Recent advances in analytical techniques for high throughput experimentation

Abstract

High throughput experimentation is a growing and evolving field that allows to execute dozens to several thousands of experiments per day with relatively limited resources. Through miniaturization, typically a high degree of automation and the use of digital data tools, many parallel reactions or experiments at a time can be run in such workflows. High throughput experimentation also requires fast analytical techniques capable of generating critically important analytical data in line with the increased rate of experimentation. As traditional techniques usually do not deliver the speed required, some unique approaches are required to enable workflows to function as designed. This review covers the recent developments (2019-2020) in this field and was intended to give a comprehensive overview of the current "state-of-the-art."

Keywords: high throughput analysis; liquid chromatography; mass spectrometry; supercritical fluid chromatography; ultrafast analysis.

FIGURE 1

Modeling and initial studies using dyes from microplates spotted on TLC plates using a staggered spotting approach. A, Excel modeling showing “sweet spot” of Rf ∼0.5 for most effective visualization. B, Elution of spots from a 96‐well plate containing samples of methylene blue (Rf ∼0) and methyl red (Rf ∼0.6) using 95% ethanol/water. C, Analysis of plate containing four wells spiked with additional methyl red allows easy determination of the “address” of the hits. Reprinted with permission from J. Org. Chem. 2020, 85, 15, 9447–9453. Copyright 2020 American Chemical Society

FIGURE 2

Sub second HILIC separations of structurally and functionally related analytes on bare 1.9µm SPP silica packed in 1.0 × 0.3 cm i.d. columns at flow rates of ∼8 mL min. Reprinted with permission from Anal. Chem. 2018, 90, 5, 3349–3356. Copyright 2018 American Chemical Society

FIGURE 3

Kinetic plot showing the potential gain in analysis time by increasing the pressure limit for a number of common particle diameters. Reprinted with permission from Anal. Chem. 2020, 92, 1, 554–560. Copyright 2020 American Chemical Society

FIGURE 4

Separation of enantiomers of 13‐HOTrE, 12‐HETrE, 14‐HDoHE, and 5,6‐DiHETE. Blue line shows chromatogram from a corresponding oxidized PUFA and red trace shows chromatogram from a pure standard (in case of 13‐HOTrE, 12‐HETrE and 14‐HDoHE standards were pure S enantiomers and 5,6‐DiHETE was a racemic mixture of cis‐ enantiomers). Reprinted with permission from J Chromatogr A, 2020. 1624: p. 461206. Copyright (2020) Elsevier Publishing

FIGURE 5

Experimental chromatograms, using conditions modeled in silico, depicting the SPP (A) and FPP (B) columns. These gradients are both at 3 mL/min flow rate. For the SPP column 50% acetonitrile was held for 1 min and increased to 100% acetonitrile by 1.2 min. For the FPP column 50% acetonitrile was held for 1.5 min and increased to 100% by 1.7 minu. Note the overall faster runtime for the SPP column. Reprinted with permission from J. Chromatogr. A, 2020. 1628: p. 461‐432. Copyright 2020 Elsevier Publishing

FIGURE 6

Typical ultrafast NOTLC separations. A, NOT column: 2‐µm‐i.d. × 6 cm‐length (2.7 cm effective) coated with C18; MA: 10 mM NH₄HCO₃; MB: 50% acetonitrile in 10 mM NH₄HCO₃; mixer: mixer 2 in (C); Elution pressure: 23 MPa; Injected sample volume: ∼120 pL. Sample: 1 µM gly, 3 µM tyr, 3 µM ala, 3 µM arg, 10 µM trp and 2.5 µM phe. Reprinted with permission from Xiang, P.; Yang, Y.; Zhao, Z.; Chen, M.; Liu, S. Anal. Chem. 2019, 91, 10738−10743. Copyright 2019 American Chemical Society

FIGURE 7

Turbulent flow SFC open tubular column separation of 7 polycyclic aromatic hydrocarbons. Reprinted with permission from Anal. Chem. 2020, 92, 11, 7409–7412. Copyright 2020 American Chemical Society

FIGURE 8

Identification of novel unspecific peroxygenase chimeras and unusual YfeX axial heme ligand by a versatile high‐throughput GC‐MS approach. Reprinted with permission from ChemCatChem, 2020. 12(19): p. 4788‐4795). Copyright (2020) Wiley‐VCH Verlag GmbH & Co KGaA

FIGURE 9

Schematic diagram of the low thermal mass fast GC module installed on an available FID port atop an Agilent 7890 GC configured for GC‐MS. Note the capillary column combination in fast LPGC‐MS operation. Reprinted with permission from J. Chromatogr. A, 2020. 1612: p. 460691. Copyright (2020) Elsevier Publishing

FIGURE 10

Fast GC‐FID analyses (top) of a test mixture of 10 ng/ µL each of n‐C 16 H 34 , methyl stearate, cholesterol, and n‐C 32 H 66 injected with 9:1 split (1 ng each on column). The fast GC‐FID trace is compared with GC‐MS analysis of the same test mixture (bottom) using a standard 30 m, 0.25 mm i.d. column. The 34 min GCMS analysis was reduced to ≈1 min full analysis cycle time using the fast GC‐FID method. Reprinted with permission from J. Chromatogr. A, 2020. 1612: p. 460691. Copyright 2020 Elsevier Publishing

FIGURE 11

Decision tree for determining if a small molecule reaction mixture is suitable for MALDI analysis. Reprinted with permission from Tetrahedron, 2020. 76(36): p. 131434. Copyright (2020) Elsevier Publishing

FIGURE 12

A, Liquid AP‐MALDI extracted ion chromatogram (m/z 530.79) of 96 sample wells with bradykinin as analyte (1 µL total sample volume spotted, 25 pmol analyte on target) at 5 mm/s stage movement speed. B, Mass spectrum of all scans acquired for the first sample. C, Mass spectrum of all scans acquired for the last sample. Reprinted with permission from Anal. Chem. 2020, 92, 2931‐2936 (https://pubs.acs.org/doi/abs/10.1021/acs.analchem.9b05202). Copyright 2020 American Chemical Society. Further permissions related to the material excerpted should be directed to the ACS

FIGURE 13

Heat map of 1536 reactions (768 in droplet/thin film and 768 in bulk microtiter at three time points) from round 2 of the SNAr HTE using MeOH with 1% FA as the DESI spray solvent and NMP as the reaction solvent. (A) Droplet/thin film and bulk microtiter at 150°C. (B) Droplet/thin film and bulk microtiter at 200°C. Blue cells represent successful reactions (average product intensity ≥ 150 counts). Red cells represent unsuccessful reactions (average product intensity < 150 counts). Reprinted with permission from ACS Comb. Sci. 2020, 22, 4, 184–196. Copyright 2020 American Chemical Society

FIGURE 14

Acoustic droplet ejection‐open port interface. Reprinted with permission from Med. Chem. Lett. 2020, 11, 6, 1101–1110. Copyright 2020 American Chemical Society

FIGURE 15

Illustration of microfluidic elements that enabled stable analysis of droplets at nL/min flow rates. (A) Overview of entire droplet generator and nESI‐MS system. From left to right, pictured are the syringe pump for driving flow, which leads to the droplet generation chip on a microscope for monitoring droplet formation, followed by transfer to nESI‐MS analysis. (B) Conventional microfluidic droplet generator device setup with 750 µm o.d. inlet and outlet tubing interfaced perpendicular to microfluidic channels. (C) Modified droplet generator with insertion of 150 µm o.d. inlet and outlet capillaries in‐line with microfluidic channels. For (B,C), blue arrows indicate inlet lines, whereas red arrows show outlets. Channels had widths and heights of 100 µm and were filled with green food color to aid in visualization. Reprinted with permission from Anal Chem, 2019. 91(10): p. 6645‐6651. Copyright 2019 American Chemical Society

FIGURE 16

Conceptual representation of a droplet microchip design. Reprinted with permission from Anal. Chem. 2020, 92, 18, 12605–12612. Copyright 2020 American Chemical Society

FIGURE 17

Total ion chromatogram (TIC) acquired during infusion of droplets (∼2.1 nL) containing leucine enkephalin (LeuEnk, ∼1.3 mM solution) at an infusion rate of approximately 5 droplets/s (Hz). Each individual peak indicates one droplet reaching the Agilent 6560 IM‐Q‐TOF detector. Mass spectrum (m/z range 500−600) acquired from one droplet containing LeuEnk ([LeuEnk + H]+ = 556.27 Da. Reprinted with permission from Anal. Chem. 2020, 92, 18, 12605–12612. Copyright 2020 American Chemical Society