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  The CHROMacademy Essential Guide to Electrospray Ionization (ESI) for LC-MS (Part 1)
  Webcast - Understanding Electrospray Ionisation (ESI) for LC-MS

Tony Taylor
Technical Director
Crawford Scientific.

Kevin Schug Dr Kevin Schug
Assistant Professor
University of Texas at Arlington
This month our industry experts help you to understand the fundamental basis of electrospray ionization mass spectrometry.
 

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The CHROMacademy Essential Guide to Electrospray Ionization (ESI) for LC-MS (Part 1)

This month our industry experts help you to understand the fundamental basis of electrospray ionization mass spectrometry.  Learn how to optimize the essential parameters and improve instrument response and reproducibility in your lab.  Improve your electrospray data and troubleshoot your ionization and data interpretation problems more effectively.  Learn the fundamental basis of ion suppression so you can take best steps to avoid this phenomenon and discover best practice for acquiring qualitative data.

ESI
Tony Taylor
Technical Director
Crawford Scientific
Dr Kevin Schug
Assistant Professor
University of Texas at Arlington
FREE CHROMacademy Essential Guide Webcast
Electrospray Ionization (ESI) for LC-MS (Part 1)
Thursday 24th February 2011, 11:00am EST; 16:00 GMT

»» View here

If you find this tutorial useful, you should know that CHROMacademy
also contains the following related materials:

  • Instrumentation for electrospray ionization
  • Quadrupole / Time of Flight and Ion Trap Mass Analyzers
  • APCI / APPI Techniques
  • Solvents Buffers and Additives for Electrospray
  • Interpretation for LC-MS
  • Spectrometric experiments for LC-MS

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Electrospray ionization techniques belong to a wider group of methodologies known as atmospheric pressure ionization (API) techniques, by which ions present in an HPLC mobile phase solution can be transferred to the gas phase prior to sampling into a mass analyser. As well as electrospray ionization, other API techniques include atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI). Other, popular, ambient interfacing techniques include desorption electrospray ionization (DESI), direct analysis in real time (DART) and matrix-assisted laser desorption electrospray ionization (MALDI).

The electrospray ionization process involves the application of an electric field across an interface, which acts to form a high resistance electrochemical cell in the interface. Figure 1 shows a simplified schematic of an ESI interface.

fig01
«« Figure 1: Simplified schematic diagram of an electrospray ionization (ESI) atmospheric pressure ionization (API) HPLC–MS interface (not to scale).

A high electric field is generated between the capillary tip and inlet to the mass spectrometer
 
 

Electrospray ionization, like most other API techniques, faces two major challenges in terms of its ability to interface HPLC (a solution phase technique) to mass spectrometry (a gas phase technique):

  • The analytes involved are non-volatile and need to be transferred to the gas phase as ions
  • There is a large amount of solvent to evaporate and vent prior to sampling of the gas phase ions and to prevent a vacuum compromise in the mass spectrometer.

To overcome these issues, electrospray ionization includes three important processes in order to transfer sample molecules from the HPLC eluent into gas phase ions within the mass spectrometer (Animation 1 and Figure 2):

  • Production of charged droplets at the capillary tip
  • Shrinkage of the charged droplets: leading to coulombic fission events
  • Production of gas phase ions from small / highly charged droplets
 
 
   
Animation 1: Discrete processes involved in the electrospray ionization process. Figure 2: Discrete processes involved in the electrospray ionization process.
Artwork courtesy of Shimadzu Scientific Instruments, Inc.
 
 

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Electrospray ionization is highly compatible with analytes possessing the following characteristics:

  • Moderate to highly polarity
  • Up to 100 000 Dalton
  • Ionize in solution perhaps with multiple charges (z>1)

Electrospray ionization favours the analyte in the ionized form, in eluent solution prior to introduction into the API interface, although this is not a pre-requisite for generating a response.

Analyte species that are capable of carrying charge in solution include: [1–7]

mol 1

1,2 di-chlorobenzene

Compounds containing hetero-atoms (i.e., chlorinated, brominated etc.) within the molecule are able to carry a proton in solution by association with unpaired electrons
     
     

mol 1

azobenzene

Compounds producing a charge through inductive effects (i.e., azo-compounds, phthalates, opiates etc.)
     
     
Compounds capable of holding multiple charge (i.e., proteins, peptides etc.) Multiple basic amino acid sites on these compounds are all capable of ionization in solution given correct pH conditions.
mol 1Polypeptide
     
     

mol 1

Orotic acid

Compounds that are ionizable (acids, bases etc.) Adjusting pH can cause the compound to favour the ionic state in solution.
     
     
Compounds that have a strong dipole moment but do not ionize under conditions suitable for HPLC analysis (aldehydes, some ethers etc.) ESI droplets may be charged by electrostatic effects and the compound carries charge by association with adducting species such as sodium, potassium, ammonium or protonated acetonitrile ions in solution
mol 1Digoxin (ether)
 
 

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The first stage in electrospray ion production is the production of charged eluent droplets at the tip of the ‘sprayer’. The sprayer is a capillary tube that is fed by the HPLC eluent (at a suitable flow-rate — see later) and the resulting spray is directed into the desolvation chamber of the atmospheric pressure interface.

The capillary is metallic with an internal diameter of approximately 0.1 mm. A potential difference (Vc) is applied between the capillary and the sampling cone (usually between 2 to 6 KV). The applied voltage forms an electrochemical cell in the interface which acts to accelerate charged droplets between the sprayer and the sampling orifice as well as forming the basis for an electrophoretic charge separation at the capillary tip. It’s worthy of note that the metal composition of the sprayer capillary has an effect on the oxidation and reduction processes that occur to produce charge separation and establish the current at the capillary tip. Changes in the sprayer make, type, or dimensions can produce changes in the sensitivity of the instrument under at a given sprayer potential!

According to the nature of the analyte of interest, positive or negative ionization mode can be selected.

In the positive ion mode the capillary is the positive electrode (anode) and the sampling aperture plate (at the rear of the interface) is the negative electrode (cathode). Movie 2 shows positive ions within the eluent solution (including analytes, matrix components and background electrolyte / buffer ions) being repelled from the inner walls of the sprayer needle and moving electrophoretically into the body of the droplet formed at the capillary tip. This mode causes positive ions to predominate the sprayed droplet and is used where the analytes (such as bases for example) form cations in solution. Oxidation and reduction processes at the capillary wall either produce or consume electrons that sustain the current within the system.

 

«« Animation 2: Charge separation at the capillary tip to form a predominantly positively charged droplet known as positive ion mode.

 
 
In negative ion mode the reverse situation occurs. Figure 4 shows the capillary as the negative electrode (cathode) and the sampling aperture plate is the positive electrode (anode). This mode causes negative ions to predominate within the sprayed droplet and is used where the analytes (such as acids for example) more readily form anions in solution.
 

«« Animation 3: Charge separation at the capillary tip to form a predominantly negatively charged droplet known as negative ion mode.

 
 
When the charge density at the liquid surface is raised due to the repulsion of anions (negative ion mode) or cations (positive ion mode), the coulombic repulsion forces are also increased. As shown in Animation 4 the point at which the surface charge repulsion matches the surface tension of the eluent is termed the ‘Rayleigh Instability Limit’. When the number of ions of like charge is increased, coulombic repulsion overcomes the Rayleigh limit and the shape of the meniscus changes to a cone shape in order to relieve charge repulsion. This is referred as the ‘Taylor Cone’ and upon formation of the cone a stream of droplets containing a vast excess of either cations or anions will emerge from its surface. This process is termed ‘Electrospray’ or sometimes referres to as Cone Jet Emission.
 

«« Animation 4: Surface charge repulsion overcomes the surface tension of the liquid at the capillary tip (Rayleigh Instability Limit) and a Taylor cone is formed to reduce charge repulsion effects.

 
 

From the end of the Taylor cone, a fine jet of charged droplets emerges. The droplets ultimately disperse as they travel through the interface into a ‘plume’ of droplets that is familiar to those who work with electrospray ionization. This is shown in the photograph in Figure 3.

The dispersion of a liquid into an aerosol is known as nebulisation.  The processes of charge separation, formation of a Taylor cone and emission of charged droplets into a plume, as in ESI, are considered to be a form of electrostatic nebulisation.  In many conventional ESI sources, nebulization is pneumatically-assisted with the addition of a flow of nitrogen gas, coaxially with the electrospray.

 

«« Figure 3: The electrospray showing the Taylor cone emitting a jet of fine droplets that disperse into the familiar electrospray ‘plume’.

 
 

Tutorial Discussion

Tony: Is it right that analytes don’t need to be ionized in the eluent solution prior to spraying to obtain optimum MS sensitivity using this technique?

Kevin: The direct answer to the question is that an analyte does NOT need to be ionized in the eluent solution in order to obtain optimum MS sensitivity.

Ion generation in ESI is generally attributed as a solution phase process, although gas phase processes once the analyte ion has left the droplet are important to consider in some cases. Ionization typically occurs as some combination of acid-base or charge-transfer reaction in the electrospray droplet. Importantly, compounds that are not readily ionizable in solution can still be observed in mass spectra generated by ESI. In such cases, adduction of ions, such as sodium, ammonium or potassium (or chloride in the negative ionization mode), could be the predominant forms of ions observed. Analytes that are not readily ionizable in bulk solution can still migrate to the surface of the ESI droplet and acquire a charge to generate strong signal.

Another interesting thing is the notion that acidic compounds can still be ionized with good sensitivity in an acidic environment; similarly for basic compounds in a basic environment. Conventional wisdom and standard acid-base chemistry tells us that ionization of basic analytes would be suppressed in a basic environment. However, there is a growing body of literature, which demonstrates higher sensitivity for some such cases (search “wrong-way-round ionization”). On an initial pass, I would still go for the use of an acidic mobile phase modifier to efficiently ionize compounds in the positive ionization mode. And even if the compound did not have a nice amine group sitting there to be protonated, I could still be pretty confident that some cationization would be possible.

Of course, for optimum sensitivity, or in cases where some atypical mobile phase additives are needed to affect separation, a thorough investigation of the effect of mobile phase additives and pH on ion response should be made. It is important to initially monitor a wide mass range, so that different ion forms, such as adduct and dimer ions, can be tracked.

 

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The formation of a stable nebulization aerosol is dependant upon several factors including:

  • The applied potential difference (Vc)
  • The flow-rate, surface tension and electrolyte concentration of the HPLC eluent
  • The flow-rate and temperature of any nebulizing gas that is applied concentrically to the capillary to assist droplet formation.

Establishing a reproducible electrospray is of primary importance in practical LC–MS and the factors mentioned above act interactively. Let’s look first at the impact of the applied potential difference on the nature and reproducibility of the electrospray.

 

The effectiveness and stability of the nebulization process is found to be proportional to the magnitude of the potential difference across the system (Vc). As the potential difference across the system is increased, the droplet size reduces and their motion acquires a horizontal component.

Above a certain applied potential difference (experimentally dependant), the Taylor cone is formed and small charged droplets are formed from its tip. This is known as the Axial Spray mode. This spray mode will contain the optimum voltage for the experiment (empirically determined).

Further increasing the applied potential will cause a sudden transition to take place — the liquid cone vanishes and a fine mist of droplets is produced from a number of points on the edge of the capillary tip. This is known as Rim Emission mode. Whilst an instrument signal will still be produced, it will be not be optimal in terms of response and will be irreproducible.

A second transformation occurs at still higher capillary potentials. A corona discharge is established between the needle tip and the sampling plate. [8] Discharge is not a stable or reproducible spray state and the noisy baselines produced results in reduced signal-to-noise (S/N) and hence reduced instrument sensitivity.

A summary of the transition through these modes in shown in Animation 5.

Tutorial Discussion

Tony: Once you have found the right capillary (sprayer) voltage for one application, this will be equally applicable for most separations – true or false? (And please expand!)

Kevin: This is actually true and false, depending on your point of view, and how well you want to optimize the method!

While it is true that you can often find a good sprayer voltage that satisfies analysis of a wide range of compounds, in positive or negative ionization modes, it may not be optimal.

If you are configuring for walk-up sample analysis, a set-it and forget-it approach is possible. A good rule of thumb is to err on the side of lower voltages, if possible. If you are looking to get the most out of your method, then rigorous optimization of experimental variables should include the sprayer voltage. At high spray voltages, especially in the negative ionization mode, the source can be prone to discharge. To alleviate these events, which will certainly hurt reproducibility and system stability, move to lower spray voltages. Besides discharges, some analytes, especially in atypical mobile phase solvents, may be prone to redox processes. Reducing the sprayer voltage can reduce contributions from unwanted side reactions that reduce signal quality. I once received some great advice on ESI, which pertains to many of its experimental parameters – ‘If a little bit works, a little bit less probably works better’. I tell this to my students all the time and it particularly pertains to concentrations of mobile phase additives, flow-rates and spray voltage.

Animation 5: Effects of increasing capillary voltage (Vc) on an electrospray: Up to 2kV — increasing voltage gives an increasing horizontal moment to the sprayed droplets / 2–3 kV — onset of axial spray, the optimum potential for maximum and reproducible spray lies in this voltage range / 4 kV — onset of rim emission resulting in unstable spray and variable instrument response / 5 kV — corona discharge, no instrument response.
 
 
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Non-pneumatically-assisted electrospray is a low flow-rate technique limited to a few microlitres per minute. The instrument response and sensitivity decreases as the eluent flow-rate is increased. This behaviour is closely related to the size of the droplets formed at the capillary tip and the number of charges within the droplet. By introducing an axially sprayed inert gas from a concentric tube around the sprayer and, therefore, the forming droplet (ESI with ‘pneumatic assistance’), the droplet size is restricted and the droplets are charged more efficiently at higher eluent flow-rates. The pneumatic-assistance also aids droplet desolvation by increasing the surface evaporation of solvent from the electrospray droplets.
 

Animation 6: »» As the flow-rate of the concentrically introduced inert gas is increased the droplet takes longer to form at the capillary tip. This constrains the droplet growth and allows time for the charge separation processes (which are fixed rate and not dependent upon eluent flow-rate), to efficiently fill the droplet with ions and the charge to droplet volume ratio will increase. Ultimately this allows the eluent to flow into the capillary at higher flow-rates whilst maintaining droplet charging efficiency.

 
 
 
The eluent flow-rates of 1 mL per minute with pneumatic assistance are possible, but the signal tends to optimize at lower flow-rates (around 200 mL/min in the example in Figure 4), depending upon the nature of the eluent (primarily the aqueous / organic ratio) and the applied capillary potential. Figure 4 shows the variation of instrument response against eluent flow-rate for penicillin in a water/acetonitrile mobile phase. [9–11]
 
«« Figure 4: Instrument response versus eluent flow-rate for penicillin in pneumatically-assisted and non-pneumatically modes of electrospray.
 
 
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Solvents with low surface tension (i.e, methanol and iso-propanol — see Table 1), allow for stable Taylor cone formation and hence a stable and reproducible electrospray. The Rayleigh limit will be overcome at lower potential (Table 2) and this will tend to lead, on average, to smaller droplets being produced that aids in the ion formation process and can lead to an increase in instrument sensitivity.

Lower potential difference across the system will be required for effective spraying when using lower eluent water content. This reduces the risk of spraying in rim emission mode and lowers the possibility of electrical discharge at the capillary tip. The addition of a small amount of methanol or iso-propanol (1–2% v/v) to a highly aqueous HPLC eluent can often bring about an increase in instrument response, as the surface tension is lowered.

 
Solvent
Surface tension
(dyn/cm)
Dipole (D)
Dielectric constant
(@ 20oC)
Viscosity (cp)
Vapour Pressure
25oC (mmHg)
Methanol
22.55
2.87
32.7
0.59
127
Isopropanol
21.79
1.69
19.92
2.40
45.4
Acetonitrile
19.10
3.44
37.50
0.38
88.8
Water
72.80
1.87
80.10
1.00
23.9

Table 1: Physical properties of selected solvents.

 

Solvent
ΔV (kV)
Isopropanol
2.0
Methanol
2.2
Acetonitrile
2.5
Water
4.0

Table 2: Potential differences to overcome Rayleigh limit for selected solvent.

 
 
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As the droplet is sprayed into the desolvation zone towards the sampling cone, it will shrink because of solvent evaporation. Solvent evaporation is aided by raising the temperature of the ambient air within the ionization chamber. This is often supplemented by the use of an inert desolvation gas (nitrogen is most common) that is pumped into the interface housing. The temperature required is dependent on the solvent properties of the sprayed droplet and the eluent flow rate.

As the droplet shrinks due to solvent evaporation, its radius (R) decreases, but its charge (Z) remains constant. This leads to a decrease in the charge separation (inter-ionic distance) at the droplet surface and an increase in repulsion between the surface charges, until the electrostatic repulsive forces become equal to the eluent surface tension (Rayleigh Instability Limit). Further decreasing the radius of the droplet will cause the Rayleigh limit to be exceeded and the droplet will undergo a coulombic fission in order to reduce the coulombic stress between the surface charges. The process is often referred to as ‘coulombic’ or ‘droplet jet fission’ (Animation 7).

 

«« Animation 7: Solvent evaporation resulting in droplet jet fission.

 
 

The rate of solvent evaporation will depend upon the volatility of the solvents in the droplet and the surface tension of the solvents will dictate the point at which droplet jet fission will occur under a given set of interface conditions. Under experimental conditions below the sprayed droplet radius (R) will be around 1.5 mm, and it will carry approximately 50 000 charges (Z).

Jet fission will lead to a reduction of approximately 2% of the parent droplet mass and around 15% of the parent droplet charge (R = 0.08 µm, Z = 7500), this is termed uneven droplet fission.[11,12] This predicts that the radius of the offspring droplet will be approximately one tenth of the parent droplet. Mass balance shows that under the experimental conditions in Figure 5, the first Rayleigh jet fission will produce around 20 offspring droplets.

As the resulting droplets hold a greater charge per mass (volume) than the original droplet, the resulting (offspring) droplets will quickly undergo further droplet jet fissions to produce further offspring droplets. The ‘cascade’ of droplet fission processes lead ultimately to a very small droplet (~10 nm) containing a small number of theoretical charges.

The whole evaporative process usually occurs within a timescale of between a few hundred microseconds and a few milliseconds — the residence time of the droplet within the desolvation zone.

 

 

«« Figure 5: Schematic diagram showing subsequent jet fission processes that occur when the surface charge repulsion overcomes the surface tension of the droplet.

 

 

 

Tutorial Discussion

Tony: As the solvent composition changes during gradient analysis wouldn’t it make sense to dynamically move the sprayer to ensure ions are continually released into the source sampling ‘sweet spot’

Kevin: While it is true that characteristics of the electrospray change with solvent composition, it is impractical to consider dynamic modification of the sprayer position during an analysis. There are a couple of reasons for this. First, the sprayer voltage and position are set such that a reproducible and stable spray is generated from the electrospray nozzle. A drastic adjustment of the position of the needle would change the magnitude of the potential gradient between the spray capillary and the counter-electrode, which could alter the spray stability. Some interesting studies of spray modes generated under different operating conditions have been published. An additional consideration is that different analyte types (e.g., proteins vs. small molecules) are believed to reside preferentially in different sized droplets generated by ESI. The dynamics of an uneven droplet fission process relative to the surface activity of different analytes could sensibly lead to such differences. This, coupled with likely differences in how/when ions are released from droplets (e.g., as a charged residue vs. by ion evaporation), means that the distribution of different analytes through the spray plume is variable.
The position of the sprayer is a parameter that can and should be optimized, but its optimal setting is likely to be one which is viable for providing high signal quality across a wide range of analytes.

Tony: HILIC is a popular technique. How is the ESI process affected by working in a highly organic environment?

Kevin: One of the main reasons that HILIC has become popular is because it involves mobile phase conditions that are favorable for sensitive ESI-MS analysis. We published a review on this topic not too long ago (J. Sep. Sci., 31, 1465–1480, 2008) and in this review, we included a detailed section on the operation of ESI in low-aqueous and non-aqueous solvent systems. A couple of conditions are especially important. First, the solvent environment needs to have a sufficiently high dielectric constant to allow for separation of charge. Water is, of course, very good in this regard, but it is still limited in terms of a second important parameter and vapour pressure. In HILIC, a high percentage of acetonitrile provides an optimal environment for ESI. Not only is good charge separation accommodated by the incorporation of some, even a small, percentage of water (the dielectric constant of acetonitrile is of reasonable magnitude, as well), but also, the high vapour pressure of acetonitrile ensures that ESI droplets will desolvate and shrink rapidly to allow efficient release of gas phase ions. Acetonitrile also has a relatively low surface tension (less than half that of water). Onset voltages for generation of stable sprays are lower in mobile phases with lower surface tension. Additionally, the point at which droplets subdivide to release smaller droplets will be reached more quickly in droplets having lower surface tension. These aspects lead to higher efficiency in gas phase ion generation during the ESI process. Finally, it doesn’t hurt that many of the analytes being analysed by HILIC are highly polar and/or ionic in nature. This can facilitate ion generation in the ESI droplet.

 
 
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Animation 8: Ion evaporation processes associated with
Iribarne & Thompson, leading to solvated gas phase cluster ions.

An offspring droplet radius of around 10 nm, there are two popular theories on the mechanism by which Coulombic stress is relieved and gas phase ions are formed.

Mechanism 1
Dole et. al. proposed further droplet fissions until very small droplets containing a single ion each are produced. [13] Solvent evaporation from these droplets will lead to the formation of gas phase ions (known as the charged residue theory). This model is thought to be favoured for large molecules, although experimental evidence most strongly supports Mechanism 2, outlined below, in a general sense.

Mechanism 2
Iribarne and Thompson propose that below a droplet radius of 10 nm an ion is able to ‘evaporate’ from within the droplet. [14,15]

The main supporting evidence comes from ion mobility studies, [14] which show the production of significant amounts of gas phase ions at times where most of the charged droplets are expected to have relatively large radii and multiple charges. This observation does not support the charged residue model. These ion mobility studies also revealed highly mobile gas phase ions that were spectroscopically identified as ion-solvent cluster molecules of the type M+(H2O)n where n = 3–7 and M is the analyte species as shown in Animation 8. This model is favoured for larger molecules such as proteins or polymers and those analytes capable of multiple charging.

 
 

Figure 6: Schematic diagram showing the two possibilities for gas phase ion production and
their likely relative positions within the desolvation zone in the electrospray interface.

 
 

Figure 7: Ion evaporation is a complex interplay between the variables outlined above.

The rate of ion production (and the position of ion production onset within the desolvation zone) increases with the number of ions N within the droplet, and decreases with the radius of the droplet R. Decreasing N and/or increasing R is equivalent to a reduction of the surface repulsion forces that are experienced by the droplet (i.e., the droplet surface charges are moving further apart). It is less likely that the energy barrier to ion ejection (ΔG‡) will be overcome, as the attractive forces holding the ion cluster within the droplet will be dominant.

The production of gas phase ions is in fact a complex interplay between the competing processes described above that can be represented as shown in Figure 7.

 
 

The rate constant for ion emission (evaporation) to the gas phase is dependent on

  • Coulombic forces acting on the escaping ions (i.e., the drive to reduce surface charge due to repulsion between surface ions)
  • Repulsive forces arising from interactions with ions of the same charge within the droplet and attractive forces due to interaction with ions of opposite charge in the droplet as well as partition constant of the analyte (solvophobic/solvophilic characteristics)
  • The radius of the hydrated ion cluster (d), that describes the distance of the ion charge centre from the droplet surface (sometimes known as charge shielding)

The diameter of the hydration cluster will dictate the distance between the charged ion and the droplet surface. Those droplets with larger spheres of hydration will be less likely to undergo ion evaporation. Figure 8 »»

 
 

Strongly solvated ions such as Na+ (i.e., small radius with high charge to mass ratio), are associated with a larger number of solvent molecules, thus moving the charge centre further away from the droplet surface and the likelihood of ion evaporation.

The larger the value of d, the fewer gas phase ions will be produced because increasing the size of the sphere of hydration will

  • Move the charge further away from the droplet surface (where the electrostatic forces repelling the ions into the gas phase are lowest)
  • Shield the charge on the ion.

Table 3 shows that the strongly solvated ions Li+ and Na+ have larger transfer energies. The Iribarne and Thompson theory predicts larger activation barriers for evaporation, that is small rate constants k. [15,16]

M+
Li+
Na+
k+
Cs+
NH4+
(CH3)4N+
(C2H5)4N+
ΔGOSol(M+)
122
98.2
80.6
67.5
81
(54)
(49)
ΔGOSol[M+ (H2O)m]
61.2
56.5
55.8
54
55.6
(54)
(49)
m
7
7
6
5
~6
~0
Rion
3.82
3.58
3.30
3.29
3.30
(2)
ΔG
13.1
9.30
9.70
7.90
9.50
7.9
k(s-1)
2.0x103
1.0x104
4.9x105
9.4x106
6.8x105
9.8 x 106
 
ΔGOSol(M+) Transfer free energy from gas phase to solution for the M+ ion [kcal/mol].
ΔGOSol[M+ (H2O)m]

Transfer free energy from gas phase to solution for the hydrated ion cluster [kcal/mol].

m Number of water molecules in [M+ (H2O)m]
Rion Radius of hydrated ions, equal to d parameter in Iribarne equation [Å].
ΔG Activation energy for ion evaporation [kcal/mol].
k

rate constant [s-1]

 

Table 3: Energetic parameters for selected solvated ions.

It is obvious from the table that the tetraethylammonium ion, (C2H5)4N+is much more likely to undergo ion evaporation than the sodium ion, Na+. The sodium ion has a larger sphere of hydration (Rion) which affects the solution mobility of the hydrated ion (i.e., rate of movement of the hydrated ion from the droplet bulk to the droplet surface) as well the degree of charge shielding. Furthermore, the sodium ion is more hydrophilic and will, therefore, prefer to reside within the bulk of the droplet rather than migrating to the droplet surface, unlike the less polar tetraethylammonium ion (lower dielectric constant), which prefers to partition to the droplet surface.

From a pure practical perspective, the ion evaporation process is affected by:

  • Efficiency of droplet desolvation (temperate of the ‘drying gas’/distance between the sprayer and the sampling orifice / initial production of small efficiently charged droplets)
  • Nature of the solvents which comprise the droplet
  • Presence of ions other than the analyte ion (sometimes called ‘background electrolytes’).

These effects are studied in greater detail in the next section.

 
 
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In order to determine if the analyte or co-electrolyte species will have efficient ion evaporation characteristics it is important to know what factors influence the size of the sphere of hydration, which governs not only the possibility of ion evaporation but also the rate of movement of the hydrated ion from the bulk to the droplet surface. Initial work into experimentally determined sensitivity constants (k) were based on the assumption [15]

 

when [A+] = [B+]

 

: Intensity of the specie A.

 

: Sensitivity constant for the specie A.

 

: Concentration of the specie A+.

 

That is, having equal concentrations of the two electrolytes A and B, the ion intensity ratio will be equal to the sensitivity coefficient ratio. Experimentally, the spectrometrically determined ion intensity ratio IA/IB is measured when equal concentrations of electrolytes A+X- and B+X- are present in the electrosprayed solutions as shown in Figure 9.

 

«« Figure 9: Relative sensitivity of various ions relative to Cs+, determined experimentally.

The charge on the caesium ion is not delocalized; the ion is highly hydrophilic and has a large sphere of hydration. The charge on the tetrapentyl ammonium ion is much more delocalized; the ion is more hydophobic and has a small sphere of hydration — making ion evaporation from the droplet surface much more favourable

As is generally observed in electrospray LC–MS, singly-charged ions that have hydrophobic groups, also tend to have high experimental sensitivity coefficients. Ion evaporation theory predicts that this is due to the lower degree of hydration of the more hydrophobic ions (allowing them to more closely approach the repulsive electrostatic forces at the droplet surface), and a lower solvation energy ΔGOSol , leading to a lower energy barrier to transition into the gas phase.

 
 

Surface Activity

The likelihood of ion evaporation is also dependent upon the ion of interest migrating from the bulk to the surface of the droplet. The relative ease of migration of the hydrated ion to the droplet surface is known as the ‘surface activity’.

Tang and Kebarle [9] introduced surface activity of the respective ions into their experimental data analysis and this is an indication of the ions ability to migrate towards the surface of the electrosprayed droplet. It depends upon the hydrophobicity of the ion, as well as the number of charges the ion carries. This is demonstrated in Animation 9.

 

«« Animation 9: Surface activity describes the ability of a hydrated ion cluster to move from the bulk droplet solution to the droplet surface and is dependent upon the hydrophobicity of the ion as well as the size of the sphere of hydration.

 

 
 

The sensitivity coefficient (k) can be shown to depend not only on the Iribarne ion evaporation rate constant (kI) but also bulk to surface ion equilibrium constant (Ks), as defined by the surface activity.



Ions that show high surface activity also stand the greatest chance of transfer to smaller droplets during droplet jet fission, as well as being liberated into the gas phase via ion evaporation processes.

Ions that show high surface activity also stand the greatest chance of transfer to smaller droplets during droplet jet fission, as well as being liberated into the gas phase via ion evaporation processes.

fig 10

Figure 10: Experimental sensitivity of various parabens clearly demonstrating that surface activity and sensitivity constant increases with the length of the alkyl substituent group – reflecting an increase in hydrophobicity and accompanying reduction in the radius of the hydrated ion.

It can be concluded then that many factors affect the electrospray sensitivity including

    • The number of charges on the ion
    • Effective mass-to-charge ratio (which influences the size of the hydration sphere)
    • Ion’s ability to migrate through the droplet bulk to the droplet surface
    • Surface area of the sprayed droplet
    • The proximity of the hydrated ion cluster to the droplet surface (i.e., charge shielding).
 
 

In the presence of a second electrolyte species, the intensity of the analyte can be suppressed. This phenomenon is termed ‘ion suppression’. This phenomenon can lead to the artificial and irreproducible reduction in analyte signal when determinations of the analyte at constant concentration are performed on samples where the background electrolyte concentration varies. Perhaps the most usual presentation of this situation is the analysis in which the nature of the sample matrix changes (i.e., bioanalysis, environmental analysis, etc.) The reverse situation can also occur in which analyte response is artificially enhanced — known as ‘ion enhancement’.

Ion suppression is a highly practical significant phenomenon in LC–MS because it is so insidious. The unpredictable nature of ion suppression in examples where, for instance, analytes are contained within different matrices, can often render quantitative analysis impossible due to the lack of reproducibility. Optimization of front-end chromatography and sample preparation becomes critical for avoiding co-elution of analytes with interfering compounds, which will suppress the desired ion signal in complex matrices.

The magnitude of the suppressive effect is dependent on the sensitivity coefficient of the two ions (analyte and potentially suppressive background ions), which in turn is dependent upon surface activity, solvation energy, degree of hydration and the number of charges held by each ion.

As can be seen from Figure 11, the [Bu4N]+ and [Mor+H]+ ions (which are present at constant concentration), are increasingly ‘suppressed’ as the concentration of the
NH4+ ion (originated from the ammonium chloride buffer) is increased. This is a result of the competitive process, including surface activity and the size of the sphere of hydration, between the ions for the limited and fixed number of droplet surface sites.

Figure 11: Decrease of analyte ion intensities due to competition with added [NH4]+, at constant analyte concentrations [Bu4N]+ = [Mor+H]+ = 1 x 10-5M.[9]

 
 

Figure 12: Common HPLC eluent additives and their actions in solution.

Mobile Phase Additives
For successful electrospray it is important to consider not only the possibility of ion suppression from matrix components, but also from the additives that we typically use in reversed-phase HPLC and other modes. Ion suppression can act in the way described previously, however, signal suppression may also occur because of the formation of ion pairs in solution that can act to neutralise the charge of the analyte ion and, therefore, drastically reduce the instrument response. For some common reagents, such as trifluoroacetic acid, the ion pair will remain associated through the transition into the gas phase, which reduces the number of gas phase ions produced. Other common reagents such as the formate ion, may form an ion-pair in solution that subsequently dissociates on transition to the gas phase. Signal suppression effects in such cases are much less drastic.

 

Tutorial Discussion

Tony: Ion suppression is considered important by some workers and not so by others. Can you give us a quick overview of how this occurs and some tips on how to avoid it?

Kevin: Ion suppression comes in many flavours. In our lab, we deal with it most often in the form of matrix effects, where interferences in a sample co-elute with an analyte of interest, and change (most often, suppress) the response of the analyte relative to that from a pure standard solution. Imagine the relative content of lipids, proteins, and salts in different matrices such as food, plasma and urine. Matrix effects can introduce drastic errors in quantitative analysis if not accounted for. In other cases, samples that have been subjected to some type of detergent or surfactant will likely be prone to ion suppression. Further, the excessive use of some mobile phase additives (trifluoroacetic acid and triethylamine are notable ones) can lead to ion suppression.

Electrospray is a competitive ionization processes. Different chemical compounds in an electrospray droplet compete for a limited number of charged sites at the droplet surface. When interferences that are more surface active (e.g., detergents) than the analytes of interest are present in the droplet, these compounds outcompete the analytes for droplet surface sites. The result is ion suppression. This is the same reason that linear ranges can be limited in ESI. As higher concentrations of an analyte are reached, there may not be enough surface sites to accommodate a consistent increase in ionization commensurate with concentration. This leads to decreases in ionization efficiency and nonlinearity of calibration curves at high concentrations.

Trifluoroacetic acid (TFA) and triethylamine (TEA) can impart ion suppression in different ways. TFA should be avoided in high concentrations because, while it is a good source of protons, the trifluoroacetate moiety has a high propensity for ion pairing with positively charged species. Ion pair formation in the ESI droplet is a solution phase process and can neutralize analyte ions of interest. TEA and other amines can be problematic because they have a high gas phase proton affinity. This means that just outside of the droplet, in the gas phase, interactions can occur where such species ‘steal’ protons away from analytes of interest. This can be a good thing if you are trying to make negatively-charged ions, but it can be highly detrimental to the formation of positively charged analyte species.

In order to avoid ion suppression steps, proper sample preparation steps should be taken. Care should be taken to remove highly lipophilic species and detergents at all costs. Never use detergent-based soaps on glassware that will eventually hold solutions for ESI-MS analysis. Using more rigorous preparation techniques such as solid-phase extraction can reduce the incidence of ion suppression from matrix components relative to less selective approaches such as liquid-liquid extraction and protein precipitation. For mobile phase additives, refer back to that old adage mentioned previously — if a little bit works, a little bit less probably works better. Try to keep concentrations of additives such as TFA and TEA as low as possible. I tell my students to try to stay under 0.1% v/v concentration if we need to explore use of these or related additives.

 
 
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Electrospray ionization, in itself, produces little fragmentation because of the ‘soft’ nature of the processes involved. Analyte molecules do not generally receive sufficient energy during the ionization process for bond breakage to occur. In general, electrospray will produce protonated ions in the positive mode [M+H]+ or deprotonated ions in negative ion mode [M-H]-.

There are several options for inducing fragmentation in LC–MS, most of which involve accelerating the analyte ion through potential difference in the presence of a background gas that causes collisionally induced dissociation (CID). This will be subject of a future Resolver tutorial.

Some molecules may produce abundant adduct ions such as [M+Na]+ or [M+NH4]+ depending upon the other electrolyte ions in solution. Analysis conditions should be optimized and it is important NOT to assume that the highest mass-to-charge ratio signal is the protonated or deprotonated molecular ion. It’s also important to record mass to charge that is significantly higher than the mass of the analyte to record possible highly adducted species and dimers, etc.

Often, it is the formation of adduct ions that produces an electrospray signal, especially when the analyte ion is non-ionizable.

Tables 4 and 5 show typical adduct species encountered in both positive and negative mode and Figure 13 shows a typical adduct ion cluster around the pseudomolecular ion cluster in electrospray negative ion mode.

 
Observed Explanation Mass
[M+H]+ Protonation M+1
[M+NH4]+ Mainly seen when using CH3NH4 M+18
[M+nH2O]+ M + nH2O adduct (water cluster) M+1+(nx18)
[M+H2O]+ M + H2O adduct M+19
[M+Na]+ M + Sodium adduct M+23
[M+K]+ M + Potassium adduct M+39
[2M+H]+ Analyte dimerisation (2 x M) +1
[M+H+CH3CN]+ In presence of CH3CN M+42
[M+H+CH3CN+nH2O]+ Hydrated cluster of acetonitrile adduct M+42+(nx18)

Table 4: Typical adduct ions encountered in ESI positive ion mode.

Observed Explanation Mass
[M-H]- Deprotonation M-1
[M-H-nH2O]- Deprotonation and loss of water M-1-(nx18)
[M+Cl]- Ion attachment M+35 (37)
[M-2H+Na]- M + Sodium Adduct M+21
[M-H-CO2]- Carbon dioxide loss M-45

Table 5: Typical adduct ions encountered in ESI negative ion mode

 

Figure 13: Adduct ion signals around the pseudomolecular ion in electrospray negative ion mode.
 
 

Obtaining Good Electrospray Results

There are some very simple general rules that can be followed to obtain good results in electrospray LC–MS and these are outlined here

  • Reduce salts (such as sodium) by eliminating soap and detergent
  • Use ultra-high purity (LC-MS grade) solvents
  • Eliminate TFA and strong acids
  • Keep acid concentration low
  • Keep probe voltage low
  • Increase % of organic solvents but always have some water in the mobile phase.

Your LC–MS mantra should be ‘If a little bit works, a little bit less works better’.

Tutorial Discussion

Tony: Can you give us some tips on the most effective way to optimize source parameters for any particular application?

Kevin: Most instruments are tuned regularly for either routine operations, or in order to accommodate specific applications. An old tune file that worked well previously for the same or a similar application is always a good place to start. However, one must acknowledge that, day-to-day, optimal operation settings on an instrument can change (due to temperature, humidity, or other things). 

Most instruments these days have some sort of automatic optimization function. This can be very useful, as you can enter a mass-to-charge value of interest for your analyte, infuse a sample, and the instrument software will do a quick run though of source parameters to find settings which provide optimal signal. The operator should not rely on these settings alone. Interdependence of source variables and robustness should also be considered. 

In starting a new application, I tell my students to pick a good start point from previous operating conditions for a similar application. Next, I recommend that a systematic study of source variables be performed, starting from the outside in. This is what some software does. First, optimize the sprayer voltage, then move to transfer line variables and beyond. It is typically not necessary to adjust any parameters associated with the mass analyzer itself, since this has likely been optimized during some calibration steps or routine maintenance. 

On a day-to-day basis, signal will be mainly controlled by source and ion transfer settings. Find the settings which give a high and stable signal. Next, start over from these set points to see if further modification of the variables, again from outside in, can net any additional enhancements in signal. Do this manually or run the automatic optimization again. 

Finally, check robustness. For any variable in an experiment, for which a response curve can be generated (i.e. all voltages, flow rates, and temperatures in your ESI source), it is not necessarily good to operate on a distinct maximal setting. Small changes in that setting will manifest a loss in performance very quickly. You would rather operate on a broad maximum (plateau) in a response curve, so that if small changes in a variable do occur, this does not have a drastic effect on measured response. The ability to resist small changes in response based on small changes in experimental variables is denoted as robustness. Robustness is key to a reproducible and reliable method.

 
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References

  1. M.S. Rashed et al., Clinical Chemistry, 49(3), 499–501 (2003).
  2. K. Lemr et al., Rapid Commun. Mass Spectrom., 14, 1881–1888 (2000).
  3. P. Rapp and L.H.E. Gabriel-Jürgens, Microbiology, 149, 2879–2890 (2003).
  4. D. Garteiz, S. Din and R. Morrow. “Quantitation of Digoxin in Human Plasma Using Negative Ion Electrospray Ionization LC/MS.” VARIAN Application note, No 7, 1–2 (2003).
  5. T.J.H. Borysik et al., “Investigating the structure of monomeric and fibrillar β2m by limited proteolysis and mass spectrometry.” University of Leeds.
  6. E.T. Urbansky et al., J. Anal. At. Spectrom., 14, 1861–1866 (1999).
  7. M.S. Díaz-Cruz et al., J. Mass Spectrom., 38, 917–923 (2003).
  8. F.W. Peek in “Dielectric Phenomena in High Voltage Engineering.” McGraw-Hill, 1929. Pp 48 – 108.
  9. P. Kerbale and L. Tang, Anal. Chem., 65, 3654 (1993).
  10. M.L. Vestal, Methods and mechanisms for producing ions from large molecules, K.G. Standing and W. ENS. (eds), Plenum Press, New York, New York, 1991, 157.
  11. A. Gomez and K. Tang. Phys. Fluids, 6, 404 (1994).
  12. W.M.A. Niessen, Liquid Chromatography – Mass Spectrometry, 2nd ed. Marcel Dekker, Inc, New York, 1992, pp. 264.
  13. M. Dole et al., J. Chem. Phys., 49, 2240 (1968).
  14. J.V. Iribarne and B.A. Thompson, J. Chem. Phys., 64, 2287 (1976).
  15. B.A. Thompson and J.V. Iribarne, J. Chem. Phys., 71, 4451 (1971).


Essential CHROMacademy Reading on Electrospray Ionization Mass Spectrometry


Ionization Revisited    *** CHROMacademy Registered users only ***
MichP Balogh.(2007)

A Mass Spectrometry Primer: Part 1    *** CHROMacademy Registered users only ***
MichP Balogh.(2008)

A Study of Matrix Effects on Multiply Charged Compounds     *** CHROMacademy Registered users only ***

Rohan Thakur 03/01/2010

High-Throughput LC–MS Assay for Drugs of Abuse from Biological Matrices     *** CHROMacademy Registered users only ***

Michael McGinley,Carl Sanchez (2010)

Solvent, Chemistry, and Other Myths in LC–MS     *** CHROMacademy Registered users only ***

Michael P. Balogh (2009)

 

 

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The Theory Of HPLC
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Fundamental LC-MS
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Electrospray Ionisation Theory (6hrs)
Electrospray Ionisation Instrumentation (4hrs)
Mass Analyzers (9.5hrs)
Atmospheric Pressure Chemical Ionisation (3.5hrs)
Atmospheric Pressure Photoionisation (3hrs)
Solvents, Buffers and Additives (3.5hrs)
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MS Interpretation
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Intro to MS Proteomics Research (3.5hrs)

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