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Analytical Chemistry

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Introduction to Mass Spectrometry


Source: Laboratory of Dr. Khuloud Al-Jamal - King's College London

Mass spectrometry is an analytical chemistry technique that enables the identification of unknown compounds within a sample, the quantification of known materials, the determination of the structure, and chemical properties of different molecules.

A mass spectrometer is composed of an ionization source, an analyzer, and a detector. The process involves the ionization of chemical compounds to generate ions. When using inductively coupled plasma (ICP), samples containing elements of interest are introduced into argon plasma as aerosol droplets. The plasma dries the aerosol, dissociates the molecules, and then removes an electron from the components to be detected by the mass spectrometer. Other ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are used to analyze biological samples. Following the ionization procedure, ions are separated in the mass spectrometer according to their mass-to-charge ratio (m/z), and the relative abundance of each ion type is measured. Finally, the detector commonly consists in an electron multiplier where the collision of ions with a charged anode leads to a cascade of increasing number of electrons, which can be detected by an electrical circuit connected to a computer.

In this video, the procedure of ICP-MS analysis will be described by the detection of 56Fe as an example.


ICP-MS combines a high-temperature ICP (inductively coupled plasma) source with a mass spectrometer.

Samples need to be in ionic form prior to entering the mass analyzer in order to be detected. The digestion process of solid samples consists in the incubation of solid samples into strong and oxidizing acid at high temperature and for a prolonged period of time depending on the metal analyte. The sample is introduced as an aerosol into the ICP plasma (temperature of 6,000–10,000 K) to be converted into gaseous atoms, which are ionized.

The most commonly used mass analyzer is the quadrupole mass filter. It works as an electrostatic filter that only allows ions of a single mass-to-charge ratio (m/z) to reach the detector at a given time. It can separate up to 15,000 daltons (Da) per second and therefore is considered to have simultaneous multi-elemental analysis properties. ICP-MS is a very sensitive method that allows the detection of elements with concentrations below particle per billion (ppb), and below particle per trillion (ppt) for certain elements.

Finally, the detector system converts the number of ions striking the detector into an electrical signal. By using calibration standards (samples of known concentration for a certain element), it is possible to assess the concentration of a sample for one or several elements of interest.

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1. Cleaning of Polycarbonate Tubes

  1. Use polycarbonate tubes resistant to acidic solutions for sample digestion. In order to remove any contaminating trace of iron, fill all tubes with 5 mL of 0.1 M HCl.
  2. Place tubes in a water bath for 1 h at 50 °C.
  3. Wash the tubes with 5 mL of Milli-Q water and dry the tubes in an oven or chemical hood.

2. Sample Preparation and Digestion

  1. Place 200 µL of sample in 1.8 mL of concentrated nitric acid (65%).
  2. Place tubes in a water bath overnight at 50 °C. Adjust the protocol by increasing the temperature if a reduction of the overall digestion time is needed.
  3. Let the tubes cool down at room temperature.
  4. Dilute the samples by adding 8 mL of Milli-Q water to obtain a final nitric acid concentration bellow 20% (v/v).
  5. Centrifuge tubes at 3,000 x g for 10 min to pellet any remaining macroscopic residues.

3. Preparation of the Instrument

  1. Clean the ICP torch using ultrasonication in 5% nitric acid for 15 min. Wipe cones with 5% nitric acid. Change the peristaltic tubing. Check the pump oil level.
  2. Turn on the argon and chiller, start plasma. Start liquid flow into plasma and wait for instrument to stabilize, about 20 min.
  3. Optimize lens voltages. Run daily performance check by measuring test solutions containing Mg, In, and Ur to confirm the sensitivity of the ICP-MS instrument. Measure Ce and Ba where oxide form and double charged ions should remain below 3%. Check the mass at 8 and 220 Da to measure the background signal.
  4. The instrument is now ready for use.

4. Selection of User's Method and Sample List

  1. Select element and isotopes of interest.
  2. Select scan mode as peak hopping.
  3. Choose a dwell time of 100 ms (minimum 50) with 40 sweeps (minimum 15) per reading. Select one reading per replicate and 5 replicates (minimum 3). The total integration time is 4,000 ms. If the amount of sample is limited, reduce dwell time, number of sweeps and replicates keeping the values higher than the minimum values defined above.
  4. Use a flow rate of ammonia (NH3) at 0.7 mL/min to avoid the interference of 40Ar16O on the determination of 56Fe.
  5. Prepare calibration curve for the elements of choice.
  6. Run the samples.

Mass spectrometry is an analytical technique that enables the identification and quantification of unknown compounds within a sample, and the determination of their structure.

In mass spectrometry, gas phase ions are generated from the atoms or molecules in a sample. The ions are then separated based on their mass-to-charge ratio, symbolized by m/z.

This separation enables the determination of quantitative and qualitative information about a sample, such as their mass and structure.

This video will introduce the basic concepts and instrumentation of mass spectrometry, and demonstrate its use in element quantification.

A mass spectrometer is composed of an ionization source, a mass analyzer, and a detector. At the ionization source, the compounds are ionized, usually to a single positive charge.

Ions can be generated using various techniques, such as impact with an electron beam, plasma, or lasers, each resulting in a range of fragmentations that aid in the determination of molecular structure. These methods are loosely grouped into "hard" and "soft" ionization.

Hard ionization techniques cause extensive fragmentation, resulting in more fragments of lower mass.

Soft ionization techniques result in less, or almost no, fragmentation with a high molecular mass range.

If the fragmentation is too great, valuable structure information can be lost. If it's too little, small molecules will not be efficiently ionized. Thus, the selection of an ionization method depends on the analyte of interest and the desired degree of fragmentation.

The ions are then accelerated in an electric field as they enter the mass analyzer, where they will be separated.

The most basic mass analyzer is a magnetic sector, which is composed of a curved magnet that produces a homogeneous magnetic field. The attractive force of the magnet, plus the centrifugal force of the accelerating ions causes them to travel in a circular path through the curve.

The radius of the ions circular path depends on the accelerating voltage, the applied magnetic field, and the mass-to-charge ratio.

The voltage and magnetic field can then be selected to only allow certain mass-to-charge ratio species through the curved path. Other ions crash into the sides of the magnetic pathway and are lost. By scanning the magnetic field strength, desired ions reach the detector at different times, thereby identifying each species precisely.

Another type of mass analyzer is the quadrupole mass filter. The quadrupole consists of two pairs of parallel metal rods, with each pair of opposing rods electrically connected.

A direct current voltage is applied to the rod pairs, and their potentials continuously alternated so the pairs are always out of phase with the other.

The ion beam is then directed through the center of the four rods. Ions travel in a corkscrew-like path, due to the constant attraction and repulsion from the rods. Depending on the ions mass-to-charge ratio, the ion will either travel the full path of the quadrupole and reach the detector, or will crash into the rods.

Now that the basics of the mass spectrometer have been described, lets take a look at its use in the laboratory.

The mass spectrometer used in this experiment is an inductively coupled plasma, or ICP, ionizer, with a quadrupole filter. The instrument will be used to detect and quantify a metal component in a sample.

To begin the experiment, fill all polypropylene tubes with 5 mL of 0.1 M hydrochloric acid in order to remove any contaminating trace of iron. Place the tubes in a water bath for 1 h at 50 °C.

After incubation, wash the tubes with 5 mL of deionized water, and dry the tubes in an oven or chemical hood.

In the clean tubes, add 1.8 mL of concentrated nitric acid and 200 μL of sample containing the isotope of interest.

Follow safety precautions when using concentrated acid.

Place the tubes in a water bath overnight. The temperature can be increased to shorten digestion time, if necessary.

After the sample has been digested, let the tubes cool to room temperature.

Next, add 8 mL of deionized water to dilute the samples, and to obtain a nitric acid concentration below 20%. The final dilution of the sample is 1/50. The ideal concentration for ICP is in the parts-per-billion range. Centrifuge the tubes to pellet any remaining macroscopic residues.

ICP is a method of hard ionization that uses coupled argon plasma at about 10,000 °C that is electrically conductive to ionize the sample molecules.

Begin the instrument set up by inspecting the ICP torch to ensure that it is clean.

Then, inspect the sampler and skimmer cones to ensure they are also clean. These cones enable the sampling of only the inner portion of the ion beam generated by the ICP torch and act as a barrier to the high vacuum of the mass spectrometer.

Check the argon pressure and start the chiller. Start the plasma and liquid flow into the system. Wait 20 min for the system to warm up fully.

Next, aspirate a standard test solution, which contains various known elemental standards. The test solution should be selected to cover the expected mass range of the analyte solution.

When the solution flow is established, initialize and test the instrument according to the manufacturer's guidelines.

To run the instrument, first select the elements and isotopes of interest. Then set the scan mode to peak hopping.

Select five replicates per measurement. Set each replicate to contain 40 measurement sweeps, each sweep with a dwell time of 50 ms. The total integration time is 2,000 ms per replicate.

Prepare a calibration curve for the elements of choice by measuring pre-prepared standard solutions.

Finally, run the sample, in this case, iron-oxide nanoparticles. Determine the concentration of iron using the iron calibration curve.

Mass spectrometry is used in a wide range of applications using various ionization and mass analysis techniques.

In this example, a type of soft ionization mass spectrometry, called matrix assisted laser desorption ionization time-of-flight, or MALDI-TOF, was used to analyze high molecular weight proteins. With MALDI, molecules are stabilized with a matrix, to decrease fractionation when the large molecules are ionized.

The protein solution and matrix were both spotted on the clean MALDI plate, and dried. The MALDI plate was inserted into the instrument, and the sample analyzed.

The analysis of volatile and oxidation sensitive compounds was measured using electron ionization mass spectrometry, a hard ionization technique.

First, a lockable tube system was designed in order to enable full evacuation of the tube, followed by loading of the sample under cooling by liquid nitrogen.

The sample tube was connected to the inlet port, and the sample loaded into the instrument. The mass spectrum of the sample in this case tris(trifluoromethyl) phosphate, was then analyzed.

A molecular beam mass spectrometer coupled with synchrotron radiation was used to explore the electronic structure of gas phase molecules and clusters.

The molecular beam, integrated with synchrotron radiation, provided a selective ionization method to probe molecules in the gas phase.

The sample was loaded into the nozzle, the nozzle reloaded into the instrument, and the photon beam allowed to enter the chamber.

The mass spectrum was then collected and compared to photoionization efficiency data in order to determine the electronic structure of molecules.

You've just watched JoVE's introduction to mass spectrometry. You should now understand the basic instrumentation of mass spectrometry, and how to run a basic mass-spectrometry-based analysis.

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ICP-MS analysis of samples containing iron oxide nanoparticle is shown below. A standard curve was carried out using known concentration of 56Fe (Figure 1). The correlation coefficient being close to 1 (R2 = 0.999989) showed the good linear relationship between the sample concentrations and the intensity measured by the detector. Samples of interests showed values within the calibration range (Figure 2). The concentrations calculated by the software were then adjusted according to the dilution carried out during the protocol. The present protocol described a dilution of 1/50 following the dilution in acid (1/10) and in Mili-Q water (1/5). For example, a concentration of 51.427 µg/L was measured for the sample number 51 (Figure 2). The concentration of the original sample was 50x higher corresponding to 2.57 mg/L.

Figure 1
Figure 1. Calibration curve for 56Fe measurements. Four standard points (0.01, 0.1, 1, and 10 µg/mL) show a correlation coefficient (R2) of 0.999989. This confirms the good linear relationship between the signal intensity detected and the concentrations of reference.

Figure 2
Figure 2. Representative results following ICP-MS measurements on iron oxide nanoparticle samples. The concentration of each diluted sample is automatically calculated according to the defined calibration curve.

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Applications and Summary

The environmental and geological fields represent the first use for ICP-MS for example to measure contaminants present in water, in the soil, or in the atmosphere. The presence of contaminants at high concentration in tap water such as Fe, Cu, or Al can be monitored using ICP-MS.

The medical and forensic science fields also use ICP-MS detection. In case of suspicion of a metal poisoning such as arsenic, samples such as blood and urine can be analyzed using ICP-MS. This technique can also provide valuable information in case of pathology involving metabolic concerns or hepatological issues resulting in the poor excretion of certain elements.

ICP-MS allows the quantification of metals in any material. In Figure 3, the concentration of Fe was measured in nanoparticles and related to their magnetic resonance imaging (MRI) properties. ICP-MS provides a reliable quantification of Fe of different nanoparticles to discriminate which nanoparticles are the most efficient for imaging application.

Another application is to study the biodistribution of nanoparticles associated with metals. Figure 4 presents the organ biodistribution of nanoparticles containing iron oxide in mice following intravenous injection. At 24 h, each organ was collected and digested in concentrated nitric acid until full organ digestion was achieved. The 56Fe concentration was quantified by ICP-MS. Results show higher concentration of 56Fe in liver and spleen for mice injected with nanoparticles than in organs from naïve animals. Therefore, it was concluded that nanoparticles accumulate mostly into liver and spleen organs.

Figure 3
Figure 3. Magnetic resonance imaging (MRI) measurement of nanoparticles function of their Fe concentration. Five concentrations of iron were used (0.25, 0.5, 0.75, 1, and 1.25 mM) that were imaged for their MRI properties (relaxation rate, R2*).

Figure 4
Figure 4. Biodistribution of iron oxide nanoparticles following intravenous injection in mice. Naïve samples show the basal organ level of iron in untreated mice. Following the injection of nanoparticles containing iron oxide, the quantity of iron in certain organ increases which is associated to the accumulation of nanoparticles.

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Mass Spectrometry Analytical Technique Identification Quantification Unknown Compounds Sample Structure Determination Gas Phase Ions Mass-to-charge Ratio Quantitative Information Qualitative Information Basic Concepts Instrumentation Element Quantification Ionization Source Mass Analyzer Detector Positive Charge Electron Beam Impact Plasma Lasers Fragmentation Molecular Structure Determination Hard Ionization Techniques Soft Ionization Techniques

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