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I am currently facing a hen-egg problem. For a LC-MS method development I need to prepare reference solutions which are used to calibrate the method. Since the method is to be used for quantification in the pg/mL range, the reference solutions should have the most accurate concentrations possible. Of course I know how to prepare exact solutions in volumetric flasks, but here substance quantities are used which can be weighed easily and also quite exactly. In my case, only a few milligrams of the substance are available to me, which is why I cannot weigh high masses. The precision balance that I have at my disposal has a lower weighing limit of 1 mg, which is about the same as the mass that I would have to weigh. However, since the lower limit is very error-prone, I do not like this method. Unfortunately, I cannot think of an alternative solution that would allow me to create an exact solution without weighing. One could of course dissolve any amount of substance in the desired solvent and then determine the concentration by LC-MS, but this is exactly where the hen-egg problem mentioned above occurs. I cannot quantify via LC-MS until I have a suitable method and an internal standard. Are there any other ideas how to get exact concentrations using small amounts of the desired substance?

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  • $\begingroup$ Is what you are trying to weigh a salt? eg if a sodium salt then you could analyze the solution for sodium for which you could prepare standards by weighing a different salt and diluting. $\endgroup$ – MaxW Jun 22 at 8:25
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    $\begingroup$ No, unfortunately not. $\endgroup$ – Sam Jun 22 at 8:30
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    $\begingroup$ Is it possible for you to describe a little further about the nature of this reference substance? Molweight (roughly, say 100, 200, 500, 1000 g/mol)? What about mixing your compound with something down the road equally is diluted enough to not not interfere badly with your LC-MS, but initially eases the weight-in? E.g., murexide (en.wikipedia.org/wiki/Murexide) is mixed 1:250 or even 1:500 with Na2SO4 and still then it is a powerful indicator for heavy metals. Then an aliquote could be electrochemically analyzed (if reversibly responsive) by redox and cyclic voltammetry. $\endgroup$ – Buttonwood Jun 22 at 13:32
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    $\begingroup$ What is the nature of your sample (peptide / protein / oligomer / dye)? In similar tune to @MaxW, researcher isolating natural products / determining LC50 values work with small quantities (e.g., doi 10.1039/C3NP20106F finally mentions a prep GC to yield just 2.22 mg of Caratol (MW < 200)). Then, the folks in radiochemistry equally use tiny amounts of matter only. $\endgroup$ – Buttonwood Jun 22 at 13:52
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    $\begingroup$ @Buttonwood The reference substances are steroid hormones of an MW-range of 270 to 400 g/mol. As we have a common weighing room, the other labs won't have balances that are more precise and I even don't know anyone of my circle of acquaintances who has access to such a device. Your idea of mixing the compound could be an idea, I'll have a closer look into this. Thanks! $\endgroup$ – Sam Jun 22 at 15:12
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Are there any other ideas how to get exact concentrations using small amounts of the desired substance?

Every mass spectrometry lab should have microbalances to prepare samples and standards. A lab which can afford an LC-MS can also afford a semimicro balance or a microbalance. Such balances are meant to weigh 0.01 mg to 0.001 mg (with extreme caution).

Two simple options: (i) Since you are apparently not the first user of LC-MS system in your group, ask how other senior researchers prepared standards in your lab?

(ii) Another (crude) option is that you try weighing by difference. You will tare your analytical balance to zero. By an analytical balance I mean a balance which reads to four decimal places in grams, this is the most commonly used in research labs. Place your standard container on the balance and read the mass. Carefully transfer approximate amounts to your desired container in which the standard will be prepared, and note the weight of the standard container again. The change in mass corresponds to the amount transferred. Practice it with some commonly available substance to see if this works and if your balance is sensitive enough or not. Balances age with time.

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    $\begingroup$ I wish you were right. I've now worked in several LC-MS laboratories and have never had access to a microbalance. Normally, an ordinary analytical balance as you describe it was sufficient, but this time I would need exactly that. One more question about differential weighing: What's the difference between putting an Eppendorf tube on the scale and taring it, adding substance and reading off the mass, and doing what you describe? Are there concrete indications why one method is more sensitive than the other? $\endgroup$ – Sam Jun 22 at 15:20
  • $\begingroup$ You did not address the simplest point. Can you ask someone in your lab how they prepared standards with small amounts? I can say exactly the opposite, every mass spectrometry facility I saw, had microbalances. I am talking about research labs and good universities. Microbalances are expensive (up to $ 30,000). The pros and cons of weighing by difference are described in standard textbooks. See google.com/search?tbm=bks&q=%22weighing+by+difference%22 $\endgroup$ – M. Farooq Jun 22 at 15:34

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