Chromatography Forum

Hi ,

How best is it to calibrate a 10ul positive displacement pipette?
Struggling to meet criteria using water for % rsd < 2.0 accuracy and precision .

Taking into water temperature and weighing on microbalance using sealed vial for n= 5 weighings

Any ideas much appreciated

Dear ydna6969

I would prefer to have the calibration done by experts, but if you want to give it a try you may consult the pdf file EPPENDORF SOP that is online.

Good luck!

Fernando

Keep in mind that you are actually calibrating the pipette and its operator together. The operator is almost certainly the source of the variability and bias. Proper training and practice are critical.

On a specific technical point - what is the readability (decimal points) of the balance that you are using ?

Peter

Hi ,

Our in house SOP states calibrate before use for finn pipettes.

This is our first displacement pipette .. so do you think it requires such calibration?

I agree ,, with practice I now have accuracy of < 1.0% but Precision of 3.3 .. is normal criteria so tight at such a low volume ie +-5% acceptable?

We are using sealed vial on micro balance to 3dp ,, weighing 5ul n=5 ..seems to settle and work ok

You are trying to measure a difference in weight of 5 mg between an empty vial and the same vial containing 5 ul of water, on a balance that reads to 1 mg.

So you have only 5 increments of measurement result.

To demonstrate a 1% repeatability of the pipette you need better than 1% repeatability in the weighing - this will need a 5 figure balance if you are weighing 5 ul of water.

To see for yourself, determine the repeatability of the weighings - tare the balance, weigh the empty vial, tare again, re-weigh and repeat for 5 replicates. What is the standard deviation. Add 5 ul of water and repeat for 5 replicates. What is the standard deviation. What are the standard deviations as a fraction of 5 mg ?

Also, check the specifications of the balance for its minimum weight.

Peter

Hi ,

MAQ of balance is 2.5mg - (Lowest level we can weigh)

The repeatability of weighings is pretty much what we are doing ,, results as below ,, are these acceptable at 5ul :

Water Temperature = 19.0oC therefore density value for water at 19oC used
Density (g/mL) 0.99840
Set Volume (uL) 5.00

Measurement Corrected for Density Inaccuracy
4.8520 4.8598 -2.804
4.8600 4.8678 -2.644
4.8980 4.9058 -1.883
4.8120 4.8197 -3.606
4.7930 4.8007 -3.986


AVE 4.8430 4.8508 -2.985
SD 0.041 0.041
RSD 0.855 0.855

Inaccuracy = (Corrected for density-5) /5

You say that you have a three-figure balance but you give weights to four figures ??

Assuming that the 4.8..... weights are replicates of a vial (with or without water makes no difference here) - you have an s.d. of 4.1 mg and your are trying to weigh 5 mg approx. This will give you a repeatability of s.d. of about 80% even if you ignore the tare, so nowhere near good enough to test the pipette. In summary, the problem is with the weighing, not with the pipette.

Peter

Understood , thanks

What would be suitable tolerance levels/calibration acceptance criteria ?

sd < 10% ?

the 4 figures are calculated as water corrected for density and then relative to targert ie :

4.852/0.99840 =4.8598 then (4.8598-5)/5 *100 = -2.804

Balance has reading 0.000mg (2.5mg - 5g)

How repeatable you need the pipetting to be depends on how repeatable you need the analytical results to be, and how much the variability in pipetting contributes to the variability of the results.

If you are weighing only to three figures you cannot generate an extra decimal point of precision just by multiplying the weights by density.

Also, if you balance reads to three decimals of grams, i.e. 1.000 grams, then it cannot read to 0.000 mg.

All of the balance manufacturers have very good information on their web sites about good weighing practice and the accuracy and precision of weighing results with different balances. Take some time to read it.

Peter

misunderstood... balance used as a micro balance.

3dp to mg ..weighing range 2.500mg - 5000.000mg

The concern is not of weighing ,,only tolerance limits of accuracy and precision at 10ul

is it typically % rsd 2 or 5 ? or sd < 10

Dispensing 10 ul from a micro-syringe (which is a specific instance of a positive displacement pipette) it is possible to get an rsd of less than 1%, even with volatile organic solvents;

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.

Since water is less volatile it is easier to weigh repeatably, so your rsd could be lower.

What is the repeatability specification for the pipette ? - this will give you a best case figure to aim at.

Peter

Hi,

rsd< 1.0 is achievable .

ISO8655 limits do you mean ?

for 10ul displacement pipette it is : accuracy = 1.2 and repeatability (random error) = 0.8

I have no idea what the ISO 8655 limits are - if they are the standard that you have to work to, then you have to comply with them.

The accuracy of the pipette depends on its construction - the repeatability will depend mostly on the operator. The repeatabilities cited by the manufacturers are achieved by well trained, highly skilled operators. Depending on your analytical method and sample preparation you may not need to achieve such narrow limits, that is why nobody else can tell you what repeatability criteria you need to meet.

Peter

Hi ,

Understood Peter apps,
Thanks for your posts and advice with this.

All we can do is our best I guess.. the actual formulation is very viscous anyway ,so not sure how relevant calibration with water really is.

guess gives you confidence in your technique and pipette

The problem probably lies with the viscosity of the sample - it would have been good to know this earlier, because it makes a huge difference in the best way to proceed.

My advice is to use the pipette to dispense nominal volumes, and to measure the actual addition by weighing. Then use the actual weight of the sample when you calculate the results.

Peter