Dilution of liquids within a scientific protocol can be utilised to produce a standard curve for quantification or ensure a sample is within the linear range of detection for an analytical assay.

This technical note outlines a workflow where samples were serially diluted to produce a linear dilution range using the Dilute element. The workflow was executed on three liquid handlers: a Gilson PIPETMAX 268, a Hamilton Microlab STAR and a Tecan Freedom EVO.

This technical note has been used to validate liquid handling accuracy for the volumes of liquid (tartrazine) transferred for preparing serial dilutions, and provides a starting point for liquid handling optimization should it be required.

Within this workflow (Figure 1), a linear serial dilution of 150 µM Tartrazine was executed using reverse osmosis (RO) water as a diluent.

The six-point serial dilution was prepared in a 96-deep well plate sampling concentrations between 0 - 150 µM (0 µM, 30 µM, 60 µM, 90 µM, 120 µM and 150 µM) to a final volume of 520 µl, before transferring 100 µL to a 96-well assay plate for spectrophotometric measurement. To demonstrate intra- and interplate variability, the serial dilution and aliquots were replicated twelve times over two plates (six replicates per plate) (Figure 2). This workflow was executed over three devices, namely a Gilson PIPETMAX 268, a Hamilton Microlab STAR and a Tecan Freedom EVO.

Figure 1. Dilute workflow in Synthace. This workflow describes the method for diluting liquids to two plates, which are then aliquoted to two assay plates for measurement. The liquids used to make the required serial dilution are defined in the Define Liquids and Plates elements. The Describe layouts element is used to describe the plate layouts for both the intermediate dilution plates and the final assay plates. The Dilute element defines the linear dilution series required and the Aliquot element defines the dilution transfers to be made on the assay plate before analysis.

Figure 2. Execution Details Preview in Synthace. Users can simulate, preview and verify their experiments in silico prior to physical execution, demonstrated here on the Tecan Freedom EVO.

Labware information and estimated run times for each of the liquid handling devices are shown in Table 1. The workflow was run on all three devices on the same day.

Tartrazine absorbance was measured using a BMG Labtech CLARIOstar microplate reader at 425 nm (Tartrazine 𝜆max) and baseline subtraction performed with absorbance at 620 nm. Data was processed and graphs were generated by a python script in Jupyter Notebook and can be accessed from the Downloads section at the end of this article.

Table 1: Liquid handling information

Independent measurements, and intra- and inter plate variability for diluting tartrazine with twelve independent replicates along with linear model fits were calculated for samples prepared across the different devices tested (Figure 3).

Figure 3. Absorbance data for serial dilutions prepared across three devices. (A) Corrected absorbance values for the dilution series replicates of tartrazine on each plate were fitted with a linear regression to assess the linearity of the standard curves across the three devices tested. The coefficient of variation (% CV) for each set of concentration replicates on each plate, (B) intraplate variation, and across both plates, (C) interplate variation, are shown. The zero tartrazine concentration data points have been excluded from the % CV analysis. Dashed horizontal lines represent Synthace internal validation standards for the % CV maximum thresholds.

Each set of six standard curves created per plate on each device provided a correlation coefficient r2 value > 0.999 and each curve rejected the null hypothesis that the slope between x and y is equal to 0 with the level of statistical significance being p < .001. The average intraplate coefficient of variance (CV) across all concentrations in the serial dilution range was 1.11% (Gilson PIPETMAX 268), 1.43% (Hamilton Microlab Star), and 0.64% (Tecan Freedom EVO). The average interplate CV across all concentrations in the range was 0.60% (Gilson PIPETMAX 268), 0.99% (Hamilton Microlab Star) and 0.36% (Tecan Freedom EVO).

The following file contains the workflow that was discussed in this technical note. To download the file, right-click the button, then select Save as.

After you download the file, complete the following steps.

The following file contains the raw data that was discussed in this technical note. To download the file, right-click a button, then select Save as.

If you want to process the raw data in the way that was discussed in this technical note, download the Jupyter Notebook or Python scripts. You must edit the scripts to point at the directory that contains the raw data.