Dilutions for accurate calibration curve - best practices?

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

5 posts Page 1 of 1
Hello,

What are the best practices to make the most accurate dilutions in the ng/uL or pg/uL range? I was quite confident that it is more favorable to use serial dilutions, however this ISO9001 based procedure from Supelco (http://www.agilent.com/cs/library/certi ... C06999.pdf, page 2) has left me wondering.

Supelco standard preparation steps are summarized in quotes. I am assuming that they did not pre-measure the solvent but used a volumetric flask. Note that each solution is made from the STOCK solution
Std 1 0.010ug/ml: 0.10 ml of Stock Solution diluted with 499.9 ml solvent.
Std 2 0.025ug/ml: 0.25 ml of Stock Solution diluted with 499.75 ml solvent.
Std 3 0.100ug/ml: 1.0 ml of Stock Solution diluted with 499.5 ml solvent.
Std 4 0.500ug/ml: 1.0 ml of Stock Solution diluted with 99.0 ml solvent.
Std 5 1.000ug/ml: 2.0 ml of Stock Solution diluted with 98.0 ml solvent.

The concentrations are not the same, but if I were to start with a 5ng/uL (5000pg/uL) Stock solution I would use the serial dilution procedure below.
Std 1 10pg/ul: 10ul of Stock diluted to 5ml in volumetric flask.
Std 2 1pg/ul: 500ul of Std 1 diluted to 5ml in volumetric flask.
Std 3 0.10pg/ul: 500ul of Std 2 diluted to 5ml in volumetric flask.
Std 4 0.02pg/ul: 1000ul of Std 3 diluted to 5ml in volumetric flask
Std 5 0.01pg/ul: 2500ul of Std 4 diluted to 5ml in volumetric flask

Overall what is the more favorable procedure? Another thing that I noticed with the first set of procedures are that they use a large difference in diluent compared to the final volume, whereas I try to keep my diluent within 1-3 magnitudes of the final volume.

Thoughts?
I have used both methods and they both will work. Since I am working with volatile organic compounds I normally use the one stock solution diluted to different amounts such as in the first example, simply because I will lose my analytes if I try to do serial dilutions. When working with inorganic standards such as for ICP metals analysis we normally use serial dilutions or a hybrid of some serial and stock dilutions.

The only problem with serial dilutions is that if you make your first stock wrong, it will not be evident by the calibration curve. If you plan to make a 5ppm standard and you accidentally make a 4ppm standard, the it will be proportionally off all the way through the curve and the only way to detect the error is to compare the calibration curve to another standard made separately, which is how we run our metals calibrations, one standard set for calibration, then another source of standard as a check. If you do the stock dilutions, it will normally be very evident if you make a mistake in one of your dilutions. Of course if you make the stock wrong you still need the second source standard to find that error.

As for volumes, just use what works best for your application. If it works better to measure 100ul, 50ul, 25ul, 10ul and 5ul into 1ml volumes, then make a stock that works for that dilution series. Just keep in mind the stock should be the same solvent as the final standards, if not you could introduce bias because of different solvent concentrations in each standard.
The past is there to guide us into the future, not to dwell in.
I addition to what James said.

The Supelco procedure is horribly wasteful of solvent.

If you have to add different small volumes then try to design things so that you can do it all with one microsyringe, if you have to go up or down in syringe size it will often produce a kink in the calibration. Let all the materials and hardware come to room temp before you start.

Check weigh at each step and correct for actual additions and final volumes. 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.

Peter
Peter Apps
Thanks Pete and James for your insight. Do you happen to know if there are any standard academic, industry, or governmental references about these kinds of things (in addition to what Pete provided)? Most of what I know are just aggregates from my analytical chemistry book (Harris) and just through experience in my graduate studies.

One thing that I would like to point out however, is that I prefer serial dilutions because the error becomes systematic and propagates throughout uniformly. With the supelco method, as Pete pointed out, is that utilizing multiple volumes are likely to introduce kinks in the calibration curve.

It seems like there are multiple points of conflicting information especially given that people of various educational backgrounds are made to "just do it" without know proper technique. Many professionals even go without error propagation which I think is extremely important with the highly sensitive analysis techniques we use today.
Most of us learn the techniques through trial and error over the years. What Peter said about using a single micro syringe is very good advice.

I had an older co-worker who insisted that you could only use the mid point of any syringe to measure your volume as that would be the most accurate, and would try to make his calibration curve using 50ml volumetric flasks and 5 different syringes. His calibrations would always be barely passing at 20% RSD of the average response factor. When I made the same calibration I used a single 50ul syringe and injected into 400ml, 200ml, 100ml and 50ml volumetric flasks depending on concentration level since there were 7 standards, and would have below 5% RSD for the curve. Even if I had to use the 50ul syringe to measure 100ul with two draws. What my co-worker failed to realize was that each different size syringe had a different void volume in the needle and he was using an air gap below the plunger so with each syringe you get a slightly different extra volume added. If you assume that the marking on the barrel are accurate and set to zero at plunger bottom, measure with no air gap after multiple flushes with the standard to "dilute out" any wash solvent in the needle, then you have a positive displacement of the correct volume even with what is left in the needle.

When I first started out my calibration curves were horrible, now after 25 years, I often make a set of dilutions and don't even realize I am measuring out the volumes.
The past is there to guide us into the future, not to dwell in.
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