Is it just me who thinks that biology researchers can be a bit sloppy when it comes to making and dispensing reagents accurately? Analytical chemists would laugh at our faith measuring cylinders for anything other than fairly rough work like making up media.
When it comes to more accurate work, like making up assay standards, measuring cylinders just just don’t cut it anymore. Here are a few ways to ensure that your stock solutions/standards are as accurate as possible, allowing you to get your experiments off to the best possible start.
1. Make up solutions and standards using volumetric flasks.
For accurate and reproducible stock solutions your tool of choice should be a volumetric flask. Volumetric flasks are far more accurate than measuring cylinders and pipettes, especially if you use class A flasks, which are manufactured to extremely stringent standards. Volumetric flasks are available in various sizes from 1 mL upwards and you can find a guide to using them here.
2. Use the correct balance, correctly.
Obviously there’s no point in using a highly accurate volumetric flask to measure the solvent volume, only to weigh out the solid using a balance that is too coarse for your needs. Make sure you use a balance that is made for weighing a mass in the range you require, and that it is calibrated and on a level surface.
For small masses, it is often quite difficult to weigh out exactly the amount you require. But normally , solutions don’t have to be made up to an exact concentration – you just need to know their concentration (whatever it is) exactly.
A good approach is to weigh out the reagent as close to the target weight as possible then, note down the weighed mass and make up the solution in the volumetric flask. The actual concentration of the solution you made up can then be calculated exactly..
3. Make reagents in large batches where possible
Another way to ensure that stock solutions are reproducible from experiment to experiment is to make them up in large batches where possible. This not only means that you are working with the same solution in each experiment, but also that you are using a larger volume when making up the solution, which should help with the accuracy.
4. Take into account the strength of reagents you are using.
When making up stock solutions, the strength of the reagent is often overlooked. If the reagent is less than 100% strength i.e. less than 100% of the mass is the actual reagent (the remainder being impurities), it will normally be stated by the manufacturer on the container and you should take this into account when calculating how much of it to use.
Say, for example, you need to weigh out 20g of a reagent to make a 1M solution, but the reagent is only manufactured to 97% percent strength. Only 97% (19.4g) of that 20g will be your reagent so you actually have to add 20.61g ([100/97]*20) of the solid to give you 20g of reagent… and 0.61g of the impurit(y/ies).
Do you agree that many biology researchers could/should do things more accuracy? Shout out your opinion in the comments.
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Yes, I think biologists don’t spend enough time on their reagents. It is surprising how many experiments “suddenly start working” when the solutions are remade carefully. I took an analytical chemistry course as part of my biolgy degree and, in hindsight, it was one of the best choices I made for life in the lab.
I would also like to add that it is important to record exactly what chemicals you used (free acid or salt etc.) and how and when you measured the pH. Measure the pH every time if it is part of the recipe, even if it usually doesn’t need adjusting, it could alert you early if something is wrong with your solution.
Nick,
I am always amused at the need for analytical chemists to use volumetric flasks! These relics are best left on the shelf to collect dust (which we wouldn’t have in a Biological lab becasue our air is usually HEPA filtered).
I had an epiphany around 1996 after watching my group prepare solutions for enzyme purification. The observation was that most of the time was spent titrating solutions to the correct pH. And then the next day, they get titrated again!
So the answer is to prepare solutions gravimetrically: on the balance. It is much faster and much more precise. And never, never ever titrate the pH of a solution to some value. The first time you make a solution, you must measure the quantity of titrant required to achieve the desired pH and then always use this amount. Checking the pH after this is always a good idea, but if the pH is wrong, then something else has happened. Either the wrong or an adulterated raw material was used or something was mis-measured.
So the pH meter becomes the opiate of the Biological masses. If the meter says “7.40″ then it must be good, right? Strange how we place so much faith in arguably the single most variable measurement system in the lab. Don’t believe me? Then make a solution or two and have everyone in the lab measure this solution over the course of a week or two at some random interval. I think you will be surprised at the results.
This is because we ignore the manufactures recommendations, instead re-calibrating the slope of the pH measurement daily, or even before each measurement. You should use the “calibrate” function only once per week (at most) instead, measure the pH 7.0, 10 or 4.0 standards (ideally, something a bracketing the desired pH. If the measured values are aberrant, then the electrode should be cleaned and then re-measure the 7 and 10 standards.
I know that there will be a lot of people reading this and shaking their heads thinking I am nuts (especially those with a capacity for “validation” or Quality), but I will stand by my word.
Anyone who would like a copy of my algorithms for achieving the correct pH for Tris and phosphate buffer solutions, let me know and I shall send a copy and explain how it is used.
We must get the word out. Titration is a dirty word and entirely unnecessary!
Actually, Jim, I would love those algorithms. I am down for anything that makes my solutions more accurate and I’d love to try out what you suggest.
How about figuring out a way to post the algorithms here?
@Wendy – Good points, well made… thanks!
@Jim – Gravimetric solution prep is a good idea, but I don’t see that it is particularly faster than using a volumetric flask. The algorithms sound great though …. check your email!!
With regards to point 2, another good idea is to weigh the weigh boat after adding the solute to the flask. Then you know exactly how much went in there. There always seems to be something left over on the weigh boat after you’ve added the solute.
Good point Rob. My approach to that is normally to use the solvent to wash the weigh boat out into the beaker I am making the solution up in. But weighing the weight boat is also a good idea – I had never thought of that.
Another one from me. That’s a good point about the impurities Nick (never thought about that) but do any researchers actually take into account the impurities? If they don’t (and i’m assuming most don’t – i never have!), this would mean that slightly off concentration solutions are used by everyone to record their measurements and so for the sake of consistency we should all do the same and ignore the impurities. Any validity to that?
Hi just wondering how do we keep our frozen items correct during stocktaking, almost every month we do facing different in the weight. example April balance 10 ton than on May balance 5 ton which this stock hardly move the most we move is only 1-2 ton per month? please advise thanks
Desmond – look out for an article coming up on that soon