Adding Internal Standards

[chemboy831]

Question came up during a break today, what's the best way to spike internal standard into samples? Syringes would likely be the most accurate method. Pipettes would be faster but may lack the accuracy due to residual internal standard left behind in the pipettes tip. Any thoughts? What does everyone else do?

[Peter Apps]

The most accurate, and precise way is to weigh everything.

Peter

[DSP007]

Yes of course. Weight in balance is besten ( high precision) method. Usually use in criminal expertise. Also is any good (old , many years ago not have electronic balance).
Make internal stanard solution and use internal standart solution as solvent ( dissolve sample in volumetric flack) . Nowdays it safe in pharmacy.

[H.Thomas]

The most accurate, and precise way is to weigh everything.

Peter

Good luck in weighing sub-µg quantities

Pipettes (e.g. eppendorf) are good, if you use the proper technique. Pre-wet the pipette tip with your IS-solution 2-3 times and then pipette into your sample.

With fixed volume pipettes you can achieve precision and accuracy of <1%, with variable volume pipettes <5%. I don't think syringes are any better.

[Peter Apps]

And good luck to you with measuring volumes less than 1 ul, which is 1 mg in round numbers.

NB I was not suggesting that you make up mg/Kg solutions by directly weighing microgram quantities into grams of liquid, any more than anyone makes up the same solution by dispensing nL (which weigh about 1 ug) ito ml.

As with volumetric procedures, gravimetric dilutions start with concentrated stocks and dilute them down. At each stage, instead of dispensing volumetrically, you do it by weight. Besides the extra precision of balance vs volumetrics, because balances can go to smaller quantities than volumetrics can, you can make up smaller quantities at each stage, which is cheaper in standards and solvents, and less of a disposal problem. Also you can work in screw-top and septum-top vials that are a lot cheaper than volumetric glassware.

Peter

[kerri]

I vote for Positive displacement pipettes or small hamilton syringes

If you dissolve your internal standard in an amount of organic solvent, you will have a difficult time with repeat pipetting using an air-displacement pipette. The air displacement pipettes are accurate at pipetting aqueous samples only. Since organic solvent vapors have different compressibility than air, your second draw using the same tip will be a different volume than your first. The volumes will also change seasonally. So in the case that I need to pipette internal standard solution with an air displacement pipette, I use a new tip for each sample and dispense the first draw. Of course, you need to be very careful and methodical using this procedure (position of tip in the solution, angle you hold the pipette, force that you put on the new tip... these must be as near to the same every time). Changing the tip every time ensures that you keep the solvent vapors in the barrel to a minimum as well as help to prevent accidental cross contamination.

But everyone has their own way that works.

Kerri

[Consumer Products Guy]

Question came up during a break today, what's the best way to spike internal standard into samples? Syringes would likely be the most accurate method. Pipettes would be faster but may lack the accuracy due to residual internal standard left behind in the pipettes tip. Any thoughts? What does everyone else do?

I use Hamilton MicroLab dispensers. They are cGMP-accurate, work by the "Y" travel of the plunger that operates the syringe, and a variety of syringe sizes can easily be swapped in.

Maybe a better reason is that after setting the syringe size and volume, one just pushes the button and the system delivers the internal standard solution, then refills. So EVERYBODY dispenses the exact same amount, quickly, and accurately.

[DSP007]

The most accurate, and precise way is to weigh everything.

Peter

Good luck in weighing sub-µg quantities

Pipettes (e.g. eppendorf) are good, if you use the proper technique. Pre-wet the pipette tip with your IS-solution 2-3 times and then pipette into your sample.

With fixed volume pipettes you can achieve precision and accuracy of <1%, with variable volume pipettes <5%. I don't think syringes are any better.

Hi, H Thomas
Only if its new automatical pipette. If pipette old - plunger seal had worn out and return spring hooked.
Therefore, for the old automatic pipette does not have to talk about precision. Worse, they accuracy, " Only God knows ". Scientifically predict whether the next selection from 0.5 to 0.5 or 0.3 from 0.5 is impossible.
Talk about "the impact of the partial vapor pressure" for this situation is simply meaningless.

[H.Thomas]

If pipette old - plunger seal had worn out and return spring hooked.

I don't know what your QA-System looks like - in a regulated environment (ISO 17025, GLP...) regular maintenance and control of accuracy and precision of pipettes are are a must.

Talk about "the impact of the partial vapor pressure" for this situation is simply meaningless.

You might have heard of positive displacement dispensers like the Multipette...

[DSP007]

This is true, but why create the most problems for yourself? In this case, you need a special procedure and the person who will deal with it. I know in medicine (to the blood is not allowed to contact and accuracy, plus / minus kilometer).
Libra (balance) is now electronic, is not what it was 30 years ago, when the weights were equal-and with a dush of 1-100g weights. Glass pipette, also no problem.

[H.Thomas]

It all depends on your application. Weighing µL-amounts of volatile solvents is by no means easier or more accurate than pipetting.

[DSP007]

It all depends on your application. Weighing µL-amounts of volatile solvents is by no means easier or more accurate than pipetting.

And weighting and "hands pipetting " isn't accurance. Accurance - dissolve intarnal standart in solvent to C 10-100 mkg/ml and use this liquid "as sample solvent"
Weighting , pipetting.... (Legs, wings... best the tail ! End of the cartoon film Unfortunately a lot of advertising and generally suspicious site)

http://rutube.ru/tracks/2424372.html?v= ... 0c8e45cf60 Capture

[Peter Apps]

It all depends on your application. Weighing µL-amounts of volatile solvents is by no means easier or more accurate than pipetting.

But with the appropriate straightforward precautions intra-operator repeatabilities for weighing solvents are routinely less than 1% rsd, and bias is within the readability uncertainty of the balance. See table 9 in APPS, P.J. and ARCHER, M. 2010. Evaluation of the source of bias caused by losses of solvent vapour during sample preparation. Journal of Accreditation and Quality Control 15: 171–180.

Note also, that if you have an actual weight for additions you can use it as a correction factor, rather than having to accept the nominal value as you must with volumetrics.

Peter

[H.Thomas]

Before we continue making pointless arguments, let's look into some operating procedures. I randomly picked EPA 8081B. It says:

Prepare a solution of 5000 mg/L (5000 ng/μL) of pentachloronitrobenzene or 1-bromo-2-nitrobenzene. Spike 10 μL of this solution into each 1 mL of sample extract.

Let's try another one, Method 8270D:

Each 1-mL sample extract undergoing analysis should be spiked with 10 μL of the internal standard solution

Something different, EPA 625:

Pour the entire sample into a 2–L separatory funnel. Pipet 1.00 mL of the surrogate standard spiking solution into the separatory funnel and mix well.

We could continue this list, but this may suffice as example. I don't have the option to look up Russian standards, but I'm positive that they are similar.

So we see, spiking solutions are (usually) dosed volumetrically, most probably using (Microliter-)Pipettes.

[Peter Apps]

The original post asked "what is the best way ?". Citing standard methods using volumetrics does not address this question - I am sure that we can all agree that standard methods do not always represent the cutting edge of analytical practise.

I suspect that we might be cross posting - so I am not going to defend a peer reveiwed paper against the accusation of being "pointless".

Peter