The primary mission of the Scientific Instrumentation Service (SIS) consists in providing support to the EEZ research groups in the conduct of qualitative and quantitative analytical measurements using chromatographic and mass spectrometric techniques. The SIS offers a variety of established analytical protocols for compounds that of central interest to groups at the EEZ. Although responding to the internal demand of analysis is a priority, analysis for external entities can also be conducted. In addition, members of the SIS can provide advice on different aspects of analytical sciences and open to develop novel protocols for the analysis of other compounds.
Scientist responsible microcalorimetry and electron paramagnetic resonance spectrometry:
High Performance Liquid Chromatograph
High performance liquid chromatography (HPLC) is a technique used to separate individual components from mixtures and based on differences in the interaction between analytes and chromatographic columns.
The acronym UPLC stands for Ultra Performance Liquid Chromatography, which can be translated as Ultra High Resolution Liquid Chromatography. The UPLC equipment consists of a dual UV detector, a column oven, a refrigerated autosampler (up to 5ºC) and a quaternary pump system. The UPLC H-Class system can work in HPLC mode like a UPLC, depending on the columns and flows with which it works. The UPLC H-Class (Waters) equipment has replaced the HPLC Allience (Waters) connected to the Quattro Micro mass detector.
So far in SIC chromatographic separations have been performed using HPLC columns. These columns have packings with a particle size between 3.5 and 5.0 μm and withstand pressures of around 300 bar. UPLC uses columns packed with 1.7 µm diameter particles and pressures up to 1034 bar can be achieved. Advantages of UPLC over HPLV include faster and higher resolution analysis as well as reduced consumption of eluent. Compared to HPLC columns, UPLC columns provide higher resolution and sensitivity at the same analysis time or equivalent resolution and higher sensitivity at shorter analysis times.
In addition, Waters UPLC columns include eCordTM technology with the addition of a chip to the column that records the history of its use. The column's eCord chip interacts with the system software and records information on the injections performed. In addition to variable data on the use of the column, the eCord chip also stores a series of data on the process of its manufacture.
HPLC Waters model 1525 equipped with a PAD detector (photodiode array detector) as well as detectors of fluorescence and refraction index; coupled to a fraction collector.
HPLC with different detectors and fraction collector
Organic compounds, particularly high conjugated ones, absorb light in the visible and ultraviolet electromagnetic spectrum. . The Beer-Lambert law shows the absorbance of a compound solution correlates directly with its concentration, which implies that visible/UV spectrometry can be used to determine component concentrations in solution, making use of commercial standard solutions of known purity.
Possible types of analysis: Determination of nitrates and nitrites, size exclusion chromatography, analysis of fluorescent compounds, analysis of different sugars.
Comments: the fraction collector is used for the separation of compounds for which its identity is established using alternative techniques.
1 - Determination of trinitrotoluene and its derivatives
Wittich et al (2009) Environmental Science & Technology, 43, 2773-2776.
2 - Determination of nitrates and nitrites in soil and water
Tortosa et al. (2011). Ecological Engineering, 37, 539-548
HPLC VARIAN Prostar with diode array detector (model Prostar 335), fluorescence detector (model 9012) and refrigerated autosampler ProStar 410.
HPLC with differents detectors
The types of analysis that can be done using this instrument are similar to those of the previous one, except that this HPL is not equipped with a refractive index detector and fraction collector. The diode array detector is similar to that of the PDA detector of the above instrument..
* Mass spectrometry is an analytical technique that permits the precise measurement of masses from molecules. The mass spectrometer can be coupled to liquid chromatography (LCMS) or gas chromatography (GCMS).
* The mass spectrometer measures mass/charge ratios of ions. Different ionisation techniques exist that involve heating of compounds until vaporization occurs. Ions generate a specific signal in the detector that can be used for compound identification. Applications include drugs, products of chemical synthesis, pesticides, forensic analyses, compounds of environmental concern and all other ionizable compounds.
* The Scientific Instrumentation Service of the EEZ has currently two mass spectrometers, namely a gas chromatography instrument coupled to an ion tramp mass spectrometer (GCMS) as well as an electrospray ionization instrument equipped with triple quadrupole detector that is coupled to an HPLC (LCMS).
The instrument Vion IMS QTof (Ion Mobility Quadrupole Time-of-Flight) is a hybrid system that combines cuadrupole mass analysers with the high resolution time-of-flight (Tof) mass detector. This instrument is coupled to a Ultra Performance Liquid Chromatography (UPLC) unit that permits an increase in the resolution and sensitivity of the mass analysis.
In a QTof type of mass detector, the generated ions are grouped in the ionization source and are then submitted to a voltage causing their acceleration permitting them to “fly” through a tube of a determined length. Their velocity depends on their mass (m) and charge (z). In general, singly charged ions travel more slowly and arrive after multiply charged ions. This type of mass analyser permits to precisely determine the mass of a compound and enables de novo compound identification, hence the denomination of high resolution mass spectrometer..
Schematic view of the QTof mass spectrometer
With an instrument of these characteristics one can work either in Full Scan or MS/MS mode (in the latter a single ion is isolated and fragmented). The operation in the MS/MS mode permits Data Independent Analyses (DIA) and Data Dependent Analyses (DDA) as well as a mode in which DIA data are acquired simultaneously in Full scan and MS/MS mode.
In addition to the determination of the exact mass and the generation and analysis of compound fragments, this type of instrument also permits the analysis of ion mobility. Mass spectrometry involving the study of ion mobility, also referred to as ion mobility separation mass spectrometry, is an analytical method that separates gas phase ions as function of their interaction with a collision gas and their mass. Earl W. McDaniel was the scientific pioneer of ion mobility and first to associate this technique with a mass spectrometer. First attempts were made to associate this technique with TOF type mass spectrometry in 1963, but commercial instruments became only available at the beginning of the 21st century (2006, Synap-Waters).
Ions are introduced into ion mobility cell where they are exposed to a low pressure gas flow (N2) that occurs in the opposite direction with respect to the electric field mediated ion migration. As a consequence, ions are exposed to two opposing forces, namely their movement in the electric field and the resistance to the gas flow. As a consequence ions are separated according to their m/z ratio as well as to their structure. Typically, small ions advance more rapidly than larger ions. Based on these migration characteristics the instrument software allows the calculation of the CollisionCross Section (CCS) value, which is an additional parameter that provides information on the size, chemical and three-dimensional structure of the analyte. This additional parameter facilitates the de novo identification of compounds.
Apart from the calculation of CCS values, ion mobility mass spectrometry generates the exact mass, the retention time as well as high and low energy mass spectra and their combined use permit a precise characterization of the analyte. The possibility of deriving these parameters from a single experiment has a number of advantages as compared to a traditional ms analysis, such as:
- Simplification of data interpretation
- Enhanced rapidity in data interpretation
- Elimination of an interference of the analyte with other sample components
- Increase in the signal-to-noise ratio
In addition, ion mobility ms permits to broaden the range of applications including:
- The possibility to resolve co-eluting isomers
- Metabolite identification
- The conduct of directed analyses (quantification of a given analyte in a sample) as well as non-directed analyses (Screening)
Additional information on this instrument can be found at
Scientific literature on the use of this technique
Application example: Identification of the peptide leucine-encephalin.
LCMS: Electrospray ionization mass spectrometer with triple quadrupole detector coupled to HPLC (Waters Allience 2695) equipped with dual wavelength detector.
* Compound mixtures will be separated in the HPLC and the eluent is directly infused into the mass spectrometer where the corresponding mass/charge ratios are determined that permit compound identification. Mass measurements can only be carried out if the analyte can be converted into an ion in the gas phase. Electrospray ionization involves that a voltage is applied to the eluyent of the HPLC, which in combination with a high flow of nitrogen gas will produce an aerosol, comprising compounds to be analyzed, that is then introduced into the detector. The part of the instrument in which ionization occurs is referred to as strong>source.
* The instrument is equipped with two types of sources two types of sources: electrospray ionization source and ESI (ElectroSpray Ionization), a o surce APcI (Atmospheric Pressure Chemical Ionzation).
* The ions generated in the source are lead through several instrument parts that are under vacuum into the triple quadrupole analyzer. Link (http://www.waters.com/waters/en_US/MS---Mass-Spectrometry)
Schematic view of LCMS functioning
* The are no compound libraries available for our LCMS instrument, which hampers the analysis of unknown compounds. Analyses can be carried out in Scan mode, monitoring all mass/charge ratios of a chromatogram, although the instrument sensitivity is lower when working in this mode.
* Next to the Scan mode there are 4 other modes of analysis, namely: Daughter (Product) Ion Spectrum, Parent (Precursor) Ion Spectrum, Multiple Reaction Monitoring (MRM) y Constant Neutral Loss Spectrum.
* The most frequently used mode isMultiple Reaction Monitoring o MRM. This ionization mode is equivalent to the MS-MS mode in gas chromatography, in which a specific ion is selected (precursor ion) in quadrupole 1 (Q1) from the totality of ions generated in the ionization source. This ion will be fragmented in the argon gas containing collission chamber (Q2), and some or all fragmetns of this ion will then be introduced into the thrid cuadrupole (Q3) for detection. This mode is the approach of choice for the quantification of compounds.
Mass spectra in Scan and MRM mode of the pest-control substance dimetoate
Possible analysis types: peptides and proteins, other polar compounds, environmental pollutants, pesticides, drugs, sugars and polysaccharides. Separation by liquid chromatography implies that experimental and buffer conditions are identified that guarantee the solubility of the analyte.
* There is also the possibility to directly infuse compounds into the mass spectrometer with the help of a syringe pump. This permits the rapid analysis of macromolecules like proteins or DNA fragments. .
* Salts and buffer compounds interfere with electrospray ionization. Therefore, compounds to be introduced into the instrument have to be present in a volatile solvent.
* Identification and quantification of metabolites with anti-parasitic activities present in gland secretions of the hoopoe:
Martín-Vivaldi et al., (2010). Proceedings of the Royal Society B: Biological Sciences, 277, 123-130
* Determination of oxidised and reduced glutathione, nitrosoglutathione and ascorbic acid in different samples of plant origin..
Airaki et al.,(2011). Plant Cell Physiol, 52, 2006-2015
* Determination of plant hormones (salicylic acid, abscisic acid, indolacetic acid, jasmonic acid, cis-12-oxo-phytodienoic acid (OPDA)..
Torres-Vera et al., (2014) Molecular Plant Pathology, 15, 211-216
* Determination of pest-control substances in different simple types:
Boltner et al., (2008) Microbial Biotechnology, 1, 87-93
Delgado-Moreno y Peña (2009) Science of The Total Environment, 407, 1489-1495
Peña et al. (2011) Chemosphere, 84, 464-470
* The gas chromatograph is equipped with an injector (model 1079) that permits injections in split/splitless mode and in large volumes, due to the possibility to increase the vaporization temperature, which in turn enhances the sensitivity of analysis significantly.
* The mass spectrometer permits internal ionization by electronic impact or by chemical ionization (the first ionization mode generates more complex spectra with a large number of ions, this type of spectra is well suited for the identification of compounds, whereas the second ionization mode is softer and gives consequently rise to fewer ions. The spectra are frequently restricted to the molecular ion and can be used to determine the molecular weight of target compounds..
* The mass detector can acquire in Full Scan mode (over a wide ion range) and in mode SIS (Selected Ion Storage: only one ion or a narrow ion range is recorded) and can in addition support Ms/Ms and even Msn type of analyses (n<10).
Análisis Full Scan and SIS searching for the mass 128 m/z indicative of naphthalene
Standard GC-I and II analysed by Ms/Ms. It is shown in detail how some analytes (Clortal Dimetil y Dietofencarb; Isocarbofos y Tetraconazol), that elute almost simultaneously are identified correctly.
* The software contains a electronic impact compound library (NIST08) with more than 190.000 entries, which permits a comparison with recorded spectra and which is therefore very helpful for the de novo identification of compounds. The subsequent use of standards will confirm the identity of the compound identified. In case you require more information on GC-MS please refer to here.
Description of the autosampler.
* The instrument is also equipped with an autosampler CTC CombiPal ( http://www.palsystem.com/index.php?id=467) that enables the injection of up to 90 liquid samples stored in thermostated vials.
* This equipment also supports injection methods of Static Head Spacetype (http://www.palsystem.com/index.php?id=470) for vials with compounds in the gasphase as well as SPME (Solid Phase Micro Extraction) for compounds in the gas or liquid phase (HS-SPME and DI-SPME, respectively) (http://www.palsystem.com/index.php?id=471) .
* The use of SPME coupled to the sampler arm permits the conduct of analyses in which the steps extraction, concentration and injection occur in fully automated manner, not requiring the presence of an operator. SPME requires the development of a method and can be used to achieve high-sensitivity measurements. For further information on SPME please visit (http://www.sigmaaldrich.com/analytical-chromatography/sample-preparation/spme.html) y (http://www.nature.com/nprot/journal/v5/n1/full/nprot.2009.179.html.).
SPME process and its application in the study of volatile compounds form plant leaves making use of the sampler. Proceso de la y aplicación al estudio de volátiles en hojas de plantas mediante sistema acoplado a muestreador CombiPal (tomado de Asaph Aharoni et al., 2003, The Plant Cell, 15, 2866-2884). SPME is a process in which compound extraction and concentraion is accomplished using a fiber. Samples are typically in the gas phase or present in aqueous solution.
Possible types of analysis:
The very nature of an GC analysis implies that the compounds to be analyzed possess an elevated vapour pressure (volatile). per se.Alternatively, they can be transformed into a more volatile compound following a chemical reaction (referred to as derivatization). Due to this reason the analyte molecular weight is rarely beyond 600 uma. Two different analysis types can be distinguished .
1.) Analysis of unknown compounds. (screening).
* The method consists in the injection of a sample of which little or no information on the contained compounds is available. This method requires a significant effort in establishing the protocol for the extraction and the chromatographic analysis of the sample. The combined used of recorded spectra and a spectra library enable compound identification.
* This is primarily a qualitative approach, however, a compound quantification can be achieved by comparing recorded spectra with those of corresponding compound standards. An example of this type of analysis is the determination by SPME of volatile compounds of plants exposed to different types of stress..
SPME analysis using two different fibers of compounds derived from an infected plant (first and second chromatogram) against the corresponding sample from the non-infected plant (last chromatogram). Samples of infected plant show new or increased amounts of compounds as compared to the non-infected plant (highlighted by arrows).
2.) Target analysis.
* This type of analysis is employed when we know the analytes of interest presented in the sample. The extraction and analysis protocols can be based on existing protocols for similar compounds or on protocols employed by official control bodies (for example protocols of the European Union for the detection of different pesticides http://www.eurl-pesticides.eu/docs/public/home.asp?LabID=100&Lang=EN).
* This type of analysis is basically used for quantification purposes. Representative examples for this analysis type is the mutiresidue method to identify 130 different pesticides in animal fodder by GC-Ms/Ms or the method to determine aromatic (PHAs) and aliphatic compounds in oil polluted marine sediments.
Standards GC-I and II containing 90 different pesticides at a concentration of 300ppb analysed by the multiresidue method Ms/Ms.
* Analysis of 130 pesticides by GC-Ms-Ms from animal fodder extracted by QuEChERS
* Analysis of aromatic and aliphatic hydrocarbons from sediments or contaminated water. Sediment extraction using an liquid-liquid approach and separation by solid phase extraction columns. Extraction form water by solid phase microextraction with direct immersion. Acosta-González et al. (2013) Environmental Microbiology 15:77-92 (link: http://www.ncbi.nlm.nih.gov/pubmed/?term=Environmental+Microbiology+15(1)%3A77-92)
* Qualitative analysis of volatile compounds secreted by plants by head-space solid phase microextraction .
* Qualitative and quantitative analysis of pesticide degradation in aqueous matrices by solid-phase microextraction using head-space or direct-inmersion modes Castillo et al. (2014) Journal of Hazardous Materials 267:119-127 (2014) (link: http://www.ncbi.nlm.nih.gov/pubmed/?term=Journal+of+Hazardous+Materials+267%3A119-127+(2014)
Electronic Paramagnetic Resonance Spectrometry
Optical Emission Spectrometry
* ICP-OES (inductively coupled plasma-optical emission spectrometry) is a commonly used technique for the determination of trace concentrations of elements in samples based on atomic spectrometry. Routine determination of 70 elements at the same time can be made by ICP-OES (except noble gases, halogens, C, N and O).
Schematic view of the principle of analysis
When atoms absorb energy they become excited, but this excitation state is unstable. Thus, they decay to a less excited state, which is accompanied by the emission of photons. Every element has its own characteristic set of energy levels and thus its own unique set emission wavelengths. It is this property that makes atomic spectrometry useful for element-specific analytical techniques.
In ICP-OES, the sample is subjected to temperatures high enough to excite atoms. Once they are in excited states, they decay to lower states. The intensity of the light emitted at specific wavelengths is measured and used to determine the concentrations of the elements of interest.
Varian 720-ES ICP-OES espectrometer
Quantitative measurements of Al, As, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, Se, Si, Ti, U, V and Zn. Please, contact for the possibility of measuring other elements.
Semi-quantitative measurements of almost all the elements is periodic table. We can measure solid and liquid samples. We have protocols to solubilize solid samples from plants and soils, but we can easily implement any other. We also accept liquid samples that need digestion before measurement. Please, contact to service for more information.
How to prepare samples for the Mineral Analysis Service:
Solid samples should be in eppendorf tubes or any other type of capped tube, suitably labelled and weighed. It is very important to indicate the weight on the request for analysis. Organics: minimum 0,1; maximum: 0,5 g. Normally, 0.15 g is advisable. Soils: minimum: 0.1; maximum: 1 g. For amounts less than 0.1 g or any other questions, please contact the service manager. In the event that each sample has a different weight, this can be recorded on a separate sheet.
Liquid samples must be in tubes, suitably labelled and with a minimum volume of 10 ml. In case of smaller volumes, please contact the person in charge of the service.
It is a miniaturised gas chromatography equipment for the quantitative analysis of permanent gases and low molecular weight volatile compounds. Its main advantage is its speed (typical chromatographic path is less than 100 seconds), versatility (the use of 3 chromatographic columns in parallel allows the analysis of different gases in a single injection) and convenience (its small size allows it to adapt to the needs of sampling both in the laboratory and in the field, the latter as an option not available at the moment). Compounds are identified by prior analysis of standards, so it is not the best option for the analysis of unknowns. Its miniaturised technology allows it to inject quantities as small as 0.2 µl and its sensitivity allows it to detect concentrations in the ppm range. The 3 chromatographic columns included are Molsieve 5Å 10 m, PoraPlot U 10 m and CP-Sil 5 CB 6 m, allowing the analysis of compounds as varied as atmospheric permanent gases (separates N2 from O2), methane, CO, natural gas, biogas, hydrocarbons up to C10, aromatics, organic solvents, SO2, halogenates, SH2, ethylene, acetylene, N2O and others. A fourth chromatographic column can be added for very specific applications.
|QTOF Cuantitativo (€/hora)||19,03||22,32||51,31||56,2|
|Microcalorimetría (ITC)||Recorrido Titulacíon (€/hora)||10||11,73||20,55||30|
|Análisis de datos (€/Experimento)||3||3,51||6,16||9|
|Autoservicio (€/ hora)-Sólo personal de la EEZ||0,5|
|Análisis Mineral||Analisis por ICP-OES (€/muestra)||4,18||4,9||9,13||10|
|Digestión y Analisis por ICP-OES (€/muestra)||5,18||6,07||11,05||12,11|
*A los precios de OPIS/Unv y Empresas se añade el IVA
INFORMACIÓN PARA EL USUARIO
1) Las muestras deberán llegar disueltas en el disolvente adecuado y correctamente envasadas en viales, listas para su análisis. Como norma general, no se aceptarán muestras que requieran de ningún proceso de extracción, aislamiento, purificación o concentración. De no cumplir algunos de los requisitos anteriormente expuestos, se recomienda un contacto previo con el responsable del servicio.
2) Se deberán proporcionar patrones del analito a determinar preparados en las mismas condiciones y en un rango adecuado de concentraciones, si se quiere realizar análisis de tipo cuantitativo.
3) La “Fecha de solicitud” que aparece en el formulario de la página anterior es sólo orientativa. La fecha que se tendrá en cuenta para determinar el puesto que ocupan sus análisis entre el total del trabajo del servicio, será la fecha efectiva de llegada de las muestras. En ese momento se generará una Orden de Trabajo, con respecto a la cual usted podrá solicitar una fecha probable de finalización de los análisis.
4) Como norma general, las muestras analizadas se conservarán 6 meses, tras lo cual serán destruidas. Si se solicita podrán ser devueltas con el consiguiente cargo por el coste del envío.
The primary objective of the Scientific Instrumentation Service consists in providing analytical infrastructure, to assure its maintenance and to conduct analytical analyses for research groups of the EEZ.
Its activity is primarily centered on chromatographic analyses of organic compounds and their elemental analysis.
Requirements and limitations
- A demand must not surpass 4 weeks of work.
- Micro-demands are accepted (less than one day of work) that will be handled with priority.
- The sequence of demand handling is determined by the official record of demands at the Scientific Intrumentation Service.
- Samples to be analyzed need to be prepared correctly, i.e. impurities and interferences resulting from the preparation/extraction process have to eliminated by the client. Is this is not the case samples will need to be submitted by members of the Scientific instrumentation Service to additional treatments that will be billed in addition to standard costs
- The minimal sample amount is of 0.5 ml
- Together with the samples to be analyzed the corresponding standard solutions (present in the same solvent) have to be provided by the client.
- The samples to be analyzed have to be filtered in the presence of members of the Scientific Instrumentation Service
- Samples have to be provided with correct labelling, indicating also the date of sample preparation.
Cursos de formación organizados por SIC-EEZ: En el mes de Octubre de 2016 se celebró la II Edición del curso “Introducción a las técnicas cromatográficas: GC-MS, LC-MS“.
Centro de Instrumentación científica de la Universidad de Granada: http://cic.ugr.es/
Unidad de proteómica de la Universidad de Complutense de Madrid: http://www.ucm.es/gyp/protenomica
- Modelo de Solicitud de Análisis Mineral-ICP óptico EEZ-Granada [ES] (.doc)
- SOLICITUD Cromatografia [ES] (.pdf)
- Solicitud microcalorimetría [ES] (.docx)