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Related Concept Videos

Solvents01:12

Solvents

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A solvent is a substance, most often a liquid, that can dissolve other substances. Here, the substance being dissolved is called a solute. When a solvent and a solute combine, they form a solution - a homogenous mixture of both the solvent and the solute. Water is a universal biological solvent. Its polar structure allows it to dissolve many other polar compounds. The ability of water to dissolve is governed by a balance between water molecules binding to each other and binding to the solute.
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Base-Catalyzed Aldol Addition Reaction01:08

Base-Catalyzed Aldol Addition Reaction

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As depicted in Figure 1, base-catalyzed aldol addition involves adding two carbonyl compounds in aqueous sodium hydroxide to form a β-hydroxy carbonyl compound.
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Conjugate Addition (1,4-Addition) vs Direct Addition (1,2-Addition)01:27

Conjugate Addition (1,4-Addition) vs Direct Addition (1,2-Addition)

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α,β-Unsaturated carbonyl compounds with two electrophilic sites, the carbonyl carbon, and the β carbon, are susceptible to nucleophilic attack via two modes: conjugate or 1,4-addition and direct or 1,2-addition.
Conjugate addition results in a thermodynamically stable product. The reaction retains the stronger C=O bond at the expense of the weaker C=C π bond. The process is slow as the β carbon is less electrophilic than the carbonyl carbon.
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Titration in Nonaqueous Solvents01:16

Titration in Nonaqueous Solvents

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Most acid-base titrations are performed in an aqueous medium. In aqueous titrations, water competes with weaker acids or bases for proton donation or acceptance, leading to ambiguous endpoints in the titration curve. Water also affects the partial ionization of weak acids or bases. For example, water accepts a proton from acetic acid to form hydronium and acetate ions. The hydronium ion formed is a stronger acid than acetic acid, and the acetate ion is a stronger base than water. As a result,...
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Conjugate Addition of Enolates: Michael Addition01:08

Conjugate Addition of Enolates: Michael Addition

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The attack of a nucleophile at the β carbon of an α,β-unsaturated carbonyl compound is called conjugate addition. Conjugate addition reactions of active methylene compounds, such as β-diketones, β-keto esters, β-keto nitriles, and α-nitro ketones, are called Michael addition reactions.
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Proteomics01:33

Proteomics

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A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term...
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Sensitivity Enhancement of Soft Capacitive Pressure Sensors Using a Solvent Evaporation-Based Porosity Control Technique
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Enhancing Sensitivity of Microflow-Based Bottom-Up Proteomics through Postcolumn Solvent Addition.

Ute Distler1,2, Mateusz Krzysztof Łącki1, Sven Schumann3

  • 1Institute of Immunology , University Medical Center of the Johannes-Gutenberg University Mainz , Mainz 55131 , Germany.

Analytical Chemistry
|May 24, 2019
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Summary

Researchers enhanced microflow-liquid chromatography-mass spectrometry (LC-MS) sensitivity for proteomic analysis by adding organic solvents postcolumn. This simple method boosts peptide and protein identifications by up to 36% without compromising chromatography.

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Area of Science:

  • Biochemistry and Molecular Biology
  • Analytical Chemistry
  • Proteomics

Background:

  • Advancements in mass spectrometry (MS) enable adaptation of liquid chromatography (LC) for complex proteome analysis.
  • Microflow LC-MS offers a robust, high-throughput alternative to nanoflow LC-MS when absolute sensitivity is not critical.
  • Optimizing the electrospray process is crucial for enhancing microflow LC-MS performance with complex proteomic samples.

Purpose of the Study:

  • To investigate the effects of postcolumn solvent addition on microflow LC-MS electrospray efficiency and sensitivity.
  • To evaluate the impact of different organic solvents (2-propanol, methanol, acetonitrile, DMSO) on proteomic analysis.
  • To determine if postcolumn solvent addition affects chromatographic performance and peptide/protein identification rates.

Main Methods:

  • Microflow LC-MS experiments were conducted using complex proteomic samples.
  • Various organic solvents (2-propanol, methanol, acetonitrile, DMSO) were added postcolumn to the eluting sample.
  • Electrospray efficiency, chromatographic performance, and peptide/protein identification numbers were analyzed.

Main Results:

  • Postcolumn addition of organic solvents significantly enhanced electrospray efficiency in microflow LC-MS.
  • Sensitivity improved across the entire gradient, with up to a 10-fold increase for early eluting peptides.
  • Protein and peptide identifications increased by 28-36% without negative effects on chromatographic performance.

Conclusions:

  • Postcolumn organic solvent addition is an effective strategy to boost sensitivity in microflow LC-MS for proteomic studies.
  • This workflow improves the number of identified proteins and peptides, making it suitable for high-throughput analysis.
  • The presented microflow LC-MS method with postcolumn solvent addition is easily adaptable to existing LC-MS/MS platforms.