Volatile liquid handling with OT-2 optical center

Author Morayo Adeyibi, Ph.D.

Introduction Volatile liquids, i.e. liquids that evaporate easily at room temperature, are common in the application of various reagents such as ethanol, isopropanol, acetone and methanol, especially for various nucleic acid purification methods [1]. As temperature increases, the fluctuation rate (or evaporation rate) increases exponentially [2]. Even at room temperature, they are so difficult to use with pipettes, and the most common problem is dripping from the tip. The continuous evaporation of liquids creates challenges in pipetting volatile liquids. The higher vapor pressure causes the liquid to evaporate quickly and the liquid to eject out of the tip faster. Volatility In a liquid, an increase in temperature increases the volatility of the liquid, and the subsequent evaporation rate and vapor pressure. On the contrary, diluting the liquid reduces the volatility and the corresponding characteristics. Here we present optimization of liquid handling of typically automated volatile liquids, including ethanol and isopropanol at various dilutions.

Key Volatile Liquid Handling Techniques Prewetting Tips Mixing () Prewetting is a pipetting strategy that repeatedly aspirates and dispenses a volume of liquid. The high vapor pressure of the volatile liquid causes the air gap surface between the nozzle and the liquid to expand, forcing them to drip out of the tip. Prewetting saturates the evaporating solvent in the air gap between the nozzle and the liquid surface to help maintain low vapor pressure.

Tailspace Gap Aspiration () We can allow air to exist in the tip hole to avoid overflow. The air gap is determined by the pressure of the vapor liquid, the volume of liquid being aspirated, and most importantly by the time it takes for the tip to move from the deck position where it is aspirated to the dispensing position. Most volatile liquids are less dense than water (<997 kg/cubic meter). When air is inhaled following a volatile liquid, the liquid within the tip accelerates upward, which can cause the filter or pipette tip to become clogged. To avoid this problem, the tail air gap flow velocity must be kept very low. Liquids with higher vapor pressure, such as acetone, ether, etc. We can see the "wine tears" effect [5], where a ring of liquid passes through the air gap behind, creating an empty space between the sucked liquid and the liquid hole further dripping from the tip.

Tip exit velocity moves to () We can allow air to exist in the tip hole to avoid overflow. The air gap is determined by the pressure of the vapor liquid, the volume of liquid being aspirated, and most importantly by the time it takes for the tip to move from the deck position where it is aspirated to the dispensing position. Most volatile liquids are less dense than water (<997 kg/cubic meter). When air is inhaled following a volatile liquid, the liquid within the tip accelerates upward, which can cause the filter or pipette tip to become clogged. To avoid this problem, the tail air gap flow velocity must be kept very low. Liquids with higher vapor pressure, such as acetone, ether, etc. We can see the "wine tears" effect [5], where a ring of liquid passes through the air gap behind, creating an empty space between the sucked liquid and the liquid hole further dripping from the tip.

Postallocation delay protocol. There are two factors that are critical to accurately dispensing volatile liquid. First, due to the lower surface tension, the liquid sticks to the inside of the tip surface and slowly moves toward the tip hole as the remaining liquid forms. Second, the alcohol ring is dispensed. Or the well-known wine tears can cause liquid to stick to the surface of the liquid and slide towards the tip hole, creating dead volume. Adding a post-allocation delay can reduce dead capacity.

DoubleBlout() can be done by adding one more blowout. To perform a second blow, aspirate 1 µL of air by withdrawing the tip from the liquid (into or out of the liquid). Suction of 1µLair allows the plunger to maintain its original position in the suction state with a smaller displacement.

Optimized Strategy for Volatile Liquids Step 1 Hover to the top of the aspirator and soak the tip at a slower rate.

Step 2 Dip the tip into the liquid and pre-wet it. Choose the number of prewetting steps based on the volatility of the liquid. mix() in the Python API allows you to mix a liquid "n" times.

Step 3 Aspirate() the volatile liquid at the water calibrated flow rate.

Step 4 For most volatile liquids, the tip recovery speed must be reduced to 50 mm/s. This avoids liquid splashing on the top of the lab wallsmove_to() 50 mm/s.

Step 5: Adjust the air gap volume according to the steam pressure. pipette.flow_rate. The suction of the air gap () must maintain 1/20 times the water flow rate of the respective suction pipe.

Step 6 At a water calibrated flow rate, dispense the volatile liquid with the tip immersed 2.5mm below the liquid. This prevents dead capacity from increasing.

Step 7 Add protocol. The delay() time is 3 - 4 seconds depending on the vapor pressure of the liquid. Adding a delay allows fluid to settle at the tip before blowing out.

Step 8 Perform pipetting. At water calibrated blowout flow, blowout().

Step 9 (optional) If only, after blowing out the excess liquid left inside the tip for the first time, use mote_to(ok.top()) and suction() 1µLof of air and soak the tip (move to() ) in the liquid and perform a second blowout.

Step 9 If necessary, perform touch_tip() at a location determined by the necessary height within the labware.

Step 10 Remove the tip and move it to (ok. top()) at a slower flow rate of 50 mm/s.

Materials and Methods Three dilutions of ethanol, 70%, 80% and 99% (SigmaAldrich, catalog number 752X, E-030, 1.07017) were tested quantitatively by volume as a representative volatile liquid and compared with water. Compare. Weight measurements were performed using a microbalance (Radwag, XA6/21.4Y.M.A.P plus microbalancer). Volume testing was performed on 4 center GEN2 P20, P300 and P1000 pipettes. Vision centers P20 non-filter, P300 non-filter and P1000 non-filter tips are used for pipetting volatile liquids. All liquids were tested at an ambient temperature between 22˚ and 25˚ and a relative humidity of 50% - 60%. Reduce the maximum aspiration volume of each pipette to compare air gap effects. The optimization parameters listed in Table 1 were used for volume testing on weight settings.

All parameters are programmed using a central Python API using an in-house pipette validation protocol. The tip remained outside the liquid for approximately 15 seconds. Collect 10 replicates per volume of pipette using 4 different pipettes. The target volumes for Gen2 P20 pipettes are 1µL, 10µL, 15µL and 18µL. Target volumes for Generation 2 P300 are 20µL, 100µL and 200µL. Target volumes for Generation 2 P1000 are 100µL, 500µL and 950µL.

Results and Discussion To measure the performance ability of the OT-2 pipette to accurately handle the most commonly used representative volatile liquids, experimental correlations were studied at dilutions of ethanol, isopropanol, acetone, and methanol. Ethanol and isopropyl alcohol have similar vapor pressure ranges, so we used similar shift parameters.

Prewetting increases the volume within the pipette because the prewetted volume adheres to the tip surface after the prewetting action is complete. The benefit of prewetting can be seen as reduced dripping of volatile liquids. Without prewetting, higher concentrations of ethanol, especially 99% ethanol, are more likely to drip quickly even with trailing air gap aspiration. This results in contamination of the surrounding deck and failure to achieve target allocation volumes.

Although inhalation use cases are limited to <10 µL of volatile liquids, aspiration and dispensing can present challenges. Prewetting of these low volumes may result in over-aspiration. However, when using P20, a dead volume of ~1.5 µL was observed for all volumes tested. The dead volume consists of the evaporated liquid in the tip and the remaining liquid stuck to the tip.

Reverse pipetting, where the liquid is pre-aspirated to full volume without a target volume, can also affect accuracy for liquids with high vapor pressure. Higher air gap volumes can help reduce this effect. Since the amount of liquid that needs to be dispensed due to the loss of air gap volume is not known, reverse pipetting may lead to additional inaccuracies.

Conclusion The accuracy of the GEN2 P300 and P1000 is similar to the performance of water versus pipettes. The impact of the pre-wetting step can lead to over-aspiration. In cases where the P20 is used for volatile liquid handling, it is recommended to pre-evaluate the required target volumes for the protocol and to take into account a 1 to 2 µL dead volume, please contact our Support making appropriate recommendations based on your agreement. All optimization parameters are specific to the environmental factors in question, and any changes in temperature or humidity may affect the parameters in question. When dealing with liquids with higher vapor pressure, greater than 30 kPa depending on the ambient temperature, we recommend increasing the prewetting mixture (>2 times), increasing the appropriate air gap volume and reducing the flow rate to achieve effective liquid transfer. Dead volume depends on the vapor pressure of the volatile liquid, the volume of the pipette (e.g. P20, P300 or P100), tip size and depth of tip immersion when dispensing. Soaking the tip <2.5 mm during dispensing will reduce dead volume. Liquids with lower vapor pressure and higher density have smaller dead volumes. If contacts or blowouts are not required in the protocol, it is recommended to consider the target volume in terms of dead volume. The results showed that the volatile liquid handling performance was optimized after the volatile liquid handling parameters. Users will be able to customize parameters based on the specific volatile liquid used in the protocol.