|Agilent HPLCs||Bruker Autoflex III|
|Agilent 6890/5973||Agilent 1200/6130|
|Waters LCT||Thermo MAT 95|
|Thermo Trace GC||Waters CapLC|
Agilent 1100 Capillary HPLC or 1200 analytical HPLC
Purchased (as demo equipment) in November 2004 with funds from Professors Peters, Oakley, DiMarchi, Stone, and the Chemistry Chairperson's Office.
The MSF has two Agilent 1100 liquid chromatographs. The capillary liquid chromatograph has a thermostatted, 100-position autosampler compartment and the pump can create reproducible gradients with flow rates from 1 to 100 µL/min. The analytical scale HPLC has 40 position autosampler (no thermostat, 1-100 uL injection volume); its pump can generate gradients with flow rates between 0.1 to 2.5 mL/min. Either HPLC can be connected to a variable wavelength (200-600 nm) UV-VIS absorbance detector with a 1 µL flow cell. Only one of the HPLC pumps and autosamplers can be used for any given experiment (we only own one detector).
Customers may use their own columns if they so desire, and the mass spectrometry facility has a handful of columns available. Please be advised that for LC-MS applications, only volatile mobile phases and volatile mobile phase modifiers may be used. We typically employ columns with inner diameters of 0.3 mm, 0.5 mm, 1 mm for capillary scale separations. Columns with inner diameters of 2.1 mm or 4.6 mm are utilized for analytical scale analyses.
Both HPLC's are found on carts in Chemistry A454. Self-serve HPLC costs $6, staff-run HPLC costs $16.back to the top
Bruker Autoflex III MALDI-TOF Mass Spectrometer
Bought new in March 2009 with funds from the College of Arts and Sciences and the Chemistry Chairperson's Office
The Bruker Autoflex III is our matrix-assisted laser desorption/ionization (MALDI) reflectron time-of-flight mass spectrometer. It is commonly used to analyze peptides, proteins, polymers, oligonucleotides and oligosaccharides. If a customer's compound can absorb 355 nm light, this instrument can also be used for laser desorption/ionization (LDI) experiments. The BioTools module has been installed to assist with intepreting mass spectra from proteins, oligosaccharides, and oligonucleotides. Talk to Dr. Karty for training in this package. The MSF also owns a copy of the flexImaging software package facilitating MALDI imaging applications.
At present we request a minimum of 5 µL of a solution containing 10 µM analyte for peptide samples and 5 µL of a solution containing 50 µM analyte for protein samples. Please remove all detergents (e.g. Triton X-100, SDS), chaotropes (e.g. urea, guanidinium HCl), and involatile liquids (e.g. glycerol) from sample prior to submission as these can prevent ionization and/or detection of your analyte. Also, samples should have ionic strengths of 25 mM or less. We recommend ZipTips™ for sample cleanup prior to MALDI MS. Organic polymers should be brought up as dry powders if solvent free MALDI is to be performed.
- Source: 200 Hz frequency tripled Nd:YAG laser (355 nm) supports:
- Matrix-assisted laser desorption/ionization (MALDI, positive and negative ion modes)
- Laser desorption/ionization (LDI, positive ion mode)
- Inlets: 384-well steel plate, AnchorChip, NALDI (adapters for ABI targets can be purchased)
- Mass analyzer: linear time-of-flight or reflectron time-of-flight
- Mass Range: m/z 400-150,000 (linear, MALDI);
- m/z 200-8,000 (reflectron, MALD/LDII)
- Mass accuracy: (all assume quadratic curve fit with 4 calibrants):
- Reflectron, external calibration: ±20 ppm
- Reflectron, internal calibration: ±5 ppm
- Linear, external calibration (m/z < 3000): ±50 ppm
- Linear, internal calibration (m/z < 3000): ±20 ppm
- Linear, external calibration (m/z > 3000): ±150 ppm
- Linear, internal calibration (m/z > 3000): ±100 ppm
- Resolving Power:
- Reflectron: 12,000 @ m/z 2465
- Linear: 4,000 @ m/z 2465; 1,000 @ m/z 12,361
- Sensitivity: 1 fmol for a single peptide, low pmol for large polymers/proteins
The Autoflex III is located in the center of Chemistry A411. Current rates for Chemistry Department users are $6.00/analysis for self-serve, $16 if MSF staff perform the analysis. The Autoflex III training manual can be found here.back to the top
Agilent 6890N Gas Chromatograph and 5973 Inert Mass Selective Detector
Acquired new in June 2005 with funds from Professors Peters, Williams, Evans, the undergraduate teaching laboratories, and the Chemistry Department Chairperson's Office. 100-position autosampler tray purchased with MSF funds in July, 2006.
The Agilent 5973 Inert is a robust quadrupole mass spectrometer that can be fitted either with electron impact or chemical ionization sources. It serves as the detector for the Agilent 6890N GC. The 5973 Inert is the smaller box on the left-hand side of the photo. The Agilent 6890N is the inlet for the 5973 inert quadrupole mass spectrometer. It is the larger box on the right-hand side of the photo. The GC-MS system is fully computer controlled with ChemStation software, thus programming of the flow rates temperature gradients are quite easy. Look on the GC cabinet to find out which column and source are installed. IUMSF owns a small number of GC columns (the default column is a 30 m long, 0.25 mm i.d., 0.25 um dF DB-5MS from Agilent, or something equivalent); users may provide their own columns if they wish. Helium is the standard carrier gas in the MSF. The GC has a standard split/splitless injector and all gas flows are handled through the software. Therefore, the split ratio may be varied with ease. The 6890N is equipped with an autosampler and 100-position sample tray, enabling unattended recording of data. It also ensures that all injections are reproducible, enhancing the ability to accurately quantify components in complex mixtures.
- electron ionization, 70 V (EI)
- chemical ionization (CI, with CH4, C4H10, and NH3 available as reagent gases)
- It can analyze both positive and negative ions
- Inert source reduces analyte decomposition during ionization
- Inlet: Agilent 6890N gas chromatograph
- Mass analyzer: single quadrupole
- Mass range: m/z 1.6-800
- Mass accuracy: ±0.2 Da
- Resolution: unit (singly charged ion peaks ~0.7 Da wide)
- MSF owns the NIST 2002 EI mass spectral library (>170,000 spectra)
- Users can make their own mass spectral libraries
Be advised that this instrument is used by the A315 class in the fall (three afternoons per week all semester) and A314 in the spring (four afternoons during two weeks of the semester), so check ahead for availability during these times.
This instrument is located in Chemistry A454. Current rates for Chemistry Department users are $6.00/analysis for self-serve, $16 if MSF staff perform the analysis. The 6890/5973 training manual can be found here.back to the top
Agilent 1200 HPLC-6130 MSD
Purchased new in June, 2009 (as demo equipment) with funds from Professors Erin Carlson, Zach Aron, Silas Cook, Amar Flood, and Dennis Peters, the Chemistry Chairperson's Office, and the Mass Spectrometry Facility.
The Agilent 1200 HPLC-6130 MSD is a powerful walk-up instrument for synthetic organic chemists. The 1200 HPLC can generate binary gradients with flow rates between 0.1 and 2.5 mL/minute with 1% composition and flow rate precision. The autosampler can inject between 1 and 100 uL and can be directed to mix aliquots from different vials prior to injection as well as combine multiple aliquots from the same vial prior to starting the gradient. A 6-port column selection valve provides easy-to-use flexibility in separation chemistry with full software control. Currently port 1 is equipped with a 2.1x100 mm Zorbax C18 (3.5 um particles) column for LC-MS while port 2 has an empty loop for rapid (~1.5 minute run time) analyses of purified compounds; ports 3-6 are available for different columns. A diode array detector (DAD) allows for recording of UV-VIS absorption spectra (200-950 nm capabilities) during an experiment and up to 8 different wavelengths can be monitored simultaneously. The four mobile phases typically installed on the 1200 HPLC are:
A1: 95% H2O, 5% CH3CN, 0.1% HCOOH
A2: isopropanol (can be quickly changed for special experiments)
B1: 95% CH3CN, 5% H2O, 0.1% HCOOH
B2: methanol (intended for loop injections or HPLC-APCI-MS)
The 6130 MSD is a quadrupole mass spectrometer that can alternate between positive and negative ion modes in less than 0.5 seconds, allowing positive and negative ion data to be recorded throughout a separation in a single experiment. The 6130 is fitted with Agilent's multimode source facilitating simultaneous electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). These features allow one to perform multimode ESI/APCI alternating between positive and negative ionization modes every 2.5 seconds throughout an HPLC separation. The quadrupole can detect ions with m/z ratios between 10 and 3000 Th with 0.7 Th peaks (unit resolution). It should be noted that APCI can support normal phase LC-MS (e.g. hexane-2-propanol mobile phase).
The entire system is controlled by Agilent's Easy Access software system with vastly simplifies the interface for the casual user. Customers can use predefined methods or create their own with additional training and even delay long analyses until late evening any time day or night.
The 1200/6130 is located in Chemistry A454. Current rates for Chemistry Department users are $6.00 each for loop injections and $9.00 each for HPLC-MS analyses. The 1200/6130 training manual can be found here.back to the top
Waters/Micromass LCT Classic
Two instruments on indefinite loan from the laboratory of Dr. David Clemmer; originally purchased new in August 2005 by DEC.
The Waters/Micromass LCT Classic is an electrospray ionization/time-of-flight mass spectrometer (ESI-TOF). The MSF has two of these instruments in A411. The LCT next to the door (KC379) is set up for oligonucleotide analyses and has a Waters CapLC with autosampler as its inlet; its mobile phases are (A) 400 mM hexafluoroisopropanol (HFIP) buffered to pH 8.3 with triethylamine; (B) 1:1 mixture of mobile phase A and methanol; (C) methanol. The column installed with this system is a 0.5x250 mm Zorbax XDB-C18 packed with 5 um particles. It is intended for walk-up negative ion LC-MS analyses of oligonucleotides using a 20 minute gradient. The distribution of multiply charged ions can be interpreted using the ProTrawler software package from BioAnalyte. Users may bring their own columns and solvents
The LCT next to the refrigerator (KC366) is configured for high accuracy and small molecule measurements in the positive ion mode. It also has a Waters CapLC as its inlet. The CapLC has three solvents (A is 95% H2O, 5%CH3CN, 0.1% formic acid; B is 95% CH3CN, 5% H2O, 0.1% formic acid, and C is HPLC-grade CH3OH). These solvents can be readily changed if necessary. KC366 is also equipped with the LockSpray source which facilitates internal calibration for high precision work (e.g. formula matching for small molecules). This instrument is typically set up for flow injection analysis of small organic molecules (no column installed and pumping straight solvent C) and is intended for walk-up MS of synthetic organic compounds. A 0.5x150 mm Zorbax SB-C18 (5 um particles) column can be installed for LC-MS analyses.
- Sources: electrospray ionization (ESI), lock-spray ESI, atmospheric pressure chemical ionization (APCI)
- Inlets: Waters CapLC, Agilent 1100 CapLC, direct infusion
- Mass analyzer: orthogonal reflectron time-of-flight
- Mass range: m/z 10-4,000
- Resolving power: 5,000
- Mass Accuracy
- 50 ppm (external calibration)
- 5 ppm (internal calibration)
- aqueous 50% acetonitrile, 0.1% formic acid
- aqueous 50% methanol, 0.5% acetic acid
- 50% methanol, 50% chloroform
- aqueous 50% acetone, 0.1% formic acid
- aqueous 50% acetonitrile, 0.2% ammonia
- aqueous 50% acetonitrile, 2 mM ammonium acetate
- acetone or acetonitrile with 2 mM sodium acetate + 0.1% formic acid
- tetrahydrofuran may be used with some difficulty
- 200 mM hexafluoroisopropanol and methanol are used for ion pairing LC-MS of oligonucleotides.
Both LCTs are found in Chemistry A411. Current rates for Chemistry Department users are $6.00/analysis for self serve, $16.00/analysis when MSF staff perform the analysis and $18.00 for a staff-run, accurate mass, formula matching analysis. LC-MS with this instrument is $9 for self-serve and $25 if staff-run. The LCT training manual for small molecule MS can be found here; the training manual for large molecule LC-MS is here.back to the top
Thermo Electron Corporation MAT 95XP-Trap
Purchased new in March 2003 with funds from National Institutes of Health grant 1S10RR016657-01.
The MAT 95XP (manufactured by Thermo Electron Corp.) is our high resolution, high mass accuracy mass spectrometer. It is typically used to analyze small organic compounds synthesized by Organic Chemistry faculty research groups. It is an extremely versatile instrument with a variety of sources and inlets allowing staff to analyze everything from gasses to dendrimers. Its high mass accuracy (<5 ppm in magnetic sector scan mode aka BSCAN and <2 ppm in electric sector scan mode aka ESCAN) enables its data to be used to provide formula matches. That is, the mass spectra are precise enough to verify a chemical formula based on the sum of the mass defects from the constituent atoms. This instrument also has an ion trap that can be used in place of the detector to allow up to 10 stages of tandem MS (MS2-10) to be performed on ions isolated by its magnetic and electric sectors.
- Sources: electron impact (EI, variable energy), chemical ionization (CI, variable energy, choice of methane, isobutane, and ammonia as reagent gasses), fast atom bombardment (FAB), and electrospray ionization (ESI) (note: APCI is not currently available and the LCT is the preferred instrument for accurate mass ESI analyses)
- Inlets: direct insertion probe, high temperature desorption probe, Trace gas chromatograph, direct infusion, FAB probe, and Agilent CapLC
- Mass analyzers: double focusing magnetic sector - electric sector (reversed Nier-Johnson geometry B-E sector), 3D ion trap
- Mass range: m/z 2-900 (high resolution EI/CI); 140-3,400 (high resolution ESI/FAB); m/z 2-3,500 (low resolution EI/CI/FAB/ESI); m/z 10-2000 (ion trap)
- Mass accuracy:
- ±5 ppm (magnetic scan with internal calibration)
- ±2 ppm (electric scan with internal calibration)
- ±0.2 Da (ion trap)
- Resolving power: up to 60,000 (5,000 is routinely used)
- Capable of online, high resolution GC-MS and LC-MS
- Capable of MS10 with online ion trap (ion trap has mass accuracy and resolution similar to other the quadrupole devices in the lab)
The MAT-95 is located in Chemistry A411. Current rate for Chemistry Department users is $18.00 per EI/CI analysis and $25 for accurate mass GC-MS.back to the top
Thermo Electron Trace Gas Chromatograph
Acquired as part of the MAT-95 XP Trap system.
The Thermo Trace GC is one of the many inlet systems available for use with the MAT-95 mass spectrometer. The Trace GC is the box furthest to the left in the picture above. This gas chromatograph has electronic pressure control so setting the split ratio for the injection is simple. It has a split/splitless injector and is equipped with an Agilent DB-5 MS ((5%-phenyl)-methylpolysiloxane) 0.25 mm i.d., 0.25 µm df, 30 m long column (or something equivalent). We often use this instrument to facilitate accurate mass spectrometry of volatile samples.
The Trace GC is attached to the MAT-95 in Chemistry A411. Accurate Mass GC/MS (EI or CI sources) with the MAT-95 costs $25.00/analysis.back to the top
Waters CapLC High Performance Liquid Chromatograph
Acquired as part of the LCT purchase.
This HPLC was designed from the ground up for capillary-scale (<100 µL/min) analyses. It has capillary scale pump heads and employs high pressure mixing for gradient generation. Unlike the Agilent 1100, it does not require flow splitting for capillary flow rates. It has three buffers (A is usually 95% H2O, 5% CH3CN, 0.1% HCOOH; B is usually 95% CH3CN, 5% H2O, 0.1% HCOOH, C is usually CH3OH), and can create reproducible gradients with flow rates between 1 and 100 µL/min. The CapLC can be equipped with a Waters 2487 dual wavelength UV-VIS detector and must be controlled by MassLynx or Waters HPLC software. The MSF owns two which currently serve as the inlets for the LCTs.
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