results if I changed part-way to a 0.48mL/min and back again.
Is this kosher?
Yes. As long as your method complies with ICH Q2(r1). My concern is whether the equipment can routinely perform this procedure.
If the method qualification the validation limits then shouldn't be any problem.
You can do whatever you want as long as the result is reproducible and rugged, meaning analyzing the same vial so many times and get the same result.
As long as it complies to ICH Q2(r1), and can routinely be used in cluding durring method transfer then its okay to use, i wonder would you have had any luck with diffrent column and conditions?
the flowe rate is not the factor determining the peak separation rather the gradient programing for the analysis is the key. so get full separation of peaks you need to try many derivatization trial . also the the OPA is working nicelly in RP-HPLC and you can change the column system
Aslong as it reproducable and validatable and transferable, this should not be a problem. Howevr; have you considered a diffrent conditions on your gradiant to achive good peak shape and seperation?
also you can integrate manually the area again and you can cut the baseline of the peaks to get separated peaks.
it should comply ICH Q2(r1)
please refer http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002662.pdf
*Note: 6-year old question from new user, but in case someone finds this question with similar ideas, please read on as lots of bad advice found.
EMILY, NO ! The flow rate should be initially optimized for the method (linear rate). Under "typical" analysis conditions, if correctly set, flow rate is NOT USED AS A VARIABLE to improve separation. Do not change it during a run (or run the method at a flow which is less than the optimized linear flow rate). Slowing down the flow rate from the optimized rate will NOT improve separation (van Deemter equation), it will delay elution and introduce more diffusion. This is a basic concept of HPLC. Co-elution is what you are observing and that is caused by a LONG list of possible issues (too many to list here and you need someone on-site, with professional experience to assist you with the method, not a web-forum).
Please review the fundamentals of liquid chromatography to understand this (i.e. K prime, Resolution, Separation etc). Contact someone with professional experience in chromatography (advanced level) for assistance as AA analysis is one of the more difficult application areas in HPLC.
William Letter Actually under certain conditions changing flow rate during the gradient can be used. One such example is that a method uses MeOH-water gradient, which is most viscous (gives the highest back pressure) at 50:50. You can have a faster-than-optimal flow rate to save run time, and have a flow rate gradient to slow down near 50:50 to reduce the max pressure, while maintaining the optimal separation. Another example is after the peaks of interest are eluted, increase the flow rate in the high organic region to speed up the column flush and save some run time.
Gang, Please note that the response addresses the question posted. The poster was not an experienced user so we should emphasize following good chromatography practices by NOT recommending FLOW rate-programming to improve separation (which is not logical). If this was a more experienced user, then we could open up the discussion to how FLOW rate can be re-optimized when using columns packed with very small particles [Link: https://hplctips.blogspot.com/2021/01/speed-up-hplc-analysis-time-using.html]. However, even in those cases, we do not recommend flow-programming during an analysis, but instead OPTIMIZING the flow rate for the support used. Slowing the flow rate from the optimized linear rate does not improve the separation (it can not do so), it delays the peaks only, often with more diffusion resulting in worse results. To improve LC separation, the method must be changed (i.e. column, mobile phase, etc). Training in HPLC fundamentals is needed if someone suggests otherwise (This is a common job interview question too and as you will see, many get the answer wrong).
Gang Chen. Time would not be saved in the examples you provide because the added re-equilibration times would be longer after you changed the flow rate. Changing flow rate has a profound effect on back-pressure and baseline stability. All that time spent trying to equilbrate and stabilize the system for analysis would be lost by changing the flow rate too. In general, with a few exceptions (see below), we teach to not use flow programming during an HPLC analysis.
- If you do NOT plan on running any more samples for the day (and/or or changing a column etc,,, ) then yes, sure you could change the flow rate to quickly flush a column out (if isocratic), though it is often better to flush with a stronger solvent, not change flow rate. Again, this has nothing to do with the posted question and may confuse other new users. If you would like to learn more about how you can use higher than normal flow rates to speed up some types of applications, then please check out the linked article.
Increasing and decreasing flow rates is feasible. Decreasing the flow rate would only give an improvement in peak separation if the issue was based upon the time constant of the detector, which is highly unlikely. To gain resolution between peaks without extending the analysis time too much, it is likely that you would need to change the column or the solvent system, perhaps using a gradient elution. This depends whether the unresolved peaks are at the beginning or the end of the chromatograms. The analysis time can likely be shortened with minimal resolution impact by increasing the flow rate.
Yes, you can use variable flow rate and it may improve peak separation in your sample but this is depending on nature of compounds which you analyze
From previous research papers,
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Using a variable flow rate on HPLC can be a useful technique to optimize separations and improve peak resolution. While it is more common to use a constant flow rate during an entire chromatographic run, there are situations where changing the flow rate during the analysis can be beneficial.
By adjusting the flow rate, you can potentially improve peak separation and reduce peak blending. Lowering the flow rate can increase the retention time of the analytes, allowing for better separation and reduced overlap. However, it's important to note that changing the flow rate can also affect other parameters, such as column efficiency, system pressure, and overall analysis time.
Before implementing a variable flow rate method, it is crucial to consider the following factors:
1. Column Compatibility: Ensure that the column you are using can withstand the desired flow rate range. Some columns may have limitations on the maximum or minimum flow rates they can handle.
2. Instrument Capability: Verify if your HPLC instrument supports variable flow rate programming. Some systems may have limitations or restrictions on flow rate changes during the run.
3. Method Validation: If you plan to use a variable flow rate method, it is important to validate the method to ensure it provides accurate and reliable results. Method validation should include evaluating parameters such as precision, accuracy, linearity, and robustness.
4. System Performance: Changing the flow rate may impact system performance, including detector response, system pressure, and instrument stability. It is essential to monitor these parameters and ensure they remain within acceptable limits.
In summary, while it is possible to use a variable flow rate on HPLC, it is recommended to thoroughly evaluate the method and consider the factors mentioned above. Method optimization and validation are crucial to ensure the reliability and reproducibility of your results.
Desh Deepak A P Singh Chauhan: "From previous research papers" LOL ! ALL of the statements you copied here are filled with sweeping generalizations and misinformation. Appears to be AI generated (?). An experienced chromatographer knows better. Please do not post misinformation of this type here which may confuse new or inexperienced users.
The optimal flow rate depends on your column's inner diameter and particle size. If you improve resolution by reducing the flow rate almost to the middle, perhaps you were working at a flow rate far away from the optimal point of your column. You can verify the column's optimal flow rate in the datasheet column.
On the other hand, I have never seen a method varying the flow rate during the run. I don´t know if that is an available option in your instrument. But varying the flow rate during the run can probably lead you to obtain reproducible areas, not appropriate for quantification.
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