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Instruments

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Our ESI-FTICR MS, an Ionspec QFT-7 from Varian Inc, was installed in Oct. 2006. Equipped with a Micromass/Waters Z-Spray electrospray ion source, a Micromass/Waters Z-Spray APCI source, and a MS Horizons NSI Source, it offers high resolution measurements and is a stunning choice for ion manipulation as well as MSn.

Both electrospray ionization (ESI) and nanospray ionization (NSI or nano-ESI) allow a very gentle ionization of polar and ionic analytes with low to very high m/z. The analytes need to be dissolved completely in methanol, 2-propanol or acetonitrile. Acetone and/or THF and up to 50% of water or 20% dichloromethane or chloroform are tolerable for ESI as well, while up to 95% water can be used in NSI. The analytes should be stable against oxygen and moisture. Easily oxidized analytes may undergo oxidation during the ionization process. In positive ion mode a voltage of approx. 4 kV is applied at the spray capillary in case of ESI, and approx. 800 V in case of NSI). As a result, usually multiply charged, cationized ions of the kind [M+nH]n+ or [M+n(Cat1+)]n+ are detected. For APCI, the sample has to be volatile enough. Typically the [M+H]+ ions are generated.

Samples should be as pure as possible. Surface-active substances like SDS or inorganic salts suppress the ionization of the analytes as well as damage the instrument due to discharges. Surfactants sometimes cannot be detected due to interference with the ionization mechanism.

The quadrupole section of the mass spectrometer provides mass selection and ion storage as well as the possibility of collisional activation (CID) before the ions are transferred into the 7 Tesla superconducting magnet.

The quadrupoles also limit the mass range to a maximum of about m/z 6,500, but even higher m/z ions may be detected in the ICR cell when highly charged parent ions are submitted to tandem MS. The ICR cell (ion cyclotron resonance cell) provides a number of fragmentation methods like IRMPD, SORI-CID, ECD and the extremely time consuming BIRD. Additional leaking and pulsing valves facilitate gas phase reactions with gases and volatile liquids as well as stable-isotope exchange experiments, e.g., H/D exchange. Thus, this instrument provides us with many methods for structure elucidation, investigation of reaction mechanisms and physicochemical properties of the studied analytes.

A limited number of specially trained users can use the instrument themselves. Two PCs are located in room 13.06 for data evaluation.

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The GC/MS we use is a GC/MS system conisting of a 5977E MSD single-quadrupole mass spectrometer with a 7820A GC by Agilent Technologies. It consists of a gas chromatography equipped with a column suitable for analyte solutions in nonpolar, volatile solvents like hexane. Solutions in water or high boiling solvents will destroy the injection system as well as the column.

The samples have to be free of any salts and must contain highly volatile compounds only. As solvents for the samples, THF, diethyl ether, esters and hexane or propane are recommended. Again, aqueous solutions can´t be analyzed with this instrument.

Since the ionization is taking place after chromatographic separation, all analytes are gaseous already. Electron impact (EI) is used. The resulting mass spectra usually contain a lot of fragment ions (odd and even electron ions) as well as M*+ radical ions for the lucky ones. Just low resolution measurements are possible (R ≈ 1,000), since a single quadrupole is used as analysator. Further limiting is the mass range of approx. m/z 20 to 650. This drawback is partly compensated by the possibility of automatic comparison of the generated spectra with a data base.

Users may perform measurements on their own after some training (open-access instrument).

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There is also a HPLC/MS system in use, which is an Agilent 6210 system consisting of an Agilent 1100 HPLC with an additional DAD (diode array detector) and an ESI-MSD TOF by Agilent Technologies.

This instrument provides high resolution spectra at very high mass precision (below 5 ppm with external and below 2 ppm with internal calibration. Furthermore, internal calibration is provided by a second in-built sprayer orthogonal to the spray capillary and the first sprayer. The mass range (m/z 50 to 3,500) means that most ionic or polar compound can be detected, since ESI (electrospray ionization) is used to interface the LC system to the mass spectrometer. For higher mass analytes in positive ion mode a positive voltage of approx. 4 kV is applied at the spray capillary. Usually multiply charged, cationized ions of the kind [M+nH]n+ and/or [M+n(Cat1+)]n+ are detected. Samples should be as pure as possible. All other precautions are described in the FTICR section.

Direct infusion experiments can be performed by trained users, as long as they ensure that only filtered (4 µm filters), completely dissolved solutions are sprayed. Also, the sample concentration should be kept as low as possible to prevent carry-over and damage to the detector, a maximum concentration of 100 µmol/L is requested. Maximum number of counts per peak is 1,000,000 (106)! If you have higher counts, please dilute immediately! The saturation of the detector also leads to a dramatic loss in mass accuracy.

For LC/MS measurements, the measurements are performed by the users themselves after some training. In addition to the precautions which should be taken into account for direct infusion measurements, the user has to ensure that all compounds in the sample solution are suitable for HPLC measurements. The samples must neither react with the column nor stick to the column for eternity. The pH of all samples and the solvents used should be between ph 7 and pH 4. As buffers, all highly volatile buffers (with anions like carbonate, acetate or formate and ammonium, triethylammonium) at a maximum concentration of 10 mmol/L can be used.

Acids/bases/buffers with low volatility like, e.g., sulfuric acid, phosphate buffers or sodium hydroxide are strictly forbidden to prevent damage.

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EI-MS
EI-MS
Image Credit: Dr. A. Springer

Starting mid of July 2015, we will be able to offer a better service. The MAT 711, built in the seventies of the previous century by the company Varian MAT retired after more than forty years of service. With access to a modern instrument, an Autospec Premier (waters Co.), we can offer not only EI, but CI and FI/FD. FI/FD are realized by the possibility to change the ion source to a LIFDI source (Linden CMS). Also, analyte mixtures can be separated by gas chromatography prior to analysis by mass spectrometry.

EI

When EI (Electron Impact) is used for ionization, the mass spectra often show the M•+ radical cation. This is usually accompanied by characteristic fragment ions with even or odd numbers of electrons which can be used to derive conclusions regarding the structure of the analyzed molecule. Some analytes might form stable radical anions, which suggests acquisition of negatively charged species as well. The analytes should be volatile enough for vaporization before ionization (boiling point/point of sublimation below 350°C in vacuum).

CI

Using CI (chemical ionization), often the protonated, closed shell ions ([M+H]+) are formed, especially when a bath gas like methane is used. The ionization is softer than with EI, but usually fragment ions are detected as well. In addition to that, an increased chemical noise coming from bath gas and bath gas adducts can be found. Like in EI, the analytes have to be volatile enough, i.e. the boiling point/point of sublimation is below 350°C in vacuum. The measurement of negatively charged ions is possible as well.

FI and LIFDI

After some time to become familiar with this new possibility, we will be able to offer FI (field ionization) and a special form of FD (field desorption), LIFDI. The required alternative ion source is produced by Linden CMS. The special sample introduction is very interesting for (metal-)organic compounds which are labile to oxygen and/or humidity. Typically, the M•+ radical cations are formed like in EI. Although is much softer when compared to EI and CI, fragment ions may also be detected. Nevertheless, analysis of labile compounds is possible. Another interesting fact is that the volatility of the analytes is not as important as for EI and CI.

Common remarks to the instrument

Standard measurements are performed with low resolution (R approx. 1000). To achieve good results and to prevent damage to the instrument it is generally necessary to submit solid or liquid or solid pure compounds with sufficient volatility (see above). For measurements of this kind, only the first digit is significant.

High resolution (up to R = 80.000) measurements are possible, but more complex and time-consuming. Due to loss of sensitivity, a sufficient intensity of the ion of interest is necessary. Also, the mass range fo high resolution measurements is limited to approx. one decade. The mass accuracy typically is better than 5 ppm.

Coupling to GC

If analyte mixtures with sufficient volatility are provided, coupling to GC is possible as well (precondition: the sample has to vaporize completely at 300°C or lower temperature, low boiling solvents like e.g. hexane or ethyl acetate have to be used, solutions with water or high boiling solvents cannot be analyzed to prevent the instrument from being destroyed).

Who can use the instrument?

Due to complexity of the instrument, most measurements are carried out by the service personnel. When doing a longer research project with mass spectrometry on this instrument as a major topic, independent measurements are possible as well - but only after successfully completing a special training.

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With the ESI-IMS-MS/MS, an UPLC-Synapt G2-S HDMS by Waters Co., a new instrument has arrived at the mass spectrometry core facility.

The system consists of two instruments coupled together:

  1. An extremely powerful chromatographic system, an Acquity UPLC

    This machine consists of a binary pump system which allows generating pressures up to 1000 bar (15000 psi). With these pumps, chromatographic columns packed with extremely small particles (down to 1.7 µm) can be used. Very efficient and extremely fast separations can be performed.

    Further more it consists of an autosampler which can be cooled down to 4°C, a column oven, and a photodiode array detector (PDA), which is capable of detecting light in the visible and the UV range.

    Since capillaries with an extremely small inner diameter and tiny particles as packing material are used, only specially filtered eluents and samples can be used to prevent blockage.

  2. An ESI-IMS-MS/MS (Synapt G2-S HDMS).

    The hybrid mass spectrometer principally consists of a Q-TOF instrument with the option to perform ion mobility measurements as well.

    As options for ionization we currently have either a Z-Spray-ESI-ion source or a NSI-ion source. Both are equipped with a second sprayer (lock spray). This allows additional external calibration while measuring. Thus, an extremely good mass accuracy can be achieved (about 1 to 2 ppm). The resolution of this Time-of-Flight (TOF) mass spectrometer is very high as well - more than 40.000 (FWHM) can be achieved. The extremely high mass range (m/z > 30.000) is very interesting, especially for very large, non-covalently bound complexes like intact antibodies. A further benefit of this instrument is its ion mobility cell. The separation in this region of the instrument is not only induced by the m/z of the ions analyzed, but also by their shape. Thus, not only a very fast separation of very complex mixtures is possible, but also the separation of isobaric substances like peptides with reverse sequences. The IMS is also very important, because the collision cross sections of the ions can be derived from the drift times.

    When ESI or NSI is used, the mass spectra will show cationized ions, [M+H]+ or [M+(Catn+)]n+, as well as multiply charged ions. Even for non-covalent complexes, fragment ions are rare. Since the instrument is very sensitive, chemical noise will add up to the background spectrum. The basic requirements which are pointed out in the FTICR-MS and HPLC-MS sections are valid for this instrument as well.

    Measurements carried out by the service personnel are offered. The instrument also can be used for long-term research projects carried out by a limited number of specially trained users.

     The UPLC and the HPLC system are both equipped with an additional DAD detector. Within the service we offer a standard C-18 column of the respective instrument manufacturer. For special demands, the users can bring their own chromatographic columns. The additional nanospray ion sources which can be equipped to the ESI-IMS-MS/MS or the ESI-FTICR-MS can only be used in the offline mode, i.e., without coupling to a separation technique.

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The UPLC and the HPLC system are both equipped with an additional DAD detector. Within the service we offer a standard C-18 column of the respective instrument manufacturer. For special demands, the users can bring their own chromatographic columns. The additional nanospray ion sources which can be equipped to the ESI-IMS-MS/MS or the ESI-FTICR-MS can only be used in the offline mode, i.e., without coupling to a separation technique.

A limited number of specially trained users can use the instrument themselves. Two PCs located in room 13.06 offer the chance of data evaluation.