What Is 2-cyano-4-hydroxycinnamic acid

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Last updated: April 15, 2026

Quick Answer: 2-Cyano-4-hydroxycinnamic acid (CHC) is a synthetic derivative of cinnamic acid used primarily in mass spectrometry as a matrix in MALDI analysis. It has the molecular formula C10H7NO3 and is valued for its ability to ionize large biomolecules. Its use dates back to the 1990s in analytical chemistry applications.

Key Facts

Molecular formula: C10H7NO3
Used as a MALDI matrix since the early 1990s
Absorbs UV light at 337 nm, matching common laser wavelengths
Enhances ionization of peptides and small proteins
Typically dissolved in acetonitrile or ethanol with trifluoroacetic acid

Overview

2-Cyano-4-hydroxycinnamic acid (CHC) is a specialized organic compound widely used in analytical chemistry, particularly in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It functions as a matrix that absorbs laser energy and facilitates the ionization of analyte molecules without significant fragmentation.

CHC is a derivative of cinnamic acid with a cyano group at the 2-position and a hydroxyl group at the 4-position of the phenyl ring. This specific substitution pattern enhances its ability to absorb ultraviolet light and interact with biomolecules such as peptides and small proteins.

Molecular weight: The compound has a molecular weight of 189.17 g/mol, making it suitable for vaporization and ionization under MALDI conditions.
Solubility: It is typically dissolved in acetonitrile-water mixtures containing 0.1% trifluoroacetic acid to ensure proper crystallization with analytes.
UV absorption: CHC strongly absorbs at 337 nm, which matches the wavelength of nitrogen lasers commonly used in MALDI instruments.
Ionization efficiency: It enables efficient protonation of peptides between 500 and 5,000 Da, improving detection sensitivity in mass spectrometry.
Crystallization behavior: CHC forms homogeneous microcrystals with analytes, reducing spot-to-spot variability during sample analysis.

How It Works

CHC functions by absorbing laser energy and transferring it to analyte molecules, promoting ionization while minimizing degradation. Its chemical structure allows for effective energy transfer and proton donation.

Matrix role:CHC acts as a proton donor during laser irradiation, enabling the formation of protonated analyte ions essential for detection in positive ion mode.
Energy absorption: The conjugated aromatic system in CHC efficiently absorbs UV photons, leading to rapid heating and desorption of analyte molecules.
Suppression of fragmentation: By reducing direct laser exposure to analytes, CHC minimizes molecular breakdown, preserving structural integrity.
Co-crystallization: CHC and analyte co-crystallize uniformly on the target plate, ensuring consistent ion yield across the sample surface.
Acidic functional groups: The phenolic hydroxyl group enhances solubility in acidic solvents and contributes to proton transfer during ionization.
Low background interference: CHC produces minimal matrix-derived ions below m/z 500, reducing spectral noise in low-mass regions.

Comparison at a Glance

The following table compares CHC with other common MALDI matrices based on key performance metrics.

Matrix	Optimal Mass Range (Da)	Laser Wavelength (nm)	Common Applications	Drawbacks
CHC	500–5,000	337	Peptides, phosphopeptides	Limited for larger proteins
α-Cyano-4-hydroxycinnamic acid (α-CHCA)	700–4,000	337	Peptide mass fingerprinting	Higher background noise
SA (Sinapinic Acid)	5,000–200,000	337	Proteins, antibodies	Poor for small peptides
DHB	1,000–20,000	337	Glycans, lipids	Crystal heterogeneity
THAP	100–1,000	337	Nucleotides, small molecules	Low sensitivity for peptides

CHC stands out for its specificity toward small to medium-sized peptides, especially in phosphoproteomics. Unlike sinapinic acid, which targets larger proteins, CHC excels in detecting post-translationally modified peptides due to its clean spectral background and efficient ionization.

Why It Matters

CHC plays a critical role in advancing proteomics and biomolecular analysis, enabling researchers to identify and characterize complex biological samples with high precision. Its development has contributed to improvements in disease biomarker discovery and drug development.

Phosphoproteomics: CHC is highly effective for detecting phosphorylated peptides, aiding in the study of cellular signaling pathways.
High-throughput screening: It enables rapid analysis of peptide mixtures in clinical and pharmaceutical research settings.
Improved sensitivity: CHC-based matrices achieve detection limits in the femtomole range, enhancing assay reliability.
Compatibility: It is compatible with automated MALDI systems, supporting large-scale proteomic studies.
Reproducibility: Uniform crystallization leads to high inter-sample reproducibility, crucial for quantitative analysis.
Research applications: Used in neuroscience, oncology, and infectious disease studies to profile protein expression changes.

As mass spectrometry continues to evolve, CHC remains a vital tool for accurate and efficient biomolecular characterization, particularly in fields requiring detailed peptide analysis.