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jonco
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[*] posted on 2-5-2011 at 08:32
Measuring milligrams accurately


What would be the most accurate way to transfer 5mg of powder A from its storage container to a scale, and then from the scale into a volumetric flask, so that there is exactly 5mg of the powder in the flask.
And then transfer 1 mg of powder B from its container into the flask, and make up a solution of 100ml?
The transfer has to be done with minimal waste of powder A or B.
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Saerynide
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[*] posted on 2-5-2011 at 08:52


Milligrams is easy. Wait til you get to the nano range :D

Weigh and record weight of volumetric flask. Keep volumetric on balance and add A into flask. Rinse storage container with liquid and pour into volumetric. Repeat with as many rinses as you see fit.

Use a 10^-5 g scale. That way, your error will be ~1% of a milligram

If its more important to you that you get every last molecule of A and B, use a micropipette and forget the volumetrics. The volume of 5 and 1 mg of any material will be negligible for sure and in the limit where it is not negligible, they don't make volumetrics that small anyway lol

Or use a 10^-5/6 scale and measure out a "large" amt. Dissolve in an accurately measured amt of liquid, then aliquot and serial dilute. This method will accurately get you to nanogram/molar concentrations. Micropipette is required. Just dont try to take any short cuts like adding 1 ul to 999 ul or something like that :P

Btw, I think you understand that it is absolutely impossible to get "exactly" 5 mg. I take it that accuracy to the few 10^-9g is for all intents and purposes "exact" :D

[Edited on 5/2/2011 by Saerynide]




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jonco
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[*] posted on 2-5-2011 at 10:09


Thanks Saerynide

Do you know of any link demonstrating how to use the micropipette for such an application?

In my question, the storage container is the bulk container of the powder e.g. 100g of powder. I wouldn't want to rinse that out into the flask!
I think by storage container you mean the measuring tray that used on the scale. If I add directly from the bulk container to the vol. flask, how will I remove the extra powder if I add too much?
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Saerynide
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[*] posted on 2-5-2011 at 10:42


If you have 100 g, then losing a few mgs will hardly be significant right? :D

I thought since the 5 and 1 mgs seemed so important/expensive, the storage container must have been a tiny vial or ampoule and you wanted to confirm the amt inside before using it.

You're right. There is little hope of removing extra once its in the volumetric - the only way is to be really pro at scooping out (and not dropping) semimicro amts.

It might sound a bit retarded because it's "one shot", but this is how its done when you cant afford to waste any material whatsoever like with biologics.

For this reason, this is also why you should not tare with the flask and should instead record the weight of the empty flask. So that in the event you add too much you can take out the flask, remove some contents, rezero the scale and reweigh. Of course everytime you take something off he scale and reweigh, you are losing accuracy, so aim for getting it right the first time.


How to use a micropipette in general:
http://www.usc.edu/org/cosee-west/Jun07Resources/PipetteUset...

Serial dilution explained:
http://biology.kenyon.edu/courses/biol09/tetrahymena/seriald...

If you've never used a micropipette, you should ask someone in your lab to show you first in real life. I know someone who didnt *actually* know the difference between the first stop and second stop and measured everything all wrong until I said "this doesnt look like 10 ml... are you sure??" :P

[Edited on 5/2/2011 by Saerynide]




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[*] posted on 2-5-2011 at 12:13


1. There is no way you can accurately take 5 mg from the bulk. In fact, you should not be allowed to keep transfering powder into and out of the scale because you are likely to cause spills.
2. The volumetric flask suggest that you are going to dissolve it anyway, so...

So the proper method is by taking an amount (slightly) more than 5 mg, which you weigh accurately using a 'weighing boat' (use google images if you have no idea), and quantitatively transfer this into an accurate volumetric flask of a smaller volume first. Dissolve the powder, fill to the proper level, mix, and take from this flask the volume that contains 5 mg, using any method for transfering accurate volumes of liquid.

Micropipettes are ok for most work, but most are not that precise actually. I guess it depends on the level of accuracy that you require.




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[*] posted on 2-5-2011 at 12:16


1. There is no way you can accurately take 5 mg from the bulk. In fact, you should not be allowed to keep transfering powder into and out of the scale because you are likely to cause spills.
2. The volumetric flask suggest that you are going to dissolve it anyway, so...

So the proper method is by taking an amount (slightly) more than 5 mg, which you weigh accurately using a 'weighing boat' (use google images if you have no idea), and quantitatively transfer this into an accurate volumetric flask of a smaller volume first. Dissolve the powder, fill to the proper level, mix, and take from this flask the volume that contains 5 mg, using any method for transfering accurate volumes of liquid.

Micropipettes are ok for most work, but most are not that precise actually. I guess it depends on the level of accuracy that you require.




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jonco
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[*] posted on 2-5-2011 at 13:56


I meant 100mg, not 100g. My bad!

Dissolving more than 5mg into a smaller flask would mean losing some of the powder, right? If I had to use this method, I would need to record the mass of the powder, say 6mg, and dissolve this in a 50ml flask, and then transfer 5*50/6 ml of this solution to the 100ml flask, right? Can the remaining solution be reused if I want to transfer another 5mg?

[Edited on 2-5-2011 by jonco]
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[*] posted on 2-5-2011 at 19:49
Measuring Milligrams Accurately


I built a copy of this balance for around $10. It weighs 1/10 milligram or less quite accurately. The article says it weighs 10 micrograms. I have no reason to work with weights that small but it appears that it can probably go that low. The key is using a razor on a razor.

Here's the link:
"http://sci-toys.com/scitoys/scitoys/mathematics/microgram_balance/balance "

I think you will be impressed with the simplicity and the accuracy of this balance.

[Edited on 3-5-2011 by Ribivarin]
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[*] posted on 3-5-2011 at 04:55


Phlogiston: Are you sure?? Although a volumetric flask could be more *precise* than a micropipette (since micropipettes are not great for measuring volatile things and air displacement can be affected by temperature and user habits and all that), but there is no way a volumetric could be more accurate than a calibrated micropipette :P For one, there are no 100 ul volumetrics, and second, you are going to lose a much larger amount of material to the wall of the volumetric than using a pipette tip because you must take the solution out of the volumetric after, whereas the pipette adds solvent directly into the sample vial and the sample doesnt have to go through an extra container.


Jonco: Phlogiston's method of dissolving more into a known amt of liquid and then taking out the right amt that would contain 5 ml is the aliquot and serial dilution method I mentioned.

You measure out a "larger" amt very accurately. Then you dissolve it into a mathematically convenient amount of liquid, also measured very accurately. Then you (very accurately lol) take out the amt of soln that would contain the amt of stuff you need (this is the aliquoting part). Finally, you (very accurately) add enough solvent to the bit you took out to get the final concentration (this is serial dilution).

There are two ways to think about the uncertainty when measuring out small amounts:

1) The scale may not be accurate when small amts are placed on, meaning even if it does say +/- 0.01 mg, it doesnt *actually* mean you can accurately expect to get 0.19-0.21 mg when trying to weigh out 0.20 mg :P Even if you put on a "true" 1mg, it would probably read somewhere between 0.8-1.5mg or something like that.

For this case, you would want to measure something much bigger, like 10 mg. If you weigh out 10.01234 mg, dissolve it into 1000 ul, and take out 100 ul, you will have 1.00123 mg, whereas if you put on material until the scale says "1.00094 mg", there might actually be 1.58393 mg, which would be too much. This has nothing to do with 10 mg having more digits and thus more accuracy. It's a mechanical error due to non-linearity. This is a very common problem and aliquoting is good for this.

2) You believe the scale is accurate *ENOUGH* (keyword lol) at small amounts. In this case, there is no need to bother scooping out more only to aliquot if you are making ONE sample.

For example, if you are going to scoop out 6.12345 mg, dissolve it into 1 ml and take 816.5 ul to get 4.99979 mg, you might as well try to scoop out something between 4.99979-5.00021 mg and not waste the extra milligram. You will also have a lower error in real life by not pouring/transfering/measuring so many times. Everytime you measure something out, you introduce error either do to the measuring device or as mechanical loss to the walls of vessels or even evaporation if the liquid is very volatile or there is very little liquid.

So in this case, if you aliquot, you have the error of the scale, the error of the volumetric, the error of pipetting out liquid from the volumetric, and then the error of measuring fresh solvent. If you just (very pro-ly) scoop out ~5mg into your sample vial and micropipette in your solvent, ignoring the 5mg's worth of volume, you only have the error of the scale, and the error of measuring the solvent, and the error of 5mg's worth of volume. I can assure you the error from assuming 5 mg has negligible volume will be *significantly* less than the error from measuring liquid 2 extra times (of course, unless you are in the limit where 5mg's worth of volume is important, but in that case, you still have no other way because theres no volumetrics that size! :P)

HOWEVER, if you need to make a MORE than one sample, you should aliquot, even if the scale is accurate at very tiny amounts. This ensures every one of your samples will be the same. You will get both precision (from them all being the same) AND accuracy (from measuring a bigger amt because the scale is better at that) Even if there is an error, at least they will all have the same error and your variability will go down.

My best advice is just to get really pro at scooping out very very tiny amounts (use the smallest spatula you can find, or even better, a scalpel blade) :P This is how we all do it in our lab and we don't bother with weigh boats (for example, you are not going to put RNA into a dirty plastic weighboat - that shit goes *directly* into the microfuge tube!) :P


When I mentioned that you can get to nanomolar concentrations with reasonable accuracy, you would take 1 ul into 9 ul fresh solvent. Then take 1 ul of the resulting soln into 9 ul of fresh solvent.... And you keep going (tediously) until you get to 10 nM or whatever you are after.

[Edit]: Jonco - if you have 100 mg and you KNOW the products are stable in your solution and you KNOW (fo sho) in the future you will have to make these solutions again, you can consider dissolving all 100mg in say 10 or 100ml of solvent (or some convenient amount) so you can just aliquot in the future when you need it. Make sure your product is stable in solution first, and even then you should stick it in the -20 or -80 freezer if its safe to freeze.

[Edited on 5/3/2011 by Saerynide]




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[*] posted on 3-5-2011 at 13:21


Why does it need to be exactly 5 mg?
Weighing about 5 mg is relatively easy; weighing exactly 5 mg is a pain.

One of our procedures at work start out by weighing about 180 µg of material (yes, I mean 0.18 mg) but the tolerance is something like 150 to 200 µg.
I did once weigh out a few 10 mg aliquots that were 10 mg give or take 1% but that's just tiresome.
We have a micro-balance but, aside from that and a steady hand, it's no different from weighing out 5 g of stuff.
I use little foil pots made by folding an inch square of Al foil over the end of a pipette as weighing boats.
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[*] posted on 3-5-2011 at 14:25


Let's focus on accuracy, because the question on how to minimise losses was never asked.

And, as discussed, the solution (which has an accurately known concentration) can be stored and reused to minimise loss (provided the compound is stable)

Quote:
Phlogiston: Are you sure?? Although a volumetric flask could be more *precise* than a micropipette (since micropipettes are not great for measuring volatile things and air displacement can be affected by temperature and user habits and all that)


Well, I guess if you take inferior glassware and a great micropipette which is handled skillfully, but in general it should be true. Also, temperature should be controlled for most accurate work and 'habits' have no place here. There are well-described procedures for pipetting which you should stick to for accurate results.

It is common for good volumetric flasks to be precise to about 0.03 to 0.1% percent.

Adjustable micropipettes hardly ever do better than 0.5%, and often much worse over most of their range. As a typical example, take Eppendorfs newest pipets:

http://de.sitestat.com/eppendorf/com/s?en.download.brochure....

Common volumes (20, 100, 200, 1000 ul) can be off by as much as 5% at the low end of their range, and none do better than 0.6% anywhere in their specified range.

Quote:
but there is no way a volumetric could be more accurate than a calibrated micropipette For one, there are no 100 ul volumetrics


Im not sure it doesn't exist anywhere, but the TS never mentioned the desired volume for compound A. Volume for compound B is 100 ml, which should be no problem.

Quote:
and second, you are going to lose a much larger amount of material to the wall of the volumetric than using a pipette tip


Actually, this volume is accounted for. You take the desired volume out with a volumetric glass pipet, and the volume that comes out is accurately the desired volume. Everything that sticks to the wall is not part of the desired volume.

For micropipettes, actually you are supposed to take this into account as well. Proper pipetting techinques involves filling the tip a couple of times to the desired volume, emptying it and only then pipet the required volume 'for real'. This forms a film on the wall of the pipet tip. There is also a specified time that you should allow for the pipet to empty itself. Anything that comes out within this time is part of the volume.

For biochemistry work micropipets are certainly good enough, if they are well maintained and kept calibrated. But in the world of analytical chemistry the word 'accuracy' has a different meaning by several orders of magnitude. And the original question was 'what would be the most accurate way to...' etc




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[*] posted on 5-5-2011 at 05:54


Hmm... Interesting... I never thought glass could be that accurate, since there is so much variation piece to piece from manufacturing. I only ever reached for the volumetric glassware if whatever I was using dissolved the pipette tips but may be now I'll use it more.

So what would an analytical chemist do if he/she needs to measure 100 ul? Use those microliter glass syringes? Perhaps I should get my self a few - could be useful for volatile liquids. I hate how the measured amt disappears inside the pipette :mad: Pumping the pipette a few times to fill it with vapor works, but is still really annoying.




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[*] posted on 5-5-2011 at 09:51


Quote: Originally posted by Saerynide  
Hmm... Interesting... I never thought glass could be that accurate, since there is so much variation piece to piece from manufacturing. I only ever reached for the volumetric glassware if whatever I was using dissolved the pipette tips but may be now I'll use it more.
Class A volumetric glass is often accurate to 1-2 parts per thousand.
It's even better if you calibrate it to the contained (or delivered) water weight at temperature.
Cheapest is to buy Class B glass and calibrate it.
Quote:
So what would an analytical chemist do if he/she needs to measure 100 ul? Use those microliter glass syringes? Perhaps I should get my self a few - could be useful for volatile liquids. I hate how the measured amt disappears inside the pipette :mad: Pumping the pipette a few times to fill it with vapor works, but is still really annoying.
The micropipettes don't fare so well; about 1% error is what you can reasonably expect.
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[*] posted on 5-5-2011 at 16:18


Quote:
So what would an analytical chemist do if he/she needs to measure 100 ul? Use those microliter glass syringes? Perhaps I should get my self a few - could be useful for volatile liquids. I hate how the measured amt disappears inside the pipette Pumping the pipette a few times to fill it with vapor works, but is still really annoying.


Yeah, I hate that too. Ever pipetted ammonia?
With such small volumes, evaporation is going to be a problem anyway, limiting the accuracy if you work with it for any amount of time, so it would be best to avoid having to do rely on the accuracy of such a small volume of a volatile liquid in the first place.

The microliter syringes (we just call them 'hamiltons', after the company that makes them) are nice but only accurate to about 1%, i.e. similar to an adjustable micropipette.

The best I can think of is to calculate the weight of 100 ul using the density and then weigh it. A good balance is very accurate.

Evaporation is going to be a serious problem with such a small volume. You are going to see the weight decrease from evaporation while it is on the scale.




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[*] posted on 5-5-2011 at 18:24


Quote: Originally posted by Ribivarin  

I think you will be impressed with the simplicity and the accuracy of this balance.

[Edited on 3-5-2011 by Ribivarin]


the link should be:

http://sci-toys.com/scitoys/scitoys/mathematics/microgram_ba...

I'm impressed. So simple. I'd want to calibrate it against known weights. On the downside for me right now would be counter space. But yes, very cool.




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[*] posted on 6-5-2011 at 05:36


This is really valuable info here. I was thinking of buying a micropipette, but now I not so sure after reading the different responses. What would be the analytical chemistry way of doing it?
By the way, I checked out the homemade microbalance. It looks awesome and not too difficult to build. Either that, or some £££ for the micropipette?

[Edited on 6-5-2011 by jonco]
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[*] posted on 13-5-2011 at 09:24


Quote:
Yeah, I hate that too. Ever pipetted ammonia?
...
The best I can think of is to calculate the weight of 100 ul using the density and then weigh it. A good balance is very accurate.

Evaporation is going to be a serious problem with such a small volume. You are going to see the weight decrease from evaporation while it is on the scale.


Hahaha you mean liquid NH3?? No.... and I can't imagine why I would ever need to :P But I do have to measure acetaldehyde constantly and the lab is always above boiling point :( I keep it in the -20 freezer along with the pipette tips. Then when I need to get some, I quickly open the bottle, pump the pipette several times til the liq level is constant, then plunge the filled tip into the receiving soln. It dissolves so quickly I dont even have to eject it from tip.

The weighing method is no use for CH3CHO. It disappears before your eyes. I cant even read the scale that fast, let alone get the cap on the container before it all vanishes into thin air lol


Jonco: Micropipettes are really expensive.... Since it sounds like you are working on a research project, I'm not sure it'd be worth buying your own :P Use your lab's funding to buy it :D

The thing with micropipettes is that you cant really buy just one. You kind of need the whole range to fully maximize their utility (2-20ul, 20-200ul, 100-1000ul and useful but not necessary, the 500-5000 ul one). If you are really hardcore, the 0.1-2.5ul or the 0.5-10ul pipette can be useful, but I doubt it's ability to measure out accurately 0.1 ul - I strongly believe one should always design the experiment so one never has to measure less than 1 ul!

[Edited on 5/13/2011 by Saerynide]




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