Besides the buffer solution recommended by the manual e.g. ammonium acetate, are any others available?
Yes, other aqueous solutions may be used, such as 0.05% trifluoracetic acid, 20 mM nitric acid, 20 mM hydrochloric acid, triethyl amine, and 20 mM acetic acid. The important thing is that whatever is added to the solution it should bring the conductivity to 0.2 S/m but be completely volatile.
Can I analyze a sample prepared in saline solution?
Like ESI-MS, the MacroIMS system requires a solvent which is completely volatile and which has a controlled electrical conductivity. As in ESI-MS, any salts, buffers, surfactants, or other nonvolatile or conductivity-influencing solutes which may be present in the sample must be removed by dialysis, ultrafiltration, affinity purification, simple dilution, or some other technique. As in ESI-MS, a favored solvent is aqueous ammonium acetate, which for proper MacroIMS operation should be near 20 millimolar concentration.
Can I use gases other than air and CO2 in the MacroIMS system?
At the electrospray source, it is recommended that you use air and CO2 gas rather than nitrogen or any noble gas (helium, neon, argon, krypton, xenon) to avoid corona discharge at the nanospray tip. The gas used in the MacroIMS system is chosen to have a significant electron affinity to minimize on HV breakdown. Air has oxygen, which helps a lot, but usually isn’t quite enough to keep the corona threshold safely above the field needed for electrospray. Thus CO2 is added to push the HV breakdown threshold up a little and give the margin needed for stable operation. Alternatively, it is possible that one could use nitrogen and increase the CO2 flow but no experiments have been done to find it out. One known substitute for CO2 in this application is sulfur hexafluoride which is actually better than CO2 but much more expensive than CO2.
Can I use the electrospray source without the neutralizer?
One of the 3482/3480C’s unique design features is that it provides mainly uncharged aerosol, unlike the electrosprays used for mass spectrometry, paint spraying, and crop dusting. Electrospray fundamentally produces droplets charged to about half the Rayleigh limit (see e.g. Hinds, Aerosol Technology, Wiley-Interscience, 2nd edition, p. 334 for a discussion of this limit). If you remove the neutralizer from the electrospray, the droplets will remain charged, evaporating and undergoing Rayleigh disintegrations which change the size distribution. The resulting highly-charged fine particles are highly mobile and are attracted to any conductive wall, so very few macroions are output from the source with no neutralizer in place.
Does the MacroIMS system measure molecular mass?
The MacroIMS system fundamentally measures the electrical mobility of singly-charged macromolecular ions in air. This quantity relates directly to the physical size of the ion and not the mass. However, a strong correlation has been demonstrated between mass and size, and the dependence is just what is expected for spherical molecules if one assumes an effective density. Like gel plates and SEC columns, the MacroIMS technique can determine masses relative to a standard. But unlike these techniques, MacroIMS’s method of calibration is determined entirely by voltage, flow rate, and geometry of the analyzer, quantities which can be independently measured so that there is no need for repeated calibration against standard samples.
How does MacroIMS measure the mobility, diameter and mass of macroions?
In the MacroIMS technique, ions move under the influence of an electric field in the presence of air/gas, where the speed at which these ions move, electrically forced through a gas, is related to their mobility:Z=v/EWhere Z is the mobility [m2/Vs], v is the velocity [m/s] and E is the electric field [V/m].In the Ion Mobility Drift Cell of the macroIMS, ions are separated in the annulus between two concentric tubes. In this annular region there is flowing gas, and an electric field applied between the inner and outer tubes. The equations which govern the velocity and drift time of an ion in this annular space are as follows: vx=Q/A=(Q1+Q2)/p(r22-r12)vr=ZEE=V/d=V/rln(r2/r1)tx= x/vx = p(r22-r12)x/(Q1+Q2)tr = ò dr/vr = (r22-r12)ln(r2/r1)/2pZVwhere x is the longitudinal distance from entering the IM Drift Cell to exiting at the downstream exit slit; r1 is the inner tube radius and r2 is the outer tube radius; Q is the total flow rate through the drift annulus and Q1,Q2 are component flows; d is an equivalent distance between flat plates; vr and vx are velocities in the radial and longitudinal axis, respectively. From the equations for tx and tr, the drift time of an ion from the entrance to the exit of the IM Drift Cell can be determined by equating tx and tr:When tx=tr=(Q1+Q2)ln(r2/r1)/ 2pZV=time it takes an ion to exitFrom this equation it is evident that the drift time is inversely proportional to the ion mobility so high mobility ions travel faster than low mobility ions.It is also evident from the above equation that building an IM Drift Cell with controlled Q, r, and V, while counting the number of ions at the output of the IM Drift cell at time t, yields a quantitation of ions of mobility Z. So clearly the MacroIMS, with its IM Drift Cell and Macroion Detector are designed to directly measure the mobility of ions. Now for a macroion drifting through a gas, it experiences a viscous drag according to Stokes Relation which is corrected for gas slippage using the Cunningham slip correction factor (C).Fviscous drag = 6pμav/Cwhere μ is the gas viscosity, “a” is the ion radius, and v is the drift velocity of the ion. The Cunningham slip correction factor is well established for ions with diameters of 100 nm, and empirically fits even smaller ions.An electric force, Felectric, and the viscous drag force, Fviscous drag, act in opposite directions, so when they are both balanced, the net force is zero and the ion is traveling at the drift velocity v. Fviscous drag = Felectric6pμav/C = neEOr 3pμDv/C=neEWhere n = number of elementary charges; e = elementary unit of charge; and D is the diameter of an ion.Thus a spherical ion of diameter D, would have the same drift velocity as an ion with the mobility Z according to the following:D=neEC/3pμv=neC/3pμZThe above equations reveal that the mobility Z for macroions that behave under slip-conditions, can be correlated with a drift-velocity-equivalent diameter D. Finally, the strong empirical correlation between mobility diameter and mass allows one to convert between the two measurements.
How does the IM Drift Cell in the macroIMS system work?
Click here to take an animated tour of the 3085C Ion Mobility Drift Cell. The animation is based on the earlier Model 3085 nanoDMA and is provided courtesy of Prof. Heinz Fissan. The animation shows the flow path for nanometer-sized charged aerosols, and is also applicable to macroions.
How does the MacroIMS technique work?
The technique uses a charge-reduced electrospray ionization source to first generate singly-charged macroions. These macroions can then be selected based on a specific ion mobility, in an ion mobility drift cell, by applying an appropriate voltage in the drift cell. To generate a full mobility/size/mass spectrum, the voltage in the drift cell is scanned and synchronized with the macroion detector response. A schematic of the MacroIMS system can be found here.
How to avoid corona discharge in the IM Drift Cell and electrospray when increasing the voltage in the macroIMS system?
IM Drift Cell: A clean IM Drift Cell running in air will not exhibit breakdown over its operating range up to 10 kV. If it has been severely contaminated, it sometimes breaks down over internal insulator surfaces at values slightly below 10 kV: it is then time to clean the unit following procedures given in the manual. Electrospray: An electrospray will not operate in the cone-jet mode when a corona discharge is present. But to electrospray aqueous solutions requires fields near the 30 kV/cm breakdown value of air. In the 3480C, this potential problem is solved by providing a small admixture (~10%) of CO2 in the air stream. This suppresses the corona discharge.
Under what circumstance does the high-voltage polarity need to be reversed in the 3482 / 3480C Electrospray?
The high-voltage polarity in the electrospray does not appear to effect the types of biomolecules that can be ionized, and thus there is no need to change it. It is different from electrospray-ionization mass spectrometry (ESI-MS) in which the polarity is important. In ESI-MS, the charge is not removed from the droplets, and the resulting high voltage field ejects molecules from the surface in a process called “ion evaporation”. If the molecules inside the solution are negatively charged, negative spray is required, and vice versa. By contrast, in the 3480C, the charge is removed quickly, so that the entire droplet evaporates without losing the nonvolatile content. Thus regardless of the solution conditions, a biomolecule contained within the droplet becomes airborne when the evaporation is complete.
What is Macroion Mobility Spectrometry?
Macroion mobility spectrometry is a technique that separates and quantifies gas-phase macroions based on their electrical mobilities. The Model 3980C macroIMS system is the successor to TSI’s GEMMA Macromolecule Analyzer.
What is the largest macromolecule I can measure using the macroIMS system?
The largest macromolecule measured to date (November 2003) is the so-called “Vault,” a blood protein structure of more than 13 megadaltons, which was measured by Professor Joseph Loo (Abstract No. A031378 presented at the 2003 American Society for Mass Spectrometry conference). The macroIMS method uses aerosol particle instruments that can measure particles up to 65 nm diameter or more. The molecular mass of a 65-nm macroion would be an astronomical 80 MEGAdaltons! This makes the macroIMS technique interesting for biocomplex studies.
What is the magnification of the viewing window for the Model 3480C Electrospray?
The magnification for a compound microscope is well defined, but for a simple single-lens loupe such as this it is not. Nevertheless, the magnification for loupes is often arbitrarily defined as the ratio of an assumed 250 mm viewing distance to the focal length of the lens. The lens in the viewing port of the 3480C has a focal length of about 25 mm, thus the magnification defined in this way is about 10X. A more reliable way to define the size of objects in the view is to note that the outside capillary diameter is 150 micrometers. Since this is clearly visible, it can be used as a standard against which to judge the size of the liquid cone and other features of the spray tip.
What is the smallest macromolecule I can measure using the MacroIMS system?
The response of the present detector decreases for macroions smaller than 3 nm in electrical mobility diameter, which corresponds to about 8,000 Daltons. Thus it is recommended to use the MacroIMS system for macromolecules >8 kDa or > 3nm. However, it has been found that bovine insulin at 5.7 kDa, and an effective density of nominally 0.65 g/cc, can be efficiently analyzed with the macroIMS system at near the lower size detection limit, and below the lower mass limit.
What should be the conductivity and pH value for the sample and how to adjust them?
The conductivity for aqueous solutions should be around 0.2 S/m. The pH value is unimportant for electrospray operation itself, but it may be very important in getting results. This is determined by specific characteristics of the sample. For example, for proteins, the pH should be above the isoelectric point (pI) of the protein, generally, to prevent the adherence of the protein to the silica capillary tube wall. But for zirconium oxide nanoparticles, use of a basic pH will result in gel formation and capillary blockage, even at very low concentrations. For sucrose which sometimes is used to generate nanometer-sized aggregates, the pH does not seem to be important. You need to know the chemistry of your sample.
When preparing a sample, do we have to centrifuge it?
The only reason is to remove very large particles from the solution. If there are no large particles present, there is no need to centrifuge it. If you begin to experience capillary plugging which you believe could be due to large particles that is the time to try centrifugation.
Will the electric field alter the nature or characteristics of a protein sample?
No more than it does in electrospray-ionization mass spectrometry (ESI-MS). In fact, the charge-reduction method used in the special macroIMS electrospray unit reduces the charge on the droplet while it is drying, thus preventing the violent rupture of the droplet and the associated shear forces which tend to fragment the proteins in ESI-MS. The macroIMS's charge-reduced electrospray is less damaging to protein molecules than ESI-MS, and ESI-MS is already considered to be a "gentle" method of producing macromolecular ions.