*3.4.4. Pipette behavior affecting dispensing variation*

Most pipettor systems provide pre- and post-air aspiration functions to ensure accurate liq‐ uid transfers. Introduction of air into the tips before or after the aspiration of the sample liq‐ uid is recommended to improve volume accuracy by forcing all the liquid out of the tips. In a study performed to optimize the automated parameters to achieve a 10 µL transfer volume in a sequential transfer experiment, introduction of a 5 µL pre-air gap significantly reduced the relative volume inaccuracy along with the CV of the final transferred volume in a 96 well plate (Albert et al. 2007). While this method may help to achieve more precise results especially with small volume transfers, bubble formation in the destination wells may be in‐ evitable unless proceeded by a shaking or centrifugation step. Post-air aspiration may also be applied to create an air gap between liquids, preventing unsought contamination in the source reservoirs when multiple samples are picked up sequentially into a single tip before the delivery into the destination reservoir.

When small and repetitive volume transfers into multiple destinations are needed, it is a common practice to pick up a single large volume and deliver smaller amounts in a sequen‐ tial mode. However, with this method, it is hard to achieve accurate delivery in each step. In a study of multi-sequential dispense accuracy, it was shown that the first and last dispense steps led to relatively higher and lower transferred volumes, respectively, in addition to in‐ creased relative inaccuracy (Albert et al. 2007). Therefore, it is recommended to dispense the first and last steps into the source reservoir to enhance the precision in the destination plate. Delivery performance of the dry versus pre-wetted tips may also exhibit differences in vari‐ ability depending on the sample characteristics.

Droplet formation at the end of the pipette tips after a dispense action remains an issue for liquids with high viscosity or low densities. Besides the selection of the optimum dispense speed, a "tip touch" function is a useful feature offered in some automated pipettors, where the tips contact the well wall at the end of a dispense step to force the release of the droplet. The path of the moving pipetting arm across the deck should be carefully determined to re‐ duce the chance of contaminating other labware by hanging droplets.

Proper mixing of solutions in the source reservoir before aspiration and in the destination reservoir after dispensing may greatly affect the final assay quality due to the necessity of uniform sample concentrations. To avoid the formation of concentration gradient in wells, mixing can be performed by repetitive pipetting cycles. Mixing of the well contents by pi‐ petting up and down is proven to be a quicker and more efficient method compared to free diffusion or shaking, which are not as successful due to the correlations between well size, content volume and the exerted capillary forces (Berg et al. 2001; Shieh et al. 2010; Travis et al. 2010). Mixing is necessary when dealing with suspensions (cells, beads, etc.). For in‐ stance, cell settling creates uneven cell density in the source reservoir, which would lead to aspiration of decreasing number of cells over time (Fig. 2C)

#### *3.4.5. Routine quality assessment*

influenced by the tip shape and the sample-polypropylene interactions, the removal speed

Impurities can also leach out of the disposable tips when in contact with solvents such as DMSO. Studies have shown that bioactive compounds released from plastic labware may interfere with assay readouts causing misleading experimental results (McDonald et al. 2008; Niles and Coassin 2008; Watson et al. 2009). Consumable materials, especially polypro‐ pylene tips, tend to adsorb certain compounds, leading to unreliable concentrations in the destination plates (Harris et al. 2010). Therefore, it is recommended to test and validate the influence of consumables on an assay during assay development and whenever there is a

Pipetting viscous and "sticky" samples is challenging due to bubble formation. Among the most important parameters to consider in avoiding these issues are the speed that the tips exit the sample fluid and the aspirate/dispense rates; they should be slow enough to avoid residu‐ als at the inside and outside of the tips. Pre- and post-air pipetting options should be avoided.

Most pipettor systems provide pre- and post-air aspiration functions to ensure accurate liq‐ uid transfers. Introduction of air into the tips before or after the aspiration of the sample liq‐ uid is recommended to improve volume accuracy by forcing all the liquid out of the tips. In a study performed to optimize the automated parameters to achieve a 10 µL transfer volume in a sequential transfer experiment, introduction of a 5 µL pre-air gap significantly reduced the relative volume inaccuracy along with the CV of the final transferred volume in a 96 well plate (Albert et al. 2007). While this method may help to achieve more precise results especially with small volume transfers, bubble formation in the destination wells may be in‐ evitable unless proceeded by a shaking or centrifugation step. Post-air aspiration may also be applied to create an air gap between liquids, preventing unsought contamination in the source reservoirs when multiple samples are picked up sequentially into a single tip before

When small and repetitive volume transfers into multiple destinations are needed, it is a common practice to pick up a single large volume and deliver smaller amounts in a sequen‐ tial mode. However, with this method, it is hard to achieve accurate delivery in each step. In a study of multi-sequential dispense accuracy, it was shown that the first and last dispense steps led to relatively higher and lower transferred volumes, respectively, in addition to in‐ creased relative inaccuracy (Albert et al. 2007). Therefore, it is recommended to dispense the first and last steps into the source reservoir to enhance the precision in the destination plate. Delivery performance of the dry versus pre-wetted tips may also exhibit differences in vari‐

Droplet formation at the end of the pipette tips after a dispense action remains an issue for liquids with high viscosity or low densities. Besides the selection of the optimum dispense

should be slow enough to diminish droplet generation.

*3.4.4. Pipette behavior affecting dispensing variation*

the delivery into the destination reservoir.

ability depending on the sample characteristics.

change in labware.

*3.4.3. Foaming*

190 Drug Discovery

Verification of transferred volumes and routine quality control (QC) are the most important and inevitable processes when working with liquid handling devices. While the verification method should be reliable enough to quantify the pipettor performance, it should also be easy and fast to be applied routinely. The performance assessment described for bulk liquid dispensers (section 3.2.7.) can also be applied to pipettors as long as the same volume is dis‐ tributed throughout the plate for %CV and %bias' calculations.

As mentioned previously, liquid handlers are heavily used to perform serial dilutions, and suitable QC techniques should be employed to validate dilution performance, particularly when accurate compound potency is directly dependent on concentration accuracy. Dilution ratio, accuracy, precision and outlier distribution constitute the four major criteria that should be evaluated (Popa-Burke et al. 2009). Artel developed an approach to determine di‐ lution and transferred volume accuracy by using dual-wavelength photometry, where two absorbance dyes with baseline resolved spectra are mixed at various ratios using a liquid handler (Albert 2007; Dong et al. 2007). This dual-dye ratiometric method can be applied by using a multichannel verification system (MVS) equipped with the necessary instrumenta‐ tion and analysis (Bradshaw et al. 2005). Dual-dye photometry is also proven to be suitable to measure the efficiency of different mixing methods (Spaulding et al. 2006) and when pi‐ petting non-aqueous solutions (Bradshaw et al. 2007).
