Scientists have developed a new X-ray technique that can capture ultrafast molecular changes in extremely dilute liquid samples - a breakthrough that could allow researchers to investigate a far broader range of biological and chemical systems than previously possible.

The study, carried out at the SCS (Spectroscopy and Coherent Scattering) instrument at the European XFEL, successfully tracked how vitamin B12 molecules in water respond after absorbing light. The findings were published in the Journal of the American Chemical Society.

Studying molecules in liquid solutions has long posed a challenge for X-ray experiments, especially when the target molecules are present only in tiny amounts. In many cases, the signal produced by surrounding water overwhelms the much weaker signal from the molecules researchers actually want to observe.

To address this issue, the international research team led by Dr. Nahid Ghodrati, Dr. Ben van Kuiken and Dr. Loic Le Guyader from European XFEL designed a new beam-splitting system capable of significantly improving measurement sensitivity. The device separates each incoming X-ray pulse into three beams: one probes the sample itself, while the other two serve as reference signals. Comparing the three simultaneously allows researchers to correct for fluctuations in the free-electron laser pulses and isolate otherwise undetectable molecular changes.

"Detecting signals from dilute samples in solution is incredibly challenging because the background from the solvent can dominate the measurement," says Zhong Yin, an associate professor at Tohoku University's SRIS. "This kind of approach provides a powerful way to improve sensitivity and could help researchers investigate many important chemical and biological systems that were previously difficult to study."

To test the method, the researchers selected vitamin B12 - a notoriously difficult sample for ultrafast X-ray measurements Vitamin B12 dissolves only sparingly in water, and the molecular changes triggered by light occur on extremely short timescales.

Despite using a relatively low concentration of around 9.5 grams of vitamin B12 per litre of water, the team succeeded in detecting signal changes as small as 0.005%. The experiment captured molecular dynamics occurring within just 100 quadrillionths of a second.

"Normalizing every shot is crucial," says SCS instrument scientist Benjamin Van Kuiken. "That's what gives us the sensitivity to work with dilute samples."

The measurements also shed light on a long-standing scientific question surrounding vitamin B12: what happens electronically inside the molecule immediately after light absorption? The results suggest that the primary change occurs near the cobalt atom at the molecule's centre, rather than through a broader redistribution of electrons across the entire structure.

"The unique ability of the SCS Instrument to study dilute samples gave us insight into the rapid evolution of the molecule's electronic structure that we could not get anywhere else," says James Penner-Hahn, professor at the University of Michigan, USA.

Researchers say the technique could significantly expand the types of experiments possible at European XFEL and other X-ray facilities worldwide. By enabling measurements on difficult low-concentration samples, the approach may open new opportunities in chemistry, biology, and materials science.