I've observed that my procedure of mobile phase preparation affects the reproducibility of results. I used volumetric flasks, cylinders (for addition of second solvent) that is not so comfortable also for everyday use as flasks have limited number of volumes and Eppendorf pippettes for addition of organic modifiers. How to make the procedure more accurate?
1. I am thinking about bottle-top dispensette for measuring volumes of solvents:
https://www.brand.de/products/liquid-handling/bottle-top-dispensers/bottle-top-dispenser-dispensetter-s/
2. I also used Glassco filtering system, hovewer after 2 months it started to leak and I could not see the reason of it (probably glass had some cracks). Nylon 0.2 um filters were used together with mentioned system.
Could you please share with your experience and advice devices (for measuring solvents volumes and vacuum filtration) that you usually use for LC mobile phases preparation?
I usually work with acetonitrile, methanol and TFA, formic, acetic acids, ammonium acetate and formate as modifiers.
As the question is about the most "accurate" way of preparing the eluents, the only acceptable answer is to weigh all the constituents of your mobile phase. However, even with weighing in all the components make sure, volatile solvents are degassed before the final composition. For the preparation of an eluent, think about how to reliably and accurately prepare any liquid mixture. In other words: Do what we learned during the first practical lessons of our lab education.
Please allow me to talk the obvious: In gradient LC it is a very practical hint never to mix 100% pure solvents. Always have a small portion of the second eluent (in case of a binary gradient) present. It avoids any unwanted salt precipitations on the column or any significant volumetric contractions (or expansions) due to the mixing.
Now, did I always use the weighing method? Nope. I only weighed in the constituents, for ultimate performance. For most of our daily work, the faster volumetric approach is more than acceptable.
What made me stumble - several times in the past - was the imprecise description of eluent preparation in articles, posters, etc. I wished that people would accurately write what they did. For example instead of "5% (v/v)", one could write "add 50 mL of solvent A to 950 mL of solvent B". A couple of letters more, and everyone can reproduce the recipe...
My two cents.
Hi,
Just to add to your train of thoughts:
How is reproducibility affected?
What HPLC system (binary/quaternary) are you using and how accurate is its mixing? How about stability of the gradient delivered by the HPLC?
It might be worthwhile to look into the pressure profiles of the runs you've observed the reproducibility issues with.
Hope this helps!
Hello Gulyaim,
I'm guessing you have fluctuations in your retention times. This could be a hardware problem with the pump, or you have a small leak somewhere. Often the reason for fluctuating retention times is an insufficient equilibration of the HPLC system. Mistakes in the preparation of the eluents can also contribute to this. We perform preparative chromatography on 4 systems in our laboratory. Our eluents are water and acetonitrile. We have systems for both acidic and alkaline conditions. In acidic systems, 0.1 % TFA is added to the water. For alkaline systems 0.5 % ammonia is added. In both cases the acetonitrile does not contain any additives. It is supplied via an automatic solvent supply. For the water, we have 50 litre storage vessels each. The additives are supplied in the form of solutions via a so-called Dispensette (Trademark Brand). The TFA solution is 50% in water, the ammonia is 33%. The volume of the added additive solution depends on how much water I want to fill the storage vessel with. The storage vessel has a graduation on a litre scale. Then the appropriate amount of additive solution is added and then the water via a pipe with demineralized water is added. This mixes homogeneously in the storage vessel. We have been doing this for years and it works without problems and is reproducible for many thousands of samples per year.
Regards
Markus
Dear Gulyaim,
A simple way is to weigh in the solvents into the bottle that is used at the LC.
For instance, I use 60% acetonitrile, 0.1% formic acid which makes for a liter I weigh in 400g of water and 471,6g acetonitrile (0.786 g/ml !) and then I add 1 ml formic acid with an Eppendorf/Gilson pipette. Balances are very accurate (
Hi Marc,
you answer is correct, but you won't get far with a litre of solution if you assume flow rates of 40-50 mL/min.
Regards
Markus
methods can vary widely for mobile phase conditions, depending on the reference document, HPLC database, or other source
Dear Gulyaim Sagandykova,
Prior to provide an answer, let e know the make and model of your HPLC. Also let me know the composition of your Mobile phase and Program of mobile phase.
regards
Piyal
You do not mention as to whether you are using gradients or are running isocratic. Fluctuations using gradients do lead to the conclusion that you are having hardware issues as Markus states.
Otherwise a graduated cylinder is probably ideal for measuring the larger volumes. A variable pipettor is fine for the smaller volumes.
As the question is about the most "accurate" way of preparing the eluents, the only acceptable answer is to weigh all the constituents of your mobile phase. However, even with weighing in all the components make sure, volatile solvents are degassed before the final composition. For the preparation of an eluent, think about how to reliably and accurately prepare any liquid mixture. In other words: Do what we learned during the first practical lessons of our lab education.
Please allow me to talk the obvious: In gradient LC it is a very practical hint never to mix 100% pure solvents. Always have a small portion of the second eluent (in case of a binary gradient) present. It avoids any unwanted salt precipitations on the column or any significant volumetric contractions (or expansions) due to the mixing.
Now, did I always use the weighing method? Nope. I only weighed in the constituents, for ultimate performance. For most of our daily work, the faster volumetric approach is more than acceptable.
What made me stumble - several times in the past - was the imprecise description of eluent preparation in articles, posters, etc. I wished that people would accurately write what they did. For example instead of "5% (v/v)", one could write "add 50 mL of solvent A to 950 mL of solvent B". A couple of letters more, and everyone can reproduce the recipe...
My two cents.
You asked, " How to make the procedure more accurate? ". Simple, do what we do in professional chromatography laboratories and develop SOP's which clearly state HOW to prepare mobile phases. In detail. There are many ways to prepare solutions, but only one of them will be reproducible. Have the SOP's reviewed by several people, esp those familiar with laboratory procedures, but not so with liquid chromatography.
Also, skip using any of those 'auto-pipettes' or bottle top dispensers. They are notoriously inaccurate when used with common HPLC solvents. Use class A glassware, esp volumetric flasks, graduated cylinders and precision pipettes.
Here is a link to a free article on the topic which may be helpful.
- "Part 1, Overlooked HPLC Chromatography Standard Operating Procedures (SOP's); Mobile Phase Preparation"; https://hplctips.blogspot.com/2018/12/part-1-overlooked-hplc-chromatography.html
I agree with William Letter. But, according to my experience, if you are maintaining verification records for auto-pipettes or bottle top dispensers it will not be a problem.
Piyal
I agree with Detlef Jensen and William Letter in this. Weighing is the most accurate method when absolute precision is required, but it is a huge pain for routine work. As William pointed out, having written procedures that everyone understands and uses is the best practice for repeatability. Professional scientists in well-run companies use SOPs all of the time, especially where there are issues of safety and proper use of expensive equipment. If you plan on having a science career in an industrial setting, you should get used to reading, following, and writing SOPs that state clearly the exact procedure for doing whatever you are trying to accomplish, in this case preparation of mobile phases for HPLC.
In our lab, we routinely measure by volume using graduated cylinders. Weighing is used only when necessary. We keep the HPLC glassware segregated from the glassware that everyone else uses. All of our "recipes" for mobile phases are written down. They have been reviewed and tested by several knowledgeable people to make sure that they work as written. Whenever language in a given procedure was deemed to be unclear or imprecise, the procedure was rewritten until everyone agreed on the language.
Dear Eef Dirksen
thank you so much for your reply, finally, reproducibility was related to other issues connected with the instrument. I appreciate a lot your recommendation :)
Dear Markus Christ
finally, we found a problem in the instrument connected to sample injection. Thank you so much for your recommendations, it is very important to consider :)
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