Calibration of Micropipettes

Calibration of Micropipettes

Objective:

1. To know different parts of Micropipettes.

2. To learn how to use Micropipettes.

3. To calibrate micropipettes used in Molecular Biology laboratory.

4. To learn how to take care of Micropipettes.

Introduction:

Micropipettes used to accurately measure small volumes of liquids (volumes typically vary from 1 to 1000 µL). The parts of typical micropipettes are shown below.

Accuracy and Precision

Pipettes and micropipettes can deliver accurate and precise volumes of solution. Our goal is to determine how accurate and how precise our pipette and micropipette are.

Accuracy is a measure of how close a measured value is to the accepted or “true” value. It is related to the percent error between the average volume of solution measured experimentally and the volume that was expected (the accepted value). Smaller percent error reflects higher accuracy. Percent error can be negative, indicating that the measured volume was smaller than the expected volume or positive, indicating that the measured volume was larger than the expected volume. For example, we are attempting to measure two different volumes of water with our micropipette and two with our graduated pipette. Perfect accuracy would have us measure the exact volume we desire each time. However, the volume of water that we actually measure will be close but probably different from these volumes. The farther away from the correct volume, the lower the accuracy of our pipettes and/or our technique will be. The formula for percent error is in the Statistical Functions portion of the Lab Manual Introduction.

Precision measures the closeness of a set of values obtained from identical measurements of the same quantity. It is the ability to repetitively measure the same volume of solution (whether it’s accurate or not). Precision is related to the standard deviation of a series of measurements of the same thing. For example, if the micropipette is set to the same volume (300 µL) and four measurements are taken at this volume, a standard deviation can be taken of these five measurements. The smaller the standard deviation, the more precise the micropipette is. We will use the standard deviation as a measure of the spread of potential errors in a given measurement. The formula for standard deviation is in the Statistical Functions portion of the Lab Manual Introduction. Standard deviation is usually reported with the average value like this:

In order to minimize the waste generated from experiments in this class, a number of the experiments involve a micropipette that can deliver between 100 and 1000 microliters (µL). The micropipettes are only to be used for this volume range. For larger volumes, a graduated pipette is in your locker (bulbs available from the stockroom or one of the side drawers).

Use of the micropipettes

When you push down gently on the plunger of the micropipette, you will feel a “stop” where the resistance increases. If you push a little harder, the plunger will move even further to a second stop. The first stop is used to suck up the correct volume. The second stop is used to completely expel the liquid you are measuring.

Liquid is never drawn into the barrel of the micropipette itself. An appropriate tip should always be placed firmly on the end. Since the principle by which the micropipette works is the creation of a vacuum in the tip, causing liquid to be drawn up, it is critical that the tip be on tight enough to make an air-tight seal. Having said this, do NOT jam the tips on so hard that they are hard to get off. The tips used for the 1000µL pipettes are usually blue.

The volume to be taken up is set by turning the plunger on the top of the micropipette and reading the numerical settings displayed. A setting of 100 µL is equal to 0.100 mL. A setting of 1000 µL is equal to 1.000 mL. Do not set the micropipette below 100 µL or above 1000 µL under any circumstances! Doing this is essentially the only way that the micropipettes can be broken.

When drawing liquid up into the micropipette, set the dial, and then pushes the plunger down to the first stop. While holding it there, put the end of the tip under the surface of the liquid to be measured and slowly, gently allow the plunger to return to its top position. If you go too fast, you will cause some liquid to spurt up into the micropipette barrel itself, which is bad for the micropipette, bad for your results and bad for your chemistry karma, whatever that is. This also lets air in, so the volume of fluid sucked up into the tip will be lower than the amount that you want. Never let the plunger snap up by it. Next, place the end of the tip where you want the liquid to go and push the down the plunger to the second stop to deliver the exact amount of fluid desired.

Although micropipettes are usually quite accurate when first purchased, they can eventually develop problems with use. We will spend some time checking the calibration of the micropipettes that we will be using throughout the semester to ensure that they are delivering a known volume of fluid. We will measure out different exact volumes of water with the micropipette, and using the density of water at the temperature of the water, we will determine the volume of fluid delivered based on the fluids mass.

There are many techniques and tips available that will optimize your pipetting performance and increase the reproducibility of your results. A brief description of each follows:

The Equipment

1) Tips - It is advocated that only high quality tips which optimize the pipette’s performance be used. A high quality tip is one that has a smooth uniform interior with straight even sides that prevents the retention of liquids and minimizes surface wetting. Also, the tip should have a clean, hydrophobic surface and a perfectly centered opening in order to ensure the complete dispensing of the sample. These tips should always securely interface with the nosecone, because if they do not fit correctly, the amount of liquid dispensed can be dramatically influenced.

2) Liquid Viscosity - Since the pipette was originally factory calibrated using water, any liquid that has a viscosity higher or lower than water will impact the volume dispensed. Viscosity differentials should be accounted for and taken into consideration in order to enhance the accuracy of the instrument.

3) Container - The material of construction for the extraction vessel is also important, since some materials tend to force water into a convex configuration while other materials force water into a concave configuration. Obviously, this can impact the amount of liquid drawn into the tip. A glass container is recommended since it tends to force water into a concave configuration which helps to reduce or eliminate variations due to this effect.

The Operator

1) Technique - Most end users have a tendency to believe that the volume delivery is completely dependent on the setting of the micrometer dial. Obviously, this is not the case, since many factors associated with pipettes come into play.

• Position - Pipettes should be held vertical during the aspiration of liquids, however, some end users often hold pipettes at many different angles during a pipetting interval. Holding a pipette 30o off vertical can cause as much as 0.7% more liquid to be aspirated due to the impact of hydrostatic pressure. Always store pipettes in an upright position when not in use.

• Pre-Wetting/Pre-Rinsing Tips -Failing to pre-wet tips can cause inconsistency between samples

since liquid in the initial samples adhere to the inside surfaces of the pipette tip, but liquid from later samples does not. Also, if a new volume is dialed in on the pipette’s micrometer, you will receive better results at the new volume by taking the old tip off and placing a new one on the shaft before you commence pipetting.

• Release of Plunger - Releasing the plunger abruptly can cause liquid to be “bumped" inside the pipette during a liquid transfer application. This can cause liquid to accumulate inside the instrument which in turn can be transferred to other samples causing variability in sample volume and the potential for cross contamination. It is recommended that a smooth, consistent pipetting rhythm be employed since it helps to increase both accuracy and precision. After the liquid has been aspirated into the tip, the pipette should be placed against the wall of the receiving vessel and the plunger slowly depressed. This will help all of the liquid in the tip to be dispensed. After a pause of about 1 second, depress the plunger to the bottom or blowout position (if equipped) and remove the pipette from the sidewall by utilizing either a sliding action up the wall or a brief movement away from the wall (called “touching off”).

• Immersion Depth - The pipette tip should only be inserted into the vessel containing the liquid of be transferred about 1-3mm. If the tip is immersed beyond this, the results could be erroneously high. This is due to the fact that liquid could adhere to the tip and be transferred along with the aliquot in the tip. If the tip is not immersed far enough then air could be drawn into the tip which could yield results that are incorrect on the low end.

• Equilibration Time – Troemner recommends that the tip, the pipette, the liquid being transferred, and the transfer container itself all be allowed to equilibrate to the same temperature. This is done to lessen the effects of thermal expansion which can dramatically impact the delivered volume.

• Thermal conductance – Thermal energy can be transferred from the operator’s hand to the air within the pipette (dead air) or even to the internal components themselves. This can have a dramatic impact on the amount of liquid dispensed due to the effects of expansion and/or contraction. To lessen this effect, it is recommended that some type of thermally insulated gloves like latex or cloth be worn.

2) Pipette Micrometer Setting – It is important to avoid significantly overdialing or underdialing the recommended range of the pipette. Volume delivery performance may change radically and may become completely undefined.

The Environment

1) Temperature – The volume delivery performance specifications of pipettes have been referenced by most manufacturers at room temperature which is defined as 20-25ºC. Any deviation from this specification can affect the amount of liquid dispensed due to the expansion or contraction of the internal components. Temperature is probably the most important factor that influences pipette performance. In fact, the density of water in a gravimetric analysis is calculated as a function of temperature.

2) Barometric Pressure – Pressure is reduced by 1.06" Hg for every 1000' of elevation, however, barometric pressure has only a small effect on the density formula, so the error encountered in not correcting for elevation is often ignored.

3) Relative Humidity – This is the percentage of moisture in the air at a measured dry bulb temperature compared to the amount of moisture that the air can hold at that temperature if the air is 100% saturated. Relative humidity exerts a major influence on taking accurate measurements of volume delivery. Under dry conditions, which are defined as less than 30% RH, it is extremely difficult to ensure an accurate measurement due to the rapid evaporation rate. Conversely, excessive humidity which is defined as greater than 75% can cause a measurement to be erroneously high due to condensation. Therefore, generally accepted guidelines for pipette volume delivery specify that relative humidity be maintained within the range of 45%-75%. Relative humidity also has an effect on the delivery of air displacement pipettes specifically. This is due to the evaporation of liquid from the upper several factors to consider when calibrating a pipette or choosing a calibration service:

1) If you require the “as found” data, it is advisable to obtain this before any parts or components are replaced since this can drastically change your results.

2) Clean and inspect the instrument for any visible signs of wear and tear. Make sure that the instrument can be autoclaved before autoclaving, since this can seriously damage the pipette.

3) Replace the pipette’s seals and o-rings and any other part that shows signs of wear. Remember

to pay special attention to the piston and replace if it seems especially worn or bent.

4) Ensure that the o-rings and seals have seated properly by performing a leak test and a vacuum test.

5) Allow the pipette to stabilize in an environmentally controlled, vibration-free room for a 24 – hour period to eliminate the effects of thermal expansion.

6) Decide which calibration technique that you wish to employ (i.e. Addition, Addition-Tare, Subtraction, or Subtraction-Tare).

7) Prepare the balance by “exercising” it and modifying it to accept a liquid containing vessel. It is our recommendation to use a glass container, so that the liquid has a concave meniscus.

8) Since most manufacturers originally calibrate their pipettes between 20-25°C while using bidistilled, degassed water, it is our recommendation that these conditions are duplicated.

9) Wear some type of thermally insulated gloves to lessen the transfer of heat from your hand to pipette. Latex or cloth seems to work the best.

10) Begin the liquid transfer stage of the calibration procedure utilizing the appropriate technique that you have chosen to employ.

11) Record the weightings, so that they can be converted into volumetric readings at the end of the calibration procedure.

12) Make the conversion taking into account all pertinent environmental conditions. Usually these conditions are used to calculate a Z-factor which is in turn used to convert from a mass reading to a volumetric reading.

Calibrating Micropipettes:

The two most common techniques of calibrating pipettes are the gravimetric and colorimetric (a.k.a. photometric) methods. Of these, the gravimetric method is the most common and the most widespread in use today. This method requires a stringently controlled environment, a high precision balance, a highly skilled pipetting technician, and a rudimentary understanding of statistics. The principle of this method is simple in that, given a certain mass of water with a known specific gravity; its volume can then be predicted. The accuracy and precision of the pipette can then be assessed by using an appropriate statistical approach. This method can be performed one of four ways: Addition, Addition-Tare, Subtraction, or Subtraction-Tare.

1) Addition is perhaps the most common mode of pipette calibration and it is performed by using the cumulative weight of a liquid to determine the volume dispensed.

2) The Addition-Tare method is performed by taring the balance each time before dispensing.

3) The Subtraction method uses the total subtracted weight of a liquid to determine the volume aspirated by the pipetting device. In this technique, you tare the balance only once, at the beginning, then you aspirate volumes of liquid from the vessel, take cumulative (negative) weights, and then calculate the volume aspirated based on the difference between the current and previous total weights.

4) The Subtraction-Tare method entails taring the balance each time before removing liquid from the vessel.

Since this method is not fool-proof, all variables must be stringently controlled and accounted for in order to produce results that are statistically accurate. The second most common type of pipette calibration process is the colorimetric or photometric method. This method involves the analysis of volumes of diluted dye in a cell of known path length. According to the Beer–Lambert Relationship, if a beam of monochromatic light passes through homogeneous solutions of equal pathlength, the absorbance measured is proportional to the dye concentration. So, with this in mind, an unknown volume of dye can be pipetted into a known volume of diluent, the resulting dye concentration can be measured photometrically, and the volume can be calculated. This method is less prone to environmental influences, but it requires the use of standardized consumables. Obviously, this means that each lot of standardized dye must be very carefully manufactured and calibrated in order to produce results of high accuracy. However, once solutions are prepared, calibrated and shown to be stable, accurate results can be obtained even at volumes less than one microliter11.

Principle:

The volume delivered by the pipettes is determined by weighing the amount delivered and dividing this by the density of water (at RT and 4°C).

Materials:

1. Balance (digital, upto 0.0001gm),

2. Micropipettes (1-10, 10-100, 100-1000µL),

3. Distilled water,

4. Aluminium foil

Procedure:

1. Turn on the balance

2. Place aluminium foil (prepared to cup shaped) on the pan of balance carefully

3. Zero the balance by tareing from its keyboard

4. Pipette out DW onto the foil and observe the weight of water on the data display

5. Zero the balance by tareing from its keyboard, again

6. Pipette out DW with the same pipette onto the foil and observe again the weight of water

7. Repeat steps 5 and 6 for more than 30 attempts

Observation:

Example: Pipetting of 10µL

Table 1: Weight (d=M/V) and corresponding volume (l) of water (d=1) delivered by micropipette

S. no.	1	2	3	4	5	6	7	…………………………………..	30
Experimental volume (µL)	11	12	09	10	13	09	08		11

Calculation/ Statistics:

Table 2: Determination of standard deviation (Sd) of volume of DW delivered by the micropipette

Experimental volume of DW (µL)-X	True Volume (µL) (expected volume)- X bar	(X-X bar)	(X-X bar)2
11
12
09
10
13
09
08
Calculate for all readings
11
S(X-X bar)2

Sd= Ö S(X-X bar)²/Ö n-1

Interpretation:

………………………………………………………………………………………………

Note: Ensure confidently for no handling error.

Annex:

Table 3: Relationship between temperature and water density

Temperature °C	Density of water gm/mL
15	0.9991026
16	0.9989460
17	0.9987779
18	0.9985986
19	0.9984082
20	0.9982071
21	0.9979955
22	0.9977735
23	0.9975415
24	0.9972995
25	0.9970479
26	0.9967867
27	0.9965162

Reference:

1. Experiment 1: Volumetric Measurement; Using Micropipettes and Graduated Pipettes Adapted from the CSUS Biochemistry (Chem 162) Lab Manual, Fall 2002.

2. The Science Learning Center at the University of Michigan-Dearborn TROEMNER Pipette Sttandards handbook Raising the standard

3. Tiwari K.B and Ghimire P. (2010) A Practical Handbook for Microbial Genetics and Molecular Biology. First Edition, Kantipur College of Medical Sciences, Sitapaila, Kathmandu.

Pipette Standards Handbook

Pipette Standards Handbook