The vacuum-drying crystallization method is a variation of the dried-droplet method in which the final analyte/matrix drop applied to the sample stage is rapidly dried in a vacuum chamber. Vacuum-drying is one of the simplest options available to reduce the size of the analyte/matrix crystals and increase crystal homogeneity by reducing the segregation effect. It is not a widespread sample preparation method, because of its mixed results and extra hardware requirements.
1. Prepare the analyte/matrix sample solution following steps 1 through 4 of the dried-droplet method.
2. Apply a 0.5 to 2 mL drop of the solution to the sample stage.
3. Immediately introduce the sample stage into a vacuum-sealed container and pump the sample down to <10-2 Torr with a vacuum pump. Wait until the solvent is completely evaporated.
4. Introduce the sample into the mass spectrometer.
Weinberger and coworkers  were the first ones to report in 1993 that vacuum drying peptide/sinapinic acid samples at ~10-2 Torr provided smaller crystals, more evenly distributed throughout the probe surface and with minimal inter-cocrystalline voids compared to identical air-dried samples. Since their original report the method has been used by several MALDI researchers with mixed results.
If nothing else, the vacuum drying method offers the fastest way to dry a MALDI sample. Vacuum drying is 20 to 30 times faster that either air or heat drying. This is a very attractive feature for users running lots of samples, requiring high sample throughput, or dealing with low volatility solvents.
When it works, vacuum-drying provides uniform crystalline deposits with small crystals. It greatly improves spot-to-spot reproducibility and minimizes the need to search for “sweet spots”. The formation of smaller crystals offers the added advantage of thinner samples and improved mass accuracy and resolution. Reductions in the amount of laser power required for ion formation have been reported for vacuum dried samples compared to similarly prepared air or heat dried samples.
The main disadvantages of vacuum-drying are that it is not guaranteed to work better than dried droplet in all cases, and it requires accessory vacuum hardware that many analytical laboratories might not have available. Peptides and proteins analyzed with the vacuum-drying method tend to exhibit extensive alkali cation adduction. This can be substantially reduced by washing the crystals directly on the probe with cold water. With evaporation times beyond 20 seconds in a vacuum system, the vacuum drying effects becomes less pronounced .