Product
Review
Electrospray continues to evolve
Electrospray MS has become more eccessible to chromatographers wiihout sacrificing performance Although it might be hard to believe, fewer than 10 years have passed since the importance of coupling electrospray to mass spectrometry was recognized. This technique, in which multiply charged ions are generated, lowers the m/z of proteins and oligonucleotides to the range that is accessible even by quadrupoles. This has made the analysis of these biomolecules easier, although the technique can't really be called mature. Three years ago, Analytical Chemistry published a product review on electrospray mass spectrometers (Anal. Chem. 1994, 66, 481A-86 A)) So, how has electrospray MS changed? "In terms of fundamental electrospray, there have not been major advances, just evolutions," says Jack Henion of Cornell University. However, after admitting that changes have been incremental, mass spectrome-
trists have little difficulty listing the changes that differ only in priority assignments. The biggest developments in the commercial market have been the coupling of electrospray to ion trap and time-of-fllght systems, and the introduction of extremely low-flow electrospray (called nanoelectrospray). The most drastic change may be that ESI-MS is being marketed more to chromatographers than mass spectrometrists, a change that manifests itself particularly with software development. Representative ESI-MS systems are listed in Tables 1 and 2. Table 1 includes research-grade instruments, whereas Table 2 describes benchtop LC/MS systems. Only complete systems are listed in the tables. Readers should note that electrospray ion sources are available separately from companies such as Analytica of Branford (Branford, CT, 203-488-8899)
and Protana, formerly known as The Protein Analysis Company (Odense, Denmark, www.protana.com). Briefly, electrospray is an atmospheric pressure ionization technique that uses a high voltage to generate a mist of droplets containing charged analytes. Most commercial instruments use a gas to aid in droplet formation in a process known as pneumatically assisted electrosprav. As the solvent evaporates, ions with a charge opposite that of theapplied field migrate to the surface of these droplets, creating a surface charge density that eventually forces the droplets
to break into mcreasingly smaller droplets. In a mechanism that is not well understood, the electric field at the droplet surface becomes large enough, at some minimum droplet size, to desorb the ions into the gas phase. In another proposed mechanism, the droplets continue to break apart until they are so small that each droplet contains one ion, at which point the remaining solvent evaporates. Doug Mclntyre, Senior Applications Chemist at Hewlett Packard, summarizes electrospray: "At the core of electrospray is getting the droplets small enough in a narrow size distribution and then getting the ions to eject from the droplets with some degree of efficiency." Mass spectrometrists have more choices of mass analyzers with electrospray than they did three years ago. At that time, quadrupoles were the dominant option even though interfaces for magnetic sectors and FT-MS instruments
Analytical Chemistry News & Features, July 1, 1197 427 A
Product
Review
Table 1 . Summary of representative products
Product
7000 series
Platform II Quattro II Micromass 100 Cummings Center Ste. 407N Beverly, MA 01915 508-524-8200 www.micromass.co.uk Single (Platform) or triple (Quattro) quadrupole
Company
Finnigan 355 River Oaks Pkwy. San Jose, CA 95134 408-433-4800
URL Type
www.finnigan.com Single (SSQ) or triple (TSQ) quadrupole
Electrospray
Other ionization methods
Micromass 100 Cummings Center Ste. 407N Beverly, MA 01915 508-524-8200 www.micromass.co.uk Quadrupole-time of flight hybrid
Nanoelectrospray (10 nL1 |iL/min); normal spray (1 u.L-1 mL/min); capillary/ skimmer CID, heated capillary desolvation
Full range of flow rates: Nanoflow 10-1000 nL/min, Megaflow 1 u.L-1 mL/min
Megaflow (5 (J.L—1 mL/min) or Nanoflow (10-1000 nL/min)
El, CI, APCI, PBI, thermospray, FAB, DC I
El, CI, APCI, SFC, PB, thermospray, static and dynamic LSIMS, GC, DCI, and solids probe
N/A
TurbolonSpray: (2-1000 |xL/min) maximum ionization efficiencies for improved sensitivity. Compatible with 4.6mm i.d. HPLC methods. MicrolonSpray: (20-2000 nL/min) microflow source for flow injection or capillary LC applications. N/A
10-2500 (SSQ) 2500/4000 (TSQ)
2-3000 (Platform II) 2-4000 (Quattro II)
8000
30-3000
< 0.01% Unit
< 0.01% Unit
5 ppm RMS 5000 mlAm (FWHM)
0.01% Unit
Data system
DEC Alpha/Unix
SSQ can be upgraded to TSQ
MassLynx NT, running under Windows NT 4.0 on 200-MHz Pentium Pro hardware; supports interactive database searching, sequencing of proteins and oligonucleotides, simulating digests, and calculating masses and compositions Triaxial, CEC, and CE/MS probes available
MassChrom data-acquisition and editing software running on RISC Power Macintosh
Options
Special features
Nonlinear octapole assembly collision cell (TSQ); intelligent "data-dependent" experiments
MassLynx NT, running under Windows NT 4.0 on 200-MHz Pentium Pro hardware; supports interactive database searching, sequencing of proteins and oligonucleotides, simulating digests, and calculating masses and compositions Extended mass range to m/z 4500 (Platform II) or m/z8000 (Quattro II); triaxial, CEC, and CE/MS probes available Dual photomultiplier detectors; prefilters on analyzer rods
INA
Benchtop instrument
Reader service no.
401
402
403
404
Performance Mass range
Mass accuracy Mass resolution
428 A
Analytical Chemistry News & Features, July 1, 1997
Q-Tof
API 165 LC/MS API 365 LC/MS/MS Perkin Elmer Sciex 761 Main Ave. Norwalk, CT 06859 203-762-1000 www.perkin-elmer.com Single (165) or triple (365) quadrupole
API 165 LC/MS can be upgraded to API 365 LC/MS/MS
Jaguar
Mariner
Esquire-LC *
JMS-AX505/RSVP
JMS-MStation
Sensar Larson-Davis 1652 West 820 North Provo, UT 84601 801-343-3775
PerSeptive Biosystems 500 Old Connecticut Path Framingham, MA 01701 508-383-7700
JEOL 11 Dearborn Rd. Peabody, MA 01960 508-535-5900
JEOL 11 Dearborn Rd. Peabody, MA 01960 508-535-5900
www.lardav.com Time of flight
www.pbio.com Time of flight
Hewlett Packard Chemical Analysis Group 2850 Centreville Rd. Wilmington, DE 19808 800-227-9770 www.hp.com/go/chem Ion trap
www.jeol.com Magnetic sector
www.jeol.com Magnetic sector
Standard
1 u.L-1 mL/min (with splitter)
To 1 mL/min, orthogonalflow sprayer with pneumatic nebulizer, highcapacity solvent drying gas system with automated user control of gas-flow rate and temperature
1 (iL-1 mL/min
1 |iL-1 mL/min
N/A
N/A
APCI
El, CI, APCI, DCI, FAB, FD, GC, LC, PB
El, CI, APCI, DCI, FAB, FD, Fl, GC, LC, MALDI
20-5000 (standard mode) 600-10,000 (extended mode) 20ppm > 1000 at 1000 m/z
40-10,000
50-2000 (standard) 100-3400 (extended) 200-6000 (high) ± 0.2 m/z (standard range) 0.2 u (FWHM maximumresolution mode)
0-1500 (standard) at 3 kV 0-2300 (extended) at 3kV
2400 at 10 kV
Simul-Pulse Recorder data system
Data Explorer program for acquisition and processing under Windows NT
HP Vectra, ESQUIRE-LC software is combined with Windows-based HP 3-D LC ChemStation software
Various electrospray head options; single, or multiple-anode data collection
Nanoelectrospray
Nanoelectrospray needle assembly
Nanoelectrospray; LinkScan MS/MS
Tandem MS/MS or hybrid TOF configuration
Designed for interfacing to CE and other microcolumn separations techniques
Benchtop configuration, database search analysis programs for rapid sample analyses
Accommodates mobilephase ranges without flow splitter; swing-out spray chamber; multipole ion trap uses nonlinear resonance and phase locking for ion excitation and fast ejection; 13000 amu/s scan speed with unit resolution; AutoMS/ MS software feature automates MS/MS analyses
INA
Scanning CCD array detector specifically designed to improve sensitivity of recording mass spectra by a factor of 50 or more over standard point detector system
405
406
407
408
409
± 1 0 ppm 5000 m/Am
INA 25,000 m/Am (10% valley) INA HP Unix workstation or PC Windows
HP Unix workstation
•Co-marketed by Hewlett Packard and Bruker Systems, 19 Fortune Dr., Manning Park, Billerica, MA 01821, 508-667-9580
Analytical
Chemistry
News & Features, July 1, 1997
429 A
Product
Review
Table 1 . Continued
Product Company
URL Type
Electrospray
Other ionization methods
Performance Mass range Mass accuracy Mass resolution Data system
Options
Special features
AutoSpec series Micromass 100 Cummings Center Ste. 407N Beverly, MA 01915 508-524-8200 www. micromass.co.uk Magnetic sector
APEX II Bruker Daltonics 15 Fortune Dr. Manning Park Billerica, MA 01821 508-667-9580 www.bruker.com FT-MS with 3.0-, 4.7-, 7.0-, 9.4-T magnets
NewStar FT/MS Finnigan 355 River Oaks Pkwy. San Jose, CA 95134 408-433-4800
Full range of flow rates: Nanoflow 10-1000 nL7 min, Megaflow 1 LIL-1 mL/min El, CI, APCI, PB, thermospray, static and dynamic LSIMS, FD/FI, MALDI, GC, DCI, and solids probe
0.5-500 Lil/min, nanospray with 5-20 nL/min
< 1 nL-1 mL/min with split
El, CI, MALDI, glow discharge, solids probe, LSIMS, and GC
MALDI, LDI, El, CI, solids probe
3000 at 8 kV typically 1-3 ppm 60,000 (10% valley) OPUS running under VMS on a DEC AlphaStation 255/233 Depending on family member, can extend mass range to 10,000 and resolution to 120,000; TOF hybrid for high-sensitivity MS/MS; magnet bypass for high-sensitivity MS; focal plane detector
66,000 < 2 ppm > 100,000 m/Am XMASS software running under UNIX on SGI02 Shielded magnets
> 30,000 2 ppm > 1,000,000 m/Am Sun Ultra SPARC/Unix
Dual-conversion dynode photomultiplier detector
Side-kick ion injection system; rf-shimmed Infinity cell
Dual cell; multiple ionization techniques simultaneously (MITS) e.g., ESI and MALDI 412
Reader service no. 410
were also available. Robert Cody of Jeol says that electrospray sources for magnetic sector instruments have evolved and are now competitive with the sources for other types of analyzers. Three years ago, he says, that wasn't true. Now, sources for ion traps and time-of-flight systems are also possibilities. Use of ion traps was already being predicted in 1994, but it has become a reality with the Finnigan LCQ and the Bruker/ Hewlett Packard Esquire LC. The primary advantage of the ion trap is its MS capability. MS(n) capabilities (as well as higher resolution and nondestructive detection) are also available with FT-MS instruments, but they're typically geared to the mass spectrometry research laboratory and are much more expensive. The ion traps, in contrast, are relative bargains at 430 A
411
www.finnigan.com FT-MS with 3- or 7-T magnet
INA
less than $200,000. Tandem MS, which can be achieved with triple quadrupole, ion trap, magnetic sector, or FTMS instruments, is particularly useful because it provides structure and sequence information in addition to the molecular weight that is found by MS alone. Let the ions fly
At first glance, electrospray and time of flight would seem completely unsuited to one another. Kelsey D. Cook of the University of Tennessee explains the initial problem: "Time of flight is intrinsically pulsed because you need an identified start time to measure the flight time. Electrospray is not intrinsically pulsed. You've got a flow of solution, and there's a period of instability at the beginning of the flow. If you're pulsing [the flow],
Analytical Chemistry News & Features, July 1, 1997
you're operating intrinsically in that unstable region." The question becomes how to keep the flow in the stable regime while giving the mass analyzer the ion packets it needs. The answer is actually quite simple: Keep the electrospray stream constant, and extract the ions orthogonally. Orthogonal acceleration provides a higher effective duty cycle and allows higher resolution than conventional pulse-forming methods in time-offlight mass spectrometry. Two electrospraytime-of-fllghtinstruments were introduced at Pittcon '97—the Mariner from PerSeptive Biosystems and the Jaguar from Sensar. Although they are the first electrospray instruments with only atime-of-flightanalyzer, they are not the first to includetime-of-flightcapabilities. That designation belongs to the Q-Tof from Micromass, which combines a single quadrupole in series with an orthogonal reflectron time-of-flight analyzer and can be used in either MS or MS/MS mode. In MS mode, the Q-Tof's quadrupole and the hexapole that follows are used in broadband mode, serving only as ion guides to the time-of-flight analyzer. For MS/MS analysis, the quadrupole resolves precursor ions and the hexapole serves as a collision cell to generate fragment ions. Lance Nicolaysen of Micromass says that the Q-Tof increases the sensitivity of fullscan MS/MS measurements by a factor of 100 because the multichannel nature reduces the number of ions that are "wasted" during detection on a scanning analyzer, such quadrupole. How to interface electrospray and time of flight is the first question. The second question is why? Cook cites the analysis speed and the ability to analyze lowcharge species. "On a microsecond timescale, you can create a time-of-flight mass spectrum of everything in the ion pulse," he says. "That is useful for LC interfaces of various kinds, including CE interfaces, where you have very narrow peaks and need to get a spectrum in a hurry, even quicker than you may be able to scan a quadrupole." According to Alan Rockwood of Sensar Larson-Davis high dataacquisition speed confers advantages beyond the obvious one of compatibility with fast separations Even lower-speed separations benefit because, in a slow separation, longer signal averagingtimesallow improved detection limits to be achieved. Ron Orlando of the Complex Carbohydrate Research Center at the University of Georgia also highlights analysis speed. He says that fast scantimesare critical for sep-
Table 2. Summary of r e p r e s e n t a t i v e benchtop LC/MS s y s t e m s .
Product
LCQ
Navigator
Company
Finnigan 355 River Oaks Pkwy. San Jose, CA 95134 408-433-4800
Finnigan 355 River Oaks Pkwy. San Jose, CA 95134 408-433-4800
URL Type Electrospray
www.finnigan.com Ion trap < 1 nL-1 mL/min
www.finnigan.co.uk Quadrupole 2 u,L-1 mL/min
Other ionization methods Performance Mass range (m/z) Mass accuracy Mass resolution
APCI (50 uL-2 mL/min) APCI (200 uL-2 mL/ min)
APCI
APCI
APCI
50-2000 > 0.01% Unit (> 10,000 with ZoomScan) LCQ data-processing and instrument control software running under Windows NT
1600 > 0.01% Unit
50-3000 ±0.13 m/z INA
2-1600 0.01% Unit
30-1200 0.01% Unit
Windows NT data system
HP ChemStation; HP LC/MSD software is fully integrated with Windows-based HP 3-D LC ChemStation software
MassLynx NT running under Windows NT 4.0 on 200-MHz Pentium Pro hardware
MassChrom data-acquisition and editing software running on Power Macintosh
Triaxial, CEC, and CE/ MS probes available; extend Quattro LC mass range to 4000 Compatible with many LC systems via contact closure; full control of Hewlett Packard pumps, autosamplers, and UV detectors, including DAD acquisition; full control of Gilson pumps and autosamplers; full control of Waters 2690 system
Data system
HP 1100 Series LC/MSD Hewlett Packard Chemical Analysis Grp. 2850 Centreville Rd. Wilmington, DE 19808 800-227-9770 www.hp.com/go/chem Quadrupole To 1 mL/min; orthogonal-flow sprayer with pneumatic nebulizer; high-capacity solvent drying gas system with automated user control of gas-flow rate and temperature; spraying system does not require mechanical adjustment
Options
100-4000 extended mass range; nanoelectrospray; CE interface
INA
Manual injection valve
Special features
Intelligent "data-dependent" experiments; SEQUEST MS/MS protein/peptide database searching software
Compatible with most HPLC systems and autosamplers; source CID; open-access LC/MS
Automatic tuning with built-in calibration standard delivery system; hinged swing-out spray chamber
414
415
Reader service no. 413
arations with narrow peaks, such as CE. "If a peak is 5-6 seconds wide and you want t1 scans, you're seriously pushing a quadrupole to scan that quickly." For example, PerSeptive's Mariner acquires 8000 time-offlight spectra per recond over r mass range of 40-4000 Da. The acquisitions are summed into as many as 10 spectra per second. Sensar Larson-Davis's benchtop Jaguar acquires 5000 spectra per second over a mass range of 20-5000 Da and sums into as many as 100 spectra per second.
Platform or Quattro LC Micromass 100 Cummings Center Ste. 407N Beverly, MA 01915 508-524-8200 www.micromass.co.uk Quadrupole Full range of flow rates: Nanoflow 10-1000 nL/min; Megaflow 1 uL-1 mL/min
416
Most species analyzed by electrospray have a distribution of chargeable basic sites that easily brings the m/z to a range analyzed by the standard quadrupole. However, the occasional "oddball" protein or fragment may have a disproportionately small number of basic sites. Time-of-flight mass analyzers tend to have a higher mass range than quadrupole instruments. Another reason a time-of-flight analyzer might be desirable is the mass resolution
API ISO MCA Perkin Elmer Sciex 761 Main Ave. Norwalk, CT 06859 203-762-1000 www.perkin-elmer.com Quadrupole TurbolonSpray: (2-1000 ul/min) maximum ionization efficiencies for improved sensitivity. Compatible with 4.6-mm i.d. HPLC methods. MicrolonSpray: (20-2000 nL/min) microflow source for flow injection or capillary LC applications.
Compatible with most commercially available LC pumps, autosamplers, manual injectors, and detectors
417
.achievable in these rapid analyses. Henion calls the mass resolution that has been demonstrated by time-of-flight analyzers "amazing" compared with earlier time-offlight instruments. The resolution is uniform across the mass range, and as the resolution increases, it becomes easier to isolate individual ion species. This permits the determination of exact mass values necessaryforstructure characterization, according to Brian Musselman of PerSeptive Biosystems.
Analytical Chemistry News & Features, July 1, 1997 431 A
Product
Review
Choosing an analyzer
Electrospray is also readily available with magnetic sector and FT-MS instruments. These instruments tend to be geared toward high-end mass spectrometry research, with price tags to prove it. Henion says, "A number of factors [should govern the choice of mass analyzer] , but primarily quadrupoles are the favorite because they have the biggest database. They work, everybody knows how to use them, they're simple to use, and there's a lot of precedence for them." On die other hand, Henion points out that compared with quadrupoles, ion traps offer the advantage of MS(n) and cost less. "With an ion trap, you get a lot of bang for your buck," he says, noting that ion traps are relatively simple devices. Henion says that it's difficult to predict what will actually drive a purchaser's decision in selecting a mass analyzer. "The neophyte, the chromatographer that just wants a mass detector, isn't going to know much about the time-of-flight [mass analyzer] . The reasons they buy what they actually buy are sometimes going to be nonscientific. How that muddies the water will be interesting in the near future, because there could be good reasons for getting a time-of-flight instrument. I don't have a time-of-flight instrument yet, but I'm getting more and more tempted." Changes at the front end
The most publicized development in electrospray sources is the introduction of miniaturized electrospray, known as nanoelectrospray. The low-flow technique, designed to consume minute quantities of sample by using flow rates on the order of tens of nanoliters per minute, is particularly useful for peptide analyses where sample may be limited. Nanoelectrospray is effective because the signal intensity in electrospray analysis is concentrationrather than quantity-dependent. Henion S3VS "[Nanoelectrospray] is primarily of interest to [researchers working] with peptide or protein isolations." Matthias Mann of EMBL-Heidelberg (Germany), who with his co-worker Matthias Wilm is widely recognized as the primary developer of the technique, says: "[Nanoelectrospray] has already revolutionized peptide sequencing by mass spectrometry, and we think it will be an indispensable source for every mass spectrometer." Mann also indicates attributes of nanoelectrospray that will make it useful in a wider range of applications, including the variety of solvents that can be used, 432 A
the fact that more buffers can be sprayed, and its general ruggedness and relative freedom from cross-contamination. Hewlett Packard has introduced a new design in which the droplets are sprayed 90° to the mass spectrometer orifice. This design prevents solvent droplets from entering the mass spectrometer where they generate noise in the mass spectrum. Hewlett Packard's Mclntyre says that previous designs have relied on spraying in the general direction of the orifice and then tweaking the angle to find a "sweet spot" that maximized the number of ions reaching the mass spectrometer but minimized the amount of solvent. He claims that the design offers a fivefold increase in sensitivity relative to earlier incarnations of the Hewlett Ptickard ion source On the brink of a revolution
Perhaps the biggest change has been one not of instrumentation but of focus. Electrospray and its cousin, atmospheric pressure chemical ionization, are poised to launch a revolution that will do for LC what MS did for GC years ago. "We are at the threshold of what I believe will be a huge proliferation of personal LC/MS systems," says Henion. "Chromatography laboratories will have multiple LC/MS systems just as [environmental laboratories] have multiple GC/MS systems. It could be even more popular than GC/MS because there will be more applications for it." With MS ready to move into chromatography laboratories, instrument companies are doing their best to make the technique more accessible for chromatographers. Even though mass spectrometer manufacturers are making the systems friendlier, they claim that they are not sacrificing performance to do so, because the issue is addressed primarily through software development. The dedicated LC/MS systems are entirely computer controlled. There SLTQ n o "physical knobs on the [LCQ] hardware," according to Ian Jardine of Finnigan. It is set up to tune and calibrate automatically The computer-controlled systems are not without their drawbacks, however. Mann comments, "Sometimes it's easy to generate good data but very difficult to do anything with diem, because you can't get at the information you know is there— which can be very frustrating." Although chromatographers may be wary of MS, remembering the days when mass spectrometers resembled "prickly stainless steel elephants", they probably
Analytical Chemistry News & Features, July 1, 1997
have a better understanding of the data generated by electrospray than of the fragmentation data generated by more traditional MS, says Nicolaysen. The instruments from Micromass are designed with the same focus on ease of use, according to Nicolaysen. The Micromass system software is divided into standard and advanced levels. At the standard setting, only the cone voltage can be adjusted. In advanced mode, other parameters, such as the needle voltage, can be adjusted. The software for the LCQ is designed so that the chromatogram is the focal point of the analysis, says Jardine. The instrument is set up so that it is controlled entirely through the software, including automatic MS/MS. Jardine "The entire system starts from the perspective that the mass spectrometer is a detector " Jardine and Micromass's Nicolaysen say that these changes in no way diminish the capabilities of the instruments Room to grow
Although electrospray has made the leap into applications laboratories, it is still a young technique with room for continued development. "Some people like to think of it as mature. I do not," says Henion. "It's maturing, but it still needs to be better because it has problems with buffers, salts, and compounds that 'don't work'. Online with LC/MS or CE/MS, we'll need significantly more sensitivity. That sensitivity can be partially gained in the electrospray process, or it can be gained by the mass spectrometers themselves with time-of-flight QJ. patter ion transmission the whole system can be tweaked." Cook agrees that the method is still developing. "It's far from a mature technology, which is good news in my view because I'm a fundamentals type. It's still not assured that you'll get a spectrum on your first try. There are parameters that are often empirically adjusted, including flow rates and voltages at different optical elements. I imagine that there will be advances in the understanding of the chemistry of the process that will result in greater efficiency and ease of application" Orlando sees the instruments becoming smaller, less expensive, and more of a "black box" as they are used in new areas. He says there will be numerous inexpensive mass spectrometer detectors for nonmass spectrometrists, a continuation of the trend that has already begun. Pretty good for a technique that's only 10 years old. Celia Henry
