What is a pipette?

Pipettes are tools for transferring or measuring small volumes of liquids from one container to another very accurately. Various models of pipettes are available. At first they were made of glass, but today they are also available in the market as plastic pipettes and they have different types. Recently, electrically controlled pipettes have also entered the market. The emphasis of this article is on fixed volume pipettes and variable volume pipettes with mechanical control. We will also talk about the maintenance and calibration of mechanical pipettes and the use of pipettes.

Pipette application

From the use of pipettes, we can mention their use in clinical and research laboratories to remove small volumes of liquids with great precision. These consumable parts are used in accuracy and speed in conducting laboratory tests, which are available in the market in different sizes and types of glass and plastic. Pipettes are usually disposable, but some of them, which are mainly made of glass, can be used more often.

Figure 1) Multi-channel pipette

Figure 2- Single channel pipette

Figure 3) Different components of a pipette

Principles of laboratory pipette operation

Mechanical or piston pipettes usually operate by applying manual pressure to the piston. A piston inside a cylinder with a fixed length moves and directs a certain volume of liquid into or out of the pipette.

There are two types of piston pipettes:

• Pipette with fixed volume:  which moves a fixed volume of liquid (NV: Nominal Volume).
• Pipette with variable volume: which moves different volumes according to the specific characteristics of the pipette.

Adjusting the variable volume on the pipette is done by moving the piston and according to the piston's operating range. In these types of pipettes, the nominal volume is the largest type of volume that the pipette can hold.

Types and applications of fixed and variable pipettes

Fixed and variable pipettes are divided into two types A and B:

Type A (or Air Displacement Pipettes): In this type, there is a volume of air between the piston head and the liquid inside the pipette cylinder.

Type B (Positive Displacement or Direct Displacement Pipettes): In this model, the piston is in direct contact with the liquid. One of the uses of type A pipette is that it is less likely to be contaminated in more uses; But unlike B-type pipettes, they are not accurate enough when they are used to remove very small volumes, because the air in them is condensed and compressed.

Figure 4- Types of pipettes

In order to identify the different volumes on the pipettes, some manufacturers of these parts use color codes. The following table represents the same color codes:

Pipette color code table

Using a pipette

In order to obtain accurate and reliable results, the user must know how to use the pipette correctly and be familiar with the full details of the steps involved. The following recommendations will help you to properly benefit from the use of pipettes:

Warning: Before using the pipette, make sure it is calibrated for volume transfer of liquids.

Recommendations and requirements for the use of pipettes

To use the pipette, the laboratory must be clean and have sufficient light.

The general terms of use are:

• Make sure that the room temperature stays constant at 20 degrees Celsius and the temperature changes ±5 degrees Celsius (between 15-30 degrees).

• Ensure the relative humidity of the room (<50%). Pipettes, samples and liquid materials must be stable under laboratory conditions. It is usually suggested that these materials reach room temperature 2-3 hours before testing.

• If toxic substances or substances that have the possibility of biological hazards are used, use appropriate safety and protective equipment.

• Make sure the pipette is in a vertical position in the solution. This mode reduces the inaccuracy in transmission!

• In general, you should not keep them under direct sunlight so that you can use the pipettes properly.
• Be aware of the manufacturer's recommendations for the minimum immersion depth of the head sampler and pipette in the liquid. This value varies according to the type and capacity of the pipette. A general guide is provided in the table below for this purpose.

The table of the minimum placement capability of the head sampler according to the capacity of the pipette volume

• After filling the head sampler, clean the drops on the tip of the head sampler. For this purpose, slowly touch the tip of the head sampler to the wall of the test tube. The use of absorbent paper may be necessary, in this case more care should be taken to prevent possible contamination.

• Empty the solution inside the head sampler into the test tube. Do this by touching the tip of the sampler to the wall of the test tube. The tip of the head sampler should be in contact with the pipe wall at an angle of 30-45 degrees and 8-10 mm above the liquid level.
• Moisten sampler heads to increase pipette accuracy. For this purpose, take a small amount of working solution several times and empty it into the waste container. This prevents the formation of air bubbles, especially in thick solutions or hydrophobic materials. It also causes uniform wetting of the inner wall of the head samplers and the piston. This action is not necessary for the use of pipettes with a volume of 10 µl or less.

The correct way to use and apply the pipette

Below are the common procedures for using type A mechanical pipettes. The user should pay attention to the manufacturer's recommendations. These steps are also used in the use of electric pipettes. Figure 5 explains these steps.

Figure 5- The steps of using a pipette

1. Connect a new head sampler according to the specifications of the pipette. Avoid contaminating the head sampler with other materials and make sure the connection of the head sampler is tight.
2. Push the piston to the first stop position (First Limit). In this position, the tip of the head sampler should not contact the liquid surface.

3. Place the head sampler tip in the liquid according to the guide table or use the manufacturer's recommendations and keep the pipette completely vertical. This corresponds to position B1 (first from left) in the figure.

4. Release the piston slowly and completely to remove the liquid (position A2). Wait at least 2 seconds before removing the head sampler from the liquid.

5. Hold the tip of the head sampler in the test tube at an angle of 30-45 degrees to the inner wall of the tube. If the test tube that receives the liquid contains a solution, avoid contact of the tip of the sampler with the solution inside the test tube (position A3).

6. Empty the contents of the head sampler by pushing the piston to the stop position (position B4). In all steps, make sure that the tip of the head sampler and the inner surface of the liquid receiving tube are in contact. Slowly move the tip of the head sampler on the inner surface at a height of 8-10 mm from the tube head so that no drop remains on the tip of the head sampler.
7. Push the piston until it reaches the second limit position (C5 position). This empties the remaining drop inside the head sampler by expelling the air. Keep the pipette in this position (position C5) until it is removed from the test tube. As soon as you remove the pipette from the test tube, return the piston to its original position.
8. Discard the head sampler by pressing the button on the end of the pipette (position 6).

Note: If variable pipettes are used, the required volume should be selected according to the manufacturer's instructions. There is usually a volume control button on the top of the pipette. It is necessary to recognize and familiarize the user with the scales for the correct use of pipettes.

Proper maintenance of the pipette to increase the use of the pipette

The usual and correct method of maintaining mechanical pipettes is given below, which includes parts of evaluation, cleaning (decontamination), calibration, as well as materials and resources related to them.

Evaluation and check

• Time period: Daily

Daily evaluation of pipettes is necessary to find failure, damage or to ensure their proper functioning. This assessment should include the following:

1. Make sure it is set and perfect. The piston should move smoothly and evenly.
2. Check the head sampler holder so that it is not damaged because it is important for the accuracy of the measurement. Make sure that the head sampler is strong.
3. Attach a head sampler to the pipette and fill it with distilled water. The pipette should not have any leakage.

Cleaning and decontamination of pipettes

1. Every day, the pipette should be cleaned with a suitable solution or detergent. The choice of detergent should be based on the manufacturer's recommendations.

2. Sterilize the pipette according to the manufacturer's recommendations. Some pipettes are sterilized in an autoclave ( read about autoclave ) at a temperature of 121 degrees Celsius for approximately 20 minutes. In some types of pipettes, it is necessary to disassemble its components so that the steam reaches the inner parts of the pipette. Pipettes should be opened according to the manufacturer's instructions. This work is possible by using a series of tools and wrenches that are provided when the pipette is sold. After sterilization, the pipette components should be reassembled at room temperature. Make sure that the pipette components are dry before doing this. To sterilize the pipettes, some manufacturers suggest using 60% isopropanol solution, washing with distilled water, drying and then connecting the parts.

3. Pay attention to the use of pipettes. If pipettes are used to transfer corrosive or harmful substances, the user is responsible for cleaning and disinfecting them before using them again or removing them from the laboratory work steps. It is suggested to prepare a report in which the type, model, serial number and contaminating materials and materials used after cleaning the pipette are recorded in it.

Figure 6- Steps to open the components of a pipette

Maintenance ( very important in the use and application of the pipette)

• Time period: twice a year

The performance of the pipette that is used daily should be checked as follows:

1. Open the pipette components according to the instructions in the manual. These steps are different according to the model and manufacturing company, but its main body is opened by a screw.

2. Clean the gaskets, piston and inside of the cylinder before oiling. If internal components are accidentally contaminated, all surfaces should be cleaned with normal washing-up liquid and distilled water .

3. If it is necessary to replace the gaskets, they should be replaced with the same gaskets with the same specifications as the original type. The type of washers is different depending on the types and application of pipettes.

4. Lubricate the piston with special pipette silica oil. Try to use the oil recommended by the manufacturer. Remove excess oil with absorbent paper.
5. Reverse the steps to mount the pip parts.
6. Calibrate the pipette before use.

Laboratory pipette calibration

The calibration of pipettes is done with the standard method and generally depends on its volume; The smaller the volume, the more expensive the calibration process will be. A brief description of the calibration steps for 20μl and 1mm pipette volumes is described below:

Necessary pipetting materials and tools

• Analytical balance

• Electronic thermometer with a resolution of 0.1 degrees Celsius or more and with a suitable temperature range and the ability to be placed in water

• A hygrometer with a standard uncertainty of 10% or less

• Pressure gauge with standard uncertainty of 0.5 KPa or less

• timer

• Micropipettes with variable volumes

• Disposable head samplers of different sizes

• Vials with flat bottoms

• Distilled water that has been distilled 2 or 3 times.

• Trained and reliable user

Accuracy in the use and application of the pipette by adjusting the calibration period

First of all, you need to know that every three years you should calibrate the laboratory pipette for correct and standard use. These steps are based on measuring the net weight of water distributed by a pipette in an empty vial and dividing it by the density of water. In practice, to prepare a series of readings, the weight of each suction (after correction by temperature, pressure and evaporation factors) is precisely recorded. With this, you can do the following:

• Comparing different types of pipettes to check their differences 

• Checking the precision of a pipette

• Checking and controlling the accuracy and precision of measuring some pipettes

• Controlling the effective factors in the use of a pipette by different users according to the application of the pipette and its types

Procedures for using a mechanical pipette

The following steps apply to mechanical pipettes:

1. Connect a new head sampler to the sampler.

2. Pipet the distilled water and empty it into another container; Repeat this at least 5 times until the air humidity inside the pipette is fixed.

3. Pour water into the weighing container until the water level reaches at least 3ml.

4. Record water temperature, ambient pressure and relative humidity.
5. If possible, close the lid of the weighing container and weigh.

6. Record the weight that the display shows on the laboratory scale or set it to zero.

7. Pipette the water inside the storage container and pour it into the weighing container. This is the same as using a pip every day. (Refer to step 7 of the correct method of using the pipette)
8. Record the new weight on the screen.
9. Repeat steps 7 and 8 nine more times and record the new weight at the end of each step.
10. Record the temperature of the water inside the weighing container at the end of step 10 and record the duration of the steps.
11. Assess the amount of water evaporation (this step is very important for very small volume pipettes). If working with these pipettes, an extra time equal to the time needed to perform steps 1-10 should be spent to be able to use the pipette and at the end a new reading should be recorded.
12. The weight of water evaporated during the extra time is divided by the total number of readings (10 readings). The obtained number is the average amount of water evaporation in each stage, which should be added to each of the readings.

Calculations (the most important use of the pipette)

Proceed using the following steps:

• Calculate the weight of the water that was poured into the weighing container by the pipette each time. For this, subtract the new reader from our penultimate reader (for each reader). If necessary, add the average amount of evaporated water to it.

• Convert the numerical value of each weight to a volume at 20°C. (weight divided by the density of water at 20 degrees Celsius)
• Calculate the average volume calculated in step 2 (the sum of the read volumes divided by the number). Apply the desired settings such as air pressure to weight (buoyancy). To do this, multiply each reading of the weight by the coefficient of the correction factor.

• Calculate the standard deviation.

• Calculate the deviation coefficient percentage.

Your frequently asked questions about solving problems with pipettes

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