Inductively coupled plasma mass spectrometry

ICP-MS (Inductively coupled plasma mass spectrometry) is a type of mass spectrometry that is highly sensitive and capable of the determination of a range of metals and several non-metals at concentrations below one part in 10¹². It is based on coupling together an inductively coupled plasma as a method of producing ions (ionization) with a mass spectrometer as a method of separating and detecting the ions. ICP-MS is also capable of monitoring isotopic speciation for the ions of choice.

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• ICP (Inductively Coupled Plasma) - A plasma is a gas that contains a sufficient concentration of ions and electrons to make the gas electrically conductive. The plasmas used in spectrochemical analysis are essentially electrically neutral, with each positive charge on an ion balanced by a free electron. In these plasmas the positive ions are almost all singly-charged and there are few negative ions, so there are nearly equal numbers of ions and electrons in each unit volume of plasma

An inductively coupled plasma (ICP) for spectrometry is sustained in a torch that consists of three concentric tubes, usually made of quartz. The end of this torch is placed inside an induction coil supplied with a radio-frequency electric current. A flow of argon gas (usually 14 to 18 liters per minute) is introduced between the two outermost tubes of the torch and an electrical spark is applied for a short time to introduce free electrons into the gas stream. These electrons interact with the radio-frequency magnetic field of the induction coil and are accelerated first in one direction, then the other, as the field changes at high frequency (usually 27.12 million cycles per second). The accelerated electrons collide with argon atoms, and sometimes a collision causes an argon atom to part with one of its electrons. The released electron is in turn accelerated by the rapidly-changing magnetic field. The process continues until the rate of release of new electrons in collisions is balanced by the rate of recombination of electrons with argon ions (atoms that have lost an electron). This produces a ‘fireball’ that consists mostly of argon atoms with a rather small fraction of free electrons and argon ions. The temperature of the plasma is very high, of the order of 10,000 K.

The ICP can be retained in the quartz torch because the flow of gas between the two outermost tubes keeps the plasma away from the walls of the torch. A second flow of argon (around 1 liter per minute)is usually introduced between the central tube and the intermediate tube to keep the plasma away from the end of the central tube. A third flow (again usually around 1 liter per minute) of gas is introduced into the central tube of the torch. This gas flow passes through the centre of the plasma, where it forms a channel that is cooler than the surrounding plasma but still much hotter than a chemical flame. Samples to be analyzed are introduced into this central channel, usually as a mist of liquid formed by passing the liquid sample into a nebulizer.

As a droplet of nebulized sample enters the central channel of the ICP, it evaporates and any solids that were dissolved in the liquid vaporize and then break down into atoms. At the temperatures prevailing in the plasma a significant proportion of the atoms of many chemical elements are ionized, each atom losing its most loosely-bound electron to form a singly charged ion.

• MS (Mass Spectrometry) the ions from the plasma are extracted through a series of cones into a mass spectrometer, usually a quadrupole. The ions are separated on the basis of their mass-to-charge ratio and a detector receives an ion signal proportional to the concentration.

The concentration of a sample can be determined through calibration with elemental standards. ICP-MS also lends itself to quantitative determinations through Isotope Dilution, a single point method based on an isotopically enriched standard.

Other mass analyzers coupled to ICP systems include double focusing magnetic-electrostatic sector systems with both single and multiple collector, as well as time of flight systems (both axial and orthogonal accelerators have been used).

Another type of spectrometer using ICP is ICP-AES (Atomic Emission Spectrometer).

Quantification of proteins and biomolecules by ICP-MS

There is an increasing trend of using ICP-MS as a tool in Speciation Analysis, which normally involves a front end chromatograph separation and an elemental selective detector, such as AAS and ICP-MS. For example, ICP-MS may be combined with size exclusion chromatography and quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) for identifying and quantifying native metal cofactor containing proteins in biofluids. Also the phosphorylation status of proteins can be analyzed.

Recently a new type of protein tagging reagents called metal coded affinity tags (MeCAT) were introduced to label proteins quantitatively with metals, especially lanthanides.^[1] The MeCAT labelling allows relative and absolute quantification of all kind of proteins or other biomolecules like peptides. MeCAT comprises a site-specific biomolecule tagging group with at least a strong chelate group which binds metals. The MeCAT labelled proteins can be accurately quantified by ICP-MS down to low attomol amount of analyte which is at least 2-3 orders of magnitude more sensitive than other mass spectrometry based quantification methods. By introducing several MeCAT labels to a biomolecule and further optimization of LC-ICP-MS detection limits in the zeptomol range are within the realms of possibility. By using different lanthanides MeCAT multiplexing can be used for pharmacokinetics of proteins and peptides or the analysis of the differential expression of proteins (proteomics) e.g. in biological fluids. Breakable PAGE SDS-PAGE (DPAGE, dissolvable PAGE), two-dimensional gel electrophoresis or chromatography is used for separation of MeCAT labelled proteins. Flow-injection ICP-MS analysis of protein bands or spots from DPAGE SDS-PAGE gels can be easily performed by dissolving the DPAGE gel after electrophoresis and staining of the gel. MeCAT labelled proteins are identified and relatively quantified on peptide level by MALDI-MS or ESI-MS.

Sample introduction

The first step in analysis is the introduction of the sample. This has been achieved in ICP-MS through a variety of means.

The most common method is the use of a nebulizer. This is a device which converts liquids into an aerosol, and that aerosol can then be swept into the plasma to create the ions. Nebulizers work best with simple liquid samples (i.e. solutions). However, there have been instances of their use with more complex materials like a slurry. Many varieties of nebulizers have been coupled to ICP-MS, including pneumatic, cross-flow, Babington, ultrasonic, and desolvating types. The aerosol generated is often treated to limit it to only smallest droplets, commonly by means of a double pass or cyclonic spray chamber. Use of autosamplers makes this easier and faster.

Less commonly, the laser ablation has been used as a means of sample introduction. In this method, a laser is focused on the sample and creates a plume of ablated material which can be swept into the plasma. This is particularly useful for solid samples, though can be difficult to create standards for leading the challenges in quantitative analysis.

Other methods of sample introduction are also utilized. Electrothermal vaporization (ETV) and in torch vaporization (ITV) use hot surfaces (graphite or metal, generally) to vaporize samples for introduction. These can use very small amounts of liquids, solids, or slurries. Other methods like vapor generation are also known.

Transfer of ions into vacuum

The carrier gas (usually argon or occasionally helium) is sent through the central channel and into the very hot plasma. The sample is then exposed to radio frequency which converts the gas into a plasma. The high temperature of the plasma is sufficient to cause a very large portion of the sample to form ions. This fraction of ionization can approach 100% for some elements (e.g. sodium), but this is dependent on the ionization potential. A fraction of the formed ions passes through a ~1mm hole (sampler cone) and then a ~0.4mm hole (skimmer cone). The purpose of which is to allow a vacuum that is required by the mass spectrometer.

The vacuum is created and maintained by a series of pumps. The first stage is usually based on a roughing pump, most commonly a standard rotary vane pump. This removes most of the gas and typically reaches a pressure of around 133 Pa. Later stages have their vacuum generated by more powerful vacuum systems, most often turbomolecular pumps. Older instruments may have used oil diffusion pumps for high vacuum regions.

Ion Optics

Before mass separation, a beam of positive ions has to be extracted from the plasma and focused into the mass-analyzer. It is important to separate the ions from UV photons, energetic neutrals and from any solid particles that may have been carried into the instrument from the ICP. Traditionally, ICP-MS instruments have used transmitting ion lens arrangements for this purpose. Examples include the Einzel lens, the Barrel lens, Agilent's Omega Lens ^[2] and Perkin-Elmer's Shadow Stop ^[3]. Another approach is to use ion guides (quadrupoles, hexapoles, or octopoles) to guide the ions into mass analyzer along a path away from the trajectory of photons or neutral particles. Yet another approach is Varian patented ^[4] 90 degrees reflecting "Ion Mirror" optics, which are claimed to provide more efficient ion transport into the mass-analyzer, resulting in better sensitivity and reduced background ^{[5] [6] [7]}

Dynamic reaction cell

Introduced by Perkin-Elmer on their Elan DRC (followed by Elan DRC II and Elan DRC-e) instrument, the Dynamic Reaction Cell is a chamber placed before the traditional quadrupole room of an ICP-MS device, for eliminating isobaric interferences.^{[8] [9] [10] [11]} The room has a quadrupole (do not confuse this quadrupole with the main one, placed after DRC and before the detector, which has longer rods and is, in generally, bigger) and can be filled-up with so-called reaction (or collision) gases (ammonia, methane, oxygen or hydrogen), with one gas type at a time or a mixture of two of them, which reacts with the introduced sample, eliminating some of the interference. The DRC is characterized by the following paramters, that can be modified: RPq (the corresponding q parameter from the Mathieu equation), RPa (the corresponding a parameter from the Mathieu equation), which refer to the voltage applied to the quadrupole rods and the gas flow of the reaction gas. Ammonia gas is the best solution for the majority of interferences, but it is far for being the perfect gas. Sometimes, for specific isotopes, other gas must be used for better results or even mathematical correction, if no gas offers a satisfactory advantage.

CRI technology

The proprietary Collisional Reaction Interface (CRI) ^[12] technology used in the Varian ICP-MS is another effective approach to destroying interfering ions. These ions are removed by injecting a collisional gas (He), or a reactive gas (H2), or a mixture of the two, directly into the plasma as it flows through the skimmer cone and/or the sampler cone. Supplying the reactive/collisional gas into the tip of the skimmer cone and/or into the tip of the sampler cone induces extra collisions and reactions that destroy polyatomic ions in the passing plasma.

Axial field technology

Axial field technology (AFT) is a patented improvement of DRC made by Perkin-Elmer, which consists in two supplementary rods placed in the DRC room, smaller than normal quadrupole's rods, with the purpose of "pushing" the ions faster to the exit by generating a supplementary electric potential, minimizing the time needed for the gas to be in the DRC and improving analysis speed. The suplimetary potential of the AFT rods does not contribute significantly to the global energy, but drastically improve ion pasage time.

Other implementations

Another implementation of this type of interference removal is an octopole (instead of a quadrupole) collision cell, implemented by Agilent's 7500 series. The ORS (Octopole Reaction System) uses only helium or hydrogen and the volume of the cell is smaller than that of a DRC. The small molecules of helium and hydrogen collide with the large, unwanted polyatomic ions formed in the plasma and break them up into other ions that can be separated in the quadrupole mass analyser. However, unlike the DRC the OCR system is based only on collision reactions and not on chemical reactions.

Plasma generation

As stated above the mode of ionisation is via an argon plasma. Argon has the advantage of being abundant (in the atmosphere, as a result of the radioactive decay of potassium). It is therefore available more cheaply than the other inert gases. Argon also has the advantage of having a higher first ionisation potential than all other elements except He, F and Ne.

The radio frequency causes the following reaction: Ar → Ar⁺ + e^-. Given the high ionisation potential as cited above reverse reaction will take electrons from any species. This recombination of Ar with an electron Ar⁺ + e^- → Ar is likely to cause the loss of an electron from a metal M → M⁺ + e^-. Group II metals may become doubly charged species due to their low second ionisation potential.

Elemental analysis

The ICP-MS allows determination of elements with atomic mass ranges 7 to 250. This encompasses Li to U. Some masses are prohibited such as 40 due to the abundance of argon in the sample. A typical ICP-MS will be able to detect in the region of nanograms per litre to 10 or 100 milligrams per litre or around 8 orders of magnitude of concentration units.

Unlike atomic absorption spectroscopy, which can only measure a single element at a time ICP-MS has the capability to scan for all elements simultaneously. This allows rapid sample processing.

Usage

ICP-MS can be used for analysis of environmental samples such as water and various other non-particulate samples. The instrument can also determine metals in urine to check for exposure to toxic metals. The instrument is very sensitive to particulate matter and high concentrations of organics will cause the instrument to cease function, requiring cleaning.

References