Lawrence Livermore National Laboratory

A technological need, widely voiced by our collaborators in recent years, has been for a user-friendlier AMS interface, including far less cumbersome sample preparation, that can reduce analysis time and cost. To facilitate sample preparation and reduce analysis times from days to minutes, we have developed an online, directly-coupled, liquid sample interface that allows analysis without first converting a sample to graphite, as has typically been required in the past.

Components of the liquid sample

Figure 1. Components of the liquid sample, moving wire interface include the (1) nickel wire; (2) cleaning, (3) drying, and (4) combustion ovens; (5) sample-depositing emitter tip apparatus from a (6) high-performance liquid chromatograph; the (7) system control panel; and (8) an accelerator mass spectrometry gas ion source.

The primary function of the liquid sample interface is to rapidly convert the carbon content of samples suspended or dissolved in a liquid to CO2 gas and to directly transport the gas to a gas-accepting ion source for real-time 14C AMS analysis Biomedical samples are prepared for bioAMS measurement by conversion to a homogeneous chemical form that is suitable for producing negative ions in a cesium sputter ion source. The overwhelming majority of bio-chemical samples measured by AMS are already in liquid form or can be readily suspended or dissolved in appropriate solvents or buffers. However, until the development of the liquid sample interface, samples were prepared offline for bioAMS analysis by converting the carbon content to CO2 via combustion, followed by reduction of CO2 to solid graphite, in a complex process that takes at least 36 hours. Graphite samples were then placed in an ion source for subsequent AMS 14C analysis.

How does the liquid Sample Interface operate?

The liquid sample interface involves depositing samples suspended or dissolved in liquid manually or via an auto-sampler or HPLC onto a periodically indented moving wire. The indentations ensure that virtually equal droplet sizes are deposited at regular intervals onto the wire for optimum interface operation. For HPLC applications, a coherent jet to increase efficiency and maintain peak resolution transfers eluate to the wire. The moving wire passes through a drying oven to evaporate the liquid carrier, and then through a combustion oven to convert the carbon content of the dried sample to CO2 gas. The combustion oven is plumbed so that 100% of the gaseous combustion products are directed in a helium stream to an exit capillary coupled to a cesium sputter, gas-accepting ion source for 14C analysis by AMS. A silent video (Video 1) showing the liquid sample interface in use illustrates operation.

Video 1.
Video (with no sound) of the liquid sample interface in operation.

The moving wire interface utilizes a high-purity nickel wire, which is fed via a tension-controlled de-spooler. The wire is first indented with a die driven by a solenoid (item 1 in Figure 2). The face of the die has a rough texture to create a region of high surface area per unit of length on the wire. Precision timing of voltage applied to the solenoid allows the indenter to strike with maximum force while not disturbing the linear motion of the wire.

The nickel wire first passes trough a 60 cm-long cleaning oven (item 2 in Figure 2) before sample deposition. In the cleaning oven, the wire is heated to 900°C to remove surface carbon and create a nickel oxide coating. The coating enables better adherence of fluids than bare nickel wire and minimizes conversion of trace amounts of carbon within the bulk wire material to CO2 during sample combustion.

Immediately after cleaning, liquid samples are directed onto the wire (item 3 in Figure 2) in a coherent stream or as single droplets. The increased surface area of wire indentations attracts fluid, causing it to form small, periodic droplets that are fixed along the length of the wire. Without periodic wire indentations, fluid tends to slide along the wire, collecting into large droplets that either do not evaporate or fall from the wire.

Samples can be deposited on the moving wire in three ways:

  • For real-time HPLC–AMS analysis, a silica-tip emitter directs a continuous, coherent jet of eluate from an HPLC onto the wire at flow rates of up to 130 µL/min.
  • For individual sample analysis, samples placed into auto-sampler vials are deposited on the wire by a silica-tip emitter via an auto-sampler injection system
  • .
  • Liquid samples can be deposited manually onto the wire as individual microliter-sized droplets as large as 3 µL from a micropipette.
The wire proceeds through a 95-cm-long drying oven (item 4 in Figure 2) held at a temperature that depends on solvent or buffer volatility and is between 90° and 140°C to evaporate the liquid solvent or buffer. A continuous flow of pre-heated helium is injected at the center of the drying oven and collected at either end. Helium increases the oven's drying capacity because of its high thermal conductivity. Complete collection of the drying gas prevents escape of semi-volatile biological or isotopically enriched material.

Schematic of a liquid-sample

Figure 2. Schematic of a liquid-sample, moving wire interface coupled to a high-performance liquid chromatograph and an accelerator mass spectrometer for online HPLC–AMS analysis.

After solvent or buffer removal, the dried sample enters a 15-cm-long combustion oven (item 5 in Figure 2) to convert the sample carbon content to CO2 gas. The entrance and exit of this oven are fitted with restrictions, and a continuous flow of helium maintains interior pressure above ambient atmospheric pressure, preventing atmospheric contamination. Interior pressures near the wire entrance and exit are balanced by connecting the points with low-impedance tubing. This pressure balancing traps and directs 100% of the gaseous combustion products to a capillary (item 6 in Figure 2) at the exit side of the oven. To ensure complete combustion of the carbon content of samples to carbon dioxide, the oven is held at 750°C, while oxygen is injected into the helium stream at a concentration of 5%, and an oxidized copper wire serves as both an additional oxygen reservoir and catalyst.

The CO2 gas is carried in the helium stream through the narrow-diameter capillary to a gas-accepting cesium sputter ion source for 14C analysis. In the source, CO2 gas is directed to the front surface of a titanium pellet that serves as an adsorbant. Cesium+ ions are focused and directed towards the titanium pellet, ionizing the CO2 and converting approximately 1% of the CO2 to a C– ion beam.

AMS quantifies the isotopic concentration by separating and identifying ions derived from a sample according to their nuclear charge and mass. AMS uses magnetic and electrostatic fields to separate the different isotopes of carbon. Interfering molecular isobars of 14C (i.e., 13CH and 12CH2) are destroyed via high-velocity collisions with either an inert, dilute gas or a thin carbon foil using an accelerator or high-voltage deck. The amount of 14C is measured relative to a more abundant isotope (i.e., 13C or 12C) in the ion beam. Absolute quantification comes from comparing the sample's measured isotope ratio to that of measured standards of known ratios that are traceable through the National Institute of Standards and Technology (referred to as NIST-traceable). From the time a sample is deposited on the wire to the time its 14C/12C ratio is measured, approximately 90 seconds have elapsed.

Advantages over AMS 14C analysis of graphite

Current AMS analysis using graphite requires samples with a carbon content of ~50 µg or greater regardless of the amount of 14C label in the sample. For our liquid sample interface, samples having a carbon content of 1 µg can readily be analyzed for 14C content at contemporary levels (14C/12C ratio of 10–12). For highly labeled material, sample sizes as small as a 1 picogram (pg) can be analyzed with the liquid sample interface. This extremely small sample size affords the important capability of readily quantifying low-abundance metabolites in metabolite profiles and improves on measurement accuracy because there is no need to add a carrier carbon-bulking agent that is often utilized to enable small-sample graphite AMS measurement.

Current AMS analysis of biological materials requires that carbonaceous biochemicals be converted to graphite. However, considerable human handling is required, and the process suffers from low sample throughput (<100 samples processed per day) and turnaround times of ~2 days. With our new method, the time required for analysis of biochemical samples by AMS is drastically reduced from days to minutes. Since liquid separation technologies are universally used in biomedical applications to separate a mixture of compounds into individual components for further study an important feature is that the new method allows for continuous flows of liquid, enabling direct coupling of an HPLC or other liquid separation technology to the spectrometer for real-time AMS analysis. In performing a single HPLC-AMS trace using graphite samples, eluant from an HPLC trace is frequently collected in sixty 30-second fractions. It takes approximately two to three days to convert the fractions to graphite and then analyze them for 14C content. In contrast, the liquid sample interface can take the output from an HPLC directly and perform 14C analysis online in the time it takes the HPLC to speciate the sample: in other words, a total of time of 30 minutes.