Understanding the concept of dead volume is important for HPLC analysts. Dead volume refers to the empty, unused space within an autosampler vial that does not contain sample. This unused volume can negatively impact HPLC analysis if not properly managed.
HPLC autosampler vials come in standardized size options, typically 1.5-2 mL and 4 mL volumes. When the actual sample volume does not fully occupy the entire vial capacity, there is inherent dead volume present. This dead space is the empty volume above the top level of the sample liquid.
Dead volume in autosampler vials can be problematic for HPLC analysis in several ways. First, it increases the exposed surface area of the sample. This heightens the risk of sample evaporation, especially for volatile analytes. Evaporation can alter the original sample concentration and composition, leading to inaccurate results.
The dead volume also affects the sample transport and injection process. The autosampler syringe must navigate through the dead volume to reach and draw up the actual sample. This dead volume is essentially “wasted” during injection, as it does not contain the analyte of interest. This can impact peak shape, retention time, and quantitation accuracy of the HPLC data.
There are a number of key strategies that HPLC analysts can employ to minimise dead volume in their autosampler vials. The most basic approach is to carefully match the vial size to the sample volume. Using the smallest vial that can hold the entire sample eliminates excessive headspace and dead volume.
Micro-volume vial inserts are also helpful for very small sample sizes in the 150-300 μL range. These specialised inserts fit into the larger 2 mL autosampler vials, effectively reducing the overall dead volume.
Proper vial sealing is another critical factor in managing dead volume. Ensuring a tight seal between the vial cap or septum and the vial itself helps maintain sample integrity and minimises evaporation from the dead volume area.
Ultimately, dead volume in HPLC autosampler vials should be minimised as much as possible. Excessive dead volume can lead to sample loss due to evaporation, altered analyte concentrations and compromised data quality and reliability. Careful vial selection, filling and sealing are essential to optimise HPLC analysis and ensure accurate, reproducible results.