The CHROMacademy Essential Guide: Optimizing LC-MS and LC-MS/MS
Thursday 24th January 2013, 11:00 AM EST, 16:00 GMT
In this session we consider the major instrument and method variables in LC-MS analysis as well as some of the
more minor ones which can sometimes make a big difference to your analysis. Eluent chemistry, interface parameters
and analyzer settings are all considered in a lively discussion format where you will have the chance to ask questions
and get answers.
Topics covered include:
What HPLC eluent factors are important in optimizing response in LC-MS
What everyday factors in HPLC can be easily changed to increase sensitivity or
improve separation in LC-MS
What API interface parameters really make a difference to LC-MS sensitivity or specificity
What MS parameters can be adjusted to improve analysis quality
What can your analyser do for you!
Ion suppression and enhancement – an insurmountable problem?
Who Should Attend:
Anyone working with LC-MS or LC-MS/MS who wants to get the very best performance from their equipment
Anyone wanting to improve their knowledge of the working principles of LC-MS analysis
Find out more about this Month's Essential Guide Webcast »
The CHROMacademy Essential Guide Tutorial Optimizing LC-MS and LC-MS/MS - January 2013
In this session, Dr Kevin Schug (Assistant Professor University of Texas at Arlington) and Tony Taylor (Technical Director, Crawford Scientific), present the “Optimizing LC-MS and LC-MS/MS Methods” Essential Guide, supported by Interactive Multi-Media content from The CHROMacademy.
The difference between a useable and optimised LC-MS method can often be differentiated by the optimisation of ‘secondary’ method parameters and attention to detail in areas perhaps not considered as of great importance. This may make the difference between obtaining usable results and obtaining the best results you can possibly get.
This tutorial will consider the working principle of a generic LCMS instrument and it also highlights the parameters which, when optimised, have a dramatic effect on sensitivity, specificity and/or robustness of the data produced. A must see for everyone using or developing methods for LC-MS and/or LC-MS/MS.
In this section, we consider the major method parameters in LC-MS which need to established prior to performing LC-MS and LC-MS/MS optimization.
For more information on the fundamental operating and theoretical principles of ESI or APCI, please visit the links below
Sample preparation is of overriding importance to achieve optimum chromatographic results.
There has been unprecedented growth in sample preparation techniques during the last few decades. In LC-MS, the term sample preparation encompasses any sample pre-treatment steps that are required to improve the selectivity of the separation, clean up the sample to prevent fouling of the column stationary phase or the LCMS interface or improve detectability of the sample.
Sample preparation begins at the point of collection and extends to sample injection on to the column. It also includes any treatment steps that are required in order to increase sample MS. Sample preparation in LC-MS is useful as it:
helps to purify the sample
increases the concentration of an analyte
increases analyte detectability by reducing matrix interference problems
improves the chromatographic behavior of the analyte
provides a robust, reproducible method that is independent of variations in the sample matrix
LC-MS is very sensitive to the presence of contaminants within the sample matrix. In particular, contaminants could appear as additional signals or manifest themselves as background.
In sample preparation for both LC-MS and LC-MS/MS, a great deal of effort is usually made in order to concentrate the analyte of interest and to remove sample components that can suppress ionization. More often than not, sample matrices contain components that need to be removed, otherwise they would compromise the chromatographic separation or increase mass spectral interference. For example, blood samples contain proteins, sugars, lipids, salts, etc. and therefore, sample preparation for blood samples must consider how to deal with such components, so LC-MS signal response is maximized for the analyte of interest. Alternatively, if the analyte has unique physic chemical characteristics compared to the bulk of the matrix, the extraction process can be deisgned such that the sorbent used is able to isolate the analyte from the matrix through, for example, a highly selective electrostatic interaction.
Sample preparation for LC-MS is usually accomplished by achieving:
the removal of unwanted matrix material
the elimination of compounds that cause ion suppression (phospholipids, TFA,…)
an increase in analyte concentration
The most widely used sample preparation techniques for LC-MS are solid phase extraction and liquid-liquid extraction.
Liquid-liquid extraction creates relatively clean extracts. It could also provide a means to concentrate the analyte of interest; however, selecting the right solvents for analyte extraction is usually a time consuming process.
Solid phase extraction is used to isolate analytes from a wide variety of samples (pharmaceutical, environmental, petrochemical, etc.). Broadly speaking, in SPE the separation depends upon analyte affinity towards the stationary phase. Advantages of using SPE for LC-MS include:
Reduced organic solvent usage
Decreased waste generation
Figure 1. SPE method selection (oversimplified).
Figure 2. Improved sample clean up with SPE.
As sample preparation provides a means to isolate and concentrate the analyte(s) of interest from your sample, it also reduces the influence of sample matrix.
Broadly speaking, electrospray (ESI) and atmospheric pressure chemical ionization (APCI) are complementary ionisation techniques as:
ESI used for medium to high polarity analytes
APCI used for medium to low polarity analytes
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 favors the analyte in the ionized form; this is, they should live in the eluent solution prior to introduction into the API interface, although this is not an absolute pre-requisite for generating a response in LC-MS, as there are secondary methods of analyte charging within the ESI interface.
APCI finds most of its applications in molecular weights below 1000 Da for medium to low polarity molecules. The analytes will need some degree of volatility and should not be thermo labile.
Under certain circumstances, the correct ionization mode is not immediately obvious (for example when dealing with moderately polar molecules); in such occasions, the following steps should be considered:
Infuse sample and evaluate ESI signal response (for both positive and negative ion modes)
Infuse sample and evaluate APCI signal response (for both positive and negative ion modes)
Choose the best ionization technique (consider signal response –intensity, linearity and time variability)
Optimize conditions (see below)
By infusing your sample and comparing signal response (in terms of intensity, linearity and time variability) obtained under different ionization techniques, it is possible to select the best choice before proceeding with the optimization process. The process can be achieved by switching either polarity (positive or negative ion mode) or ionization technique (ESI, APCI, APPI…). The strategy is illustrated in Figure 3.
Figure 3. ESI/APCI/APPI screen response. The abundance of fragment ions can be used to choose the correct ionization technique.
Alternating between ionization modes (ESI, APCI, APPI) provides a means to identify which method works best for a given sample. Please bear in mind that modern instruments are capable of switching between these two modes, this will facilitate the selection of ionization mode and polarity. Note that instruments would switch between modes during routine operation (if required), not just for deciding which is the correct ionization mode.
Some API sources are capable of operating in ‘dual mode’, and can routinely operate in both ESI and APCI mode for example. The dual mode of operation is, in some instances, the best approach for obtaining optimum response for multiple analytes within a sample – i.e. screening or qualitative work; however, this is not always the case for quantitive analysis.
There is not a general rule that allows one to predict under which circumstances which ionization mode would work best – especially for quantitative analysis. In general terms, the multimode of ionization:
does not generally give the sum signal of ionization modes used (ESI only, APCI only, APPI only)
is not always recommended, as in some applications the measured signal is not enhanced or in some cases may be reduced (i.e. the optimum response may be in either ESI OR APCI and whilst the dual mode may provide more sample information would give the best sensitivity for a particular analyte)
Figure 4. API results for an analyte (diarachidin) under different solvent conditions.
Figure 5. Multimode source allows identification of very diverse compounds in a single run. Courtesy of Agilent Technologies.
Broadly speaking reversed phase solvents (water, acetonitrile, methanol) are well suited for ESI as they favor the formation and transfer of ions from the liquid to the gas phase. Normal phase solvents (such as hexane, toluene, dichloromethane), on the other hand, cannot support ions in solution and therefore, are not suitable for ESI, but they can be used with APCI.
Mobile-phase buffers and additives are used in HPLC to control the pH and to obtain efficient and reproducible separations. They also have to be compatible with API conditions. This is, avoid nonvolatile mobile phase components as they tend to crystallize and block the ion source.
In order to optimize LC-MS signal response you should avoid the use of:
Non volatile components (NaCl, CaCl2, etc)
Surfactants (triton, sodium dodecyl sulfate, etc)
Compounds that reduce ionization/ ion formation (DMSO, TRIS, glycerol, etc.)
Corrosive agents such as inorganic acids (H2SO4, H3PO4, etc.) or alkali metal bases (NaOH, KOH, etc)
Strong ion pair reagent such as trifluoroacetic acid, as they cause ion suppression; however, TFA is seen in many earlier LCMS applications
Triethylamine/trimethylamine, however, they may enhance deprotonation for negative ion formation
Under gradient conditions the solvent composition changes with time and therefore, the properties of the spray (which in turn will affect the ion production process and the sensitivity of the whole analysis). As a consequence, caution must be exercised when using gradient LC-MS or LC-MS/MS (see later notes on using gradient HPLC in LCMS).
Surface Tension (ESI)
Solvents having higher viscosity tend to be less volatile and have higher surface tension. A high viscosity solvent will form larger, less efficiently charged droplets at the capillary tip and will require higher interface drying gas temperatures to efficiently de-solvate the electrosprayed eluent droplets. Desolvation is a critical step leading to desorption of ions. Solvents must be able to efficiently evaporate from the droplet to reduce its overall size. The ions should be solvophobic (lacking affinity for the solvent, so unable to be wetted by the solvent) to ensure the surface of the droplet has a high density of surface ions leading to efficient Rayleigh droplet jet fission.
In general, the eluent system should be designed with minimum surface tension, minimum solvation energy and reduced viscosity for optimum sensitivity in ESI-MS. Optimizing these parameters will result in correct droplet nebulisation, promote droplet desolvation and aid ion evaporation from the de-solvated droplets. In essence, the sample is infused in different eluent systems. Notice that you should perform full optimization for every eluent system (temperatures, potentials, flow rate of eluent and gases, etc.), so different signal responses are obtained. The eluent system that maximizes signal response should be selected. The process is depicted in figure 6.
Figure 6. Eluent composition versus ESI signal response.
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 and this will tend to lead, on average, to smaller droplets being produced that aid in the ion formation process and can lead to an increase in instrument sensitivity.
Table 1. Properties of selected HPLC solvents.
Surface Tension (dyn/cm)
Dielectric constant (at 20oC)
Vapor Pressure (at 25oC, mmHg)
* Potential differences to overcome Rayleigh limit for selected solvent.
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.
Solvents having higher viscosity tend to be less volatile and have higher surface tension. A high viscosity solvent will form larger, less efficiently charged droplets at the capillary tip and will require higher interface drying gas temperatures to efficiently de-solvate the electrosprayed eluent droplets.
Desolvation is a critical step leading to desorption of ions. Solvents must be able to efficiently evaporate from the droplet to reduce its overall size. The ions should also be solvophobic to ensure the surface of the droplet has a high density of surface ions leading to efficient Rayleigh droplet jet fission.
In general the eluent system should be designed with minimum surface tension, minimum solvation energy and reduced viscosity for optimum sensitivity in ESI-MS. Optimising these parameters will result in good droplet nebulisation, promote droplet desolvation and aid ion evaporation from the de-solvated droplets.
Remember: tensoactives such as surfactants and detergents tend to suppress the ionization of other compounds under ESI conditions. 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.
Under gradient conditions the solvent composition changes with time; however, it is impractical to consider dynamic modification of the sprayer position during 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. 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.
Eluent Effects on Response in ESI
Higher percentages of organic modifier within LC eluent systems tend to result in better ESI sensitivity due to lowering surface tension and solvation energies for polar compounds. Further, the increased volatility of the organic modifier allows for more efficient desolvation of the electrosprayed droplet.
Below, the signal intensity of Penicillin G against % organic modifier in the electrosprayed eluent are shown. In this example the absolute response varies between 75 and 110% relative intensity as the percentage organic modifier (in this case acetonitrile) is altered – this may result in robustness issues if the eluent system is not accurately prepared for the analysis – even in isocratic mode!
Figure 7. Response of Penicillin G with varying percentages of acetonitrile.
Further, the absolute analyte response MAY change when comparing early and later eluting species in gradient HPLC analysis. The changing nature of the eluent system (usually containing a greater percentage of an eluent component more suitable for ESI-MS i.e. the organic modifier), may result in improved response for later eluting analytes.
With this in mind it is not good practice to directly compare the response of analytes with widely differing retention times in gradient HPLC analysis. Typical examples of this type of analysis include area normalisation approaches to pharmaceutical stability analysis or quantification of pesticide residues in river water extracts etc. This is highlighted in the figure below – where the relative response ratio of ethanol to heptanol is 0.72 – whereas all analytes are present in the sample solution at equal concentration!
Figure 8. Analysis of homologous alcohols (equal concentrations). The measured ethanol (C2):heptanol(C7) ratio is 0.72 (true value 1).
Figure 9. Dynamic Drying Gas Optimization.
ESI Positive Ion Mode
The effect of changing eluent pH in positive ESI-MS is illustrated below with phenytoin (an anticonvulsant drug, 252.3 Dalton). If the pH of the eluent solution is adjusted to match the pKa of the basic functional group, 50% of the analyte molecules will be ionised at any one instant. Using an eluent pH equal to the pKa is not ideal as retention times can be variable and affected by small changes in the eluent pH. By raising the eluent pH by 2 units the basic functional group will be 99.5% non-ionised (ion-suppressed) and although its retention time will increase, the signal intensity will decrease. Conversely, by lowering the eluent pH 2 units below the pKa, the basic group will be 99.5% ionised and will show good ESI-MS positive mode sensitivity, although its retention time will be considerably shortened.
The balance between retention and sensitivity optimization in LCMS is a constant challenge and has lead to the evolution of a range of chromatographic stationary phases which show good retention of polar and ionised analytes. Such stationary phases include those designated as Aq or Aqueous compatible and the mixed mode phases which contain both hydrophobic and ionisable moieties to give a mixed retention mechanism of hydrophobic and electrostatic interactions and in which elution can be controlled using eluotropic or ionic strength.
One of the main reasons that the HILIC mode of chromatography has become popular is because it involves mobile phase conditions that are favorable for sensitive ESI-MS analysis. 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 the 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.
Compounds that have functional groups that protonate readily such as basic nitrogen atoms show good sensitivity. Those that are polar but contain no basic nitrogen atoms show moderate sensitivity. Hydrocarbons have poor positive mode ESI-MS sensitivity due to the lack of ionisable or polarisable functional groups.
Figure 10. Effect of pH on ESI(+) signal intensity.
ESI Negative Ion Mode
Solution chemistry for negative ion analysis involves the creation of [M-H]- ions in solution and the generation of these ions in solution is again a function of the sample pKa and the eluent solution pH. It is important to establish the pKa of the various acidic functional groups within the analyte molecule to allow prediction of the eluent pH that will give rise to the highest ESI-MS sensitivity. The sample will lose a proton in solution when the eluent pH is increased and become negatively charged.
The pH effect in negative ESI-MS is illustrated below with indomethacin (anti-innflamatory & pain killer, 345.5 Dalton). By raising the eluent pH, two pH units above the pKa, the acidic group will be 99.5% ionised and will show good ESI-MS negative mode sensitivity, however it’s rerention time in reverse phase HPLC will be significantly reduced. By reducing the eluent pH below the analyte pKa, the retention time of the analyte will increase but the signal intensity will be significantly reduced.
For negative ionisation, analytes with functional groups that deprotonate readily, such as carboxylic or sulfonic acids, show the best sensitivity. Analytes that are polar but contain no acidic groups show less sensitivity.
Figure 11. Effect of pH on ESI(-) signal intensity.
ESI Positive/Negative Ion Mode
For ESI positive ion mode, analytes with functional groups that protonate readily, such as amines show good sensitivity.
For ESI negative ion mode, analytes with functional groups that deprotonate readily, such as carboxylic or sulfonic acids, show the best sensitivity.
Analytes presenting polar but no highly ionisable functional groups can be analysed in either positive or negative ion mode (according to their ability to gain or lose a proton respectively), in both cases sensitivity is moderate.
Certain analytes can be monitored in both ion modes (positive and negative). The pH of the solution will determine which mode and degree of protonation/deprotonation that is observed.
Knowing the pKa of the analyte’s functional groups will allow the user to predict the required eluent solution pH for maximum sensitivity.
A basic analyte will become protonated if the eluent pH is kept below its pKa, in a similar manner an acidic analyte will protonate if the eluent pH is maintained above its pKa.
The amino acid lysine presents three functional groups (one carboxylic acid and two amines), according to the pH of the medium, this molecule can hold different charge states (+2, +1, 0, or -1) and it can be analysed under different ESI ionisation modes (positive and negative).
Figure 12. ESI Analysis of lysine.
Given all of the above discussion, it is not absolutely necessary to have the analyte 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 a strong signal, depending upon the co-electrolytes in sprayed droplet.
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, the use of an acidic mobile phase modifier to efficiently ionize compounds in the positive ionization mode is recommended and vice versa. And even if the compound did not have a nice amine group sitting there to be protonated, one might be reasonably 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 signal response should be made. It is important to initially monitor a wide mass range, so that different ionic entities, such as adducts and dimmers, can be tracked.
Figure 13 shows effects of solvent pH on the abundance of multiply charged ions of Lysozyme in ESI LCMS.
Figure 13. Solvent pH effect on the abundance of multiply charged ions of Lysozyme in ESI LCMS.
Mobile-phase buffers and additives are used in HPLC to control the pH and to obtain efficient and reliable separations. They also have to be compatible with API conditions. This is, avoid nonvolatile mobile phase components as they tend to crystallize and block the ion source. Likewise, mobile phase components should not reduce in any way analyte response.
Table 2. Buffers for LC and LC-MS.
Buffer Based System
pKa1 = 2.1
pKa2 = 7.2
pKa3 = 12.3
Trifluoroacetic Acid (0.1%)
pKa = 2.0
pKa1 = 3.1
pKa2 = 4.7
pKa3 = 5.4
Formic acid (0.1%)
pKa = 3.8
pKa = 4.8
Tris (hydroxymethyl) aminomethane
pKa = 8.3
pKa = 9.2
pKa = 9.2
pKa = 10.5
pKa = 12.0
*DBU (1,8-diazabicyclo-(5,4,0)undec-7-en) is a strong volatile base.
Buffers of trifluoroacetic acid (TFA) are widely used in LC-MS, (in particular for the analysis of proteins and peptides). Typical concentrations of TFA are in the 0.1–1% range. However, there are a couple of major drawbacks of using TFA:
It reduces signal response in ESI positive ion mode (TFA should be replaced with weaker ion pair reagents such as formic acid whenever possible)
It may completely suppress signal response in ESI negative ion mode
As a rule of thumb, a buffer concentration of 10-50 mM is adequate for small molecules. In fact, it is a good starting point to prepare your buffer at a concentration of 25 mM, this is because, if the buffer ionic strength is too low, then the buffer capacity is low as well and small changes in the eluent pH (on mixing with the sample solvent for example) can cause retention time variability. Likewise, if the buffer ionic strength is too high, then solubility problems with organic solvents may occur, and signal suppression effects are risked.
The pH within the droplet can be expected to change quite drastically during the desolvation process as the concentration of acidic or basic species become more concentrated. This phenomenon may lead to the suppression of ions (especially where the analyte has an ionisable moiety), within the droplet and therefore may change the quantitative response within the experiment. This may predicate a change in the interface parameters, the relative positioning of the capillary and sprayer and/or the eluent composition in order to optimise the instrument response.
One of the main reasons that HILIC has become popular is because it involves mobile phase conditions that are favourable for sensitive ESI-MS analysis. The high percentage of organic component in the eluent system allows efficient evaporation of the solvent (crucial to ESI). As a matter of fact, the stationary phases used with HILIC are similar to the ones used with normal phase; however, in HILIC, the eluent systems are similar to those used in reversed phase chromatography (typically acetonitrile plus an aqueous buffer), allowing ions to exist in solution.
The use of a highly organic mobile phase with low levels of salts, makes HILIC is ideal for use with electrospray ionisation mass spectrometry.
Conductivity and Ionic Strength (ESI)
The sprayer (capillary or nebulizer) current is dependent upon the conductivity of the eluent solution, which is in turn related to the concentration of ions within the eluent.
In HPLC it is usual to have several electrolyte ions present in a single eluent solution. These may include the analyte and its matrix as well as impurities from the solvents and buffer ions that may have been added. For example, HPLC grade acetonitrile contains a fairly high background level of sodium ions, which gives rise to the background signal. Buffer ions (which usually predominate), have differing conductivities. Ion conductivity is determined by the relative mobility of the electrolyte species in solution at the capillary tip and within the bulk solution of the electrosprayed droplet.
From the results shown it can be seen that even at equal concentrations in the eluent solution, HCl and NaCl electrolyte ions produce different capillary currents (and hence different ion intensities), due to their different mobilities (ability of charged particles to move through a medium in response to an electric field; basically, the faster the migration of the ions the higher the current) in an eluent system of 60%:40% water:methanol.
The practical implication is that the use of small (low molecular weight), highly charged ions as background electrolytes will give a LOW background signal. This is primarily due to the large sphere of hydration acquired by the smaller ions in solution which renders them less electrolytically mobile in the droplet.
Figure 14. ESI signal intensity vs. concentration.
Relative mobility of various electrolyte species in methanol at 25oC.
As a general rule, electrolyte ions with LOW mobilities in the eluent solution will interfere less with analyte gas phase ion production. Ions with low m/z values will give the lowest background signals in LC-MS.
The ESI interface is very sensitive to the presence of contaminants and therefore, high quality water solvents, buffers and additives should be used in order to achieve low detection limits. Likewise, the importance of proper sample preparation cannot be overemphasized.
It is not surprising that large concentrations of salts will adversely affect signal response and de-ionized water (~ 18 MΩ/cm3) is commonly used. From a practical point of view it is not possible to remove all traces of salts from the eluent system, and in fact it is undesirable, as certain analytes are known to generate detectable ions such as [M+Na]+ or [M+K]+.
Ion Pair Reagents
Ion-pair reagents may be necessary for the separation of ionic sample components. The charged sample ion forms a complex with the oppositely charged ion-pair reagent resulting in greater retention and separation.
The challenge for the analyst is to find an ion-pair system that does not interfere with the ion formation process but still yields acceptable chromatographic behavior.
Unfortunately, the most common ion-pair reagents are non-volatile and include alkyl sulfonates or tetra-alkyl ammonium salts.
API LC-MS requires that the analyte is ionic in solution (ESI) or that the analyte accepts or donates a proton in the gas phase, (APCI). Ion-pairing reagents can interfere with the ionization process due to charge neutralization in ESI-MS and by agglomerating analyte and altering proton affinities in APCI-MS.
A study of four pharmaceutically active compounds highlights the impact of the ion-pair reagent on MS response and separation as a percent of a control (ammonium acetate). The ESI signal for norepinephrine improves in the presence of the volatile ion-pair reagents (VA and PFHA). Ionization of erythromycin also improves when PFHA is present. HSA causes significant suppression for all analytes because the nonvolatile ion-pair hinders the escape of analyte from the droplets during electrospray ionization.
Figure 15. ESI signal intensity vs. concentration.
Weak and strong volatile ion-pair reagents such as valeric acid (VA) and perfluoroheptanoic acid (PFHA) can replace nonvolatile strong ion-pair reagents such as sodium heptane sulfonic acid (HSA).
Positively charged nonvolatile ion-pair reagents such as tetrabutylammonium hydroxide can be replaced with tributylamine.
When using ion-pairing reagents with LC-APCI-MS the possibility of volatility and proton transfer disruption also needs to be considered. Although it may be considered beneficial to add ion-pairing reagent to the eluent to ensure that the analyte is in a neutral form, the choice of ion-paring reagent can have profound effects on the sensitivity of the analysis, which is highly analyte specific.
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.
Tables 3 and 4 show typical adduct species encountered in both positive and negative mode and Figure 16 shows a typical adduct ion cluster around the pseudomolecular ion in electrospray negative ion mode.
Table 3. Typical adduct ions encountered in ESI positive ion mode.
Table 4. Typical adduct ions encountered in ESI negative ion mode.
Figure 16. Adduct ion signals around the pseudomolecular ion in electrospray negative ion mode.
Often, it is the formation of adduct ions that produces an electrospray signal, especially when the analyte ion is non-ionizable (see discussion below).
Ionization/ and Cationization (ESI)
Neutral molecules that have any propensity for hydrogen bonding will form adduct ions with ammonium or alkali metal ions
The detection of neutral compounds can be enhanced in electrospray mode by the addition of cationization reagents. Sodium acetate or ammonium acetate added in 20-50 mM amounts can significantly increase the signal intensity. Note that additional heptafluorobutyric acid can also increase the signal.
Cationization of certain molecules can also be achieved by adding certain organometallic salts, especially lithium, sodium, potassium, rubidium, and caesium; yielding species of the form [Analyte+Metal]+. [Analyte+2Metal]2+, etc. Alcoholic solutions of the above salts are usually prepared at a concentration of approximately 1mg/mL and then mixed with the eluent system.
Tetraethyl or tetrabutyl ammonium hydroxide had been used to increase signal response in ESI(-). In negative ion mode, signal intensity can be enhanced by the presence of certain counter ions (like sodium or ammonium) in the eluent system. The increased response is due to at least two factors: improved electrospray formation and increased ionization of the weak acid due to the higher pH.
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 practically 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.
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 a decrease in ionization efficiency and nonlinearity of calibration curves at high concentrations.
As can be seen from Figure 18, the [Bu4N]+ and [Mor+H]+ ions (which are present at constant concentration) are increasingly ‘suppressed’ as the concentration of the NH4+ ion (originating 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 18. Decrease of analyte ion intensities due to competition with added [NH4]+, at constant analyte concentrations [Bu4N]+ = [Mor+H]+ = 1 x 10-5M.
Figure 19. Ion suppression –Competing ions.
Under ESI conditions, signal response is controlled by the ion evaporation process (the process by which ions are transferred from the liquid to the gas phase). 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’)
Relative Sprayer Positioning
Differences in response for ions of varying surface activity (sensitivity constant – K) with changing relative sprayer position
Ion production (charged residue v’s ion evaporation) and relative surface activity mean ‘sweet spot’ between ions differs axially and laterally within the spray plume
Will also be affected by drying gas flow and temperature
Relative sprayer position can and should be optimized - optimal setting is likely to be viable for providing high signal quality across a wide range of analytes / parameter settings
Figure 20. Sprayer distance effect on ESI signal response.
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.
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 21.
Figure 21. 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.
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 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 Figure 22.
Figure 22. 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.
Figure 23. The sensitivity constant.
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.
Figures 24 illustrates quantitative aspects of surface activity.
Figure 24. Quantitative aspects of surface activity in the analysis of paraben.
Figure 25 illustrates an ion suppression study - Oxycodone Infusion with solvent flow. Negative control injected at ~0.1min.
Figure 25. Quantitative aspects of ion suppression.
Using more rigorous preparation techniques such as solid-phase extraction, as has been described above, can reduce the incidence of ion suppression from matrix components relative to less selective approaches such as liquid-liquid extraction and protein precipitation.
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. Additives may form 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.
Figure 26. Common HPLC eluent additives and their actions in solution.
Trifluoroacetic acid (TFA) and triethylamine (TEA) can give rise to 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 once in the gas phase, interactions can occur where such species attract protons away from analytes of interest. This can be advantageous when trying to form negative ions, but it can be highly detrimental to the formation of positively charged analyte species.
For mobile phase additives, refer to the old adage — 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.
Proton Affinity (APCI)
In order to make some rudimentary predictions about the relative sensitivities of each analyte ion and any possible suppression effects attributable to eluent components such as volatile buffers, it is important to consider the relative proton affinity of the components.
Table 5. Proton affinity of selected organic molecules for APCI.
Proton Affinity (kcal/mole)
Table 6. Reagent Gas Proton Affinity.
The APCI optimum response region represented on figure 27 is for analytes with greater proton affinity than water, but one must consider the proton affinity for ALL eluent components as the buffers and additives used may compete for protons with the analyte, potentially reducing sensitivity.
Figure 27. APCI(+) optimal response region.
Gas phase acid-base chemistry is of overriding importance in LC-APCI-MS where reactant gas mixtures are the rule rather than the exception.
The adequate selection of a suitable reagent gas permits in some cases the analysis of small polar compounds that are typically analysed by ESI.
Signal Suppression by Additives in APCI
One common problem with gas-phase acid base chemistry in negative APCI is signal quenching by acidic mobile-phase constituents.
The extent of the quenching (or signal suppression) is directly correlated to gas phase acidities and basicities. In general positive ion APCI is preferred over negative ion mode whenever this is possible. This effect is shown below.
Figure 28. Caffeine response under different APCI experimental conditions.
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 whenever possible
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’.
The practical upper limit to eluent flow in pure electrospray is 10-20 μL/min depending upon the solvent composition. Capillary design may be modified to increase the tolerance of the electrospray process to increases in eluent flow rate, liquid surface tension or electrolyte concentration.
As can be seen from Figure 29 the electrospray penicillin G signal optimises at flow rates lower than 0.05 mL/min. So, by looking at how signal response varies with the eluent flow rate (through the capillary) it is possible to select the flow that optimizes response.
Figure 29. Penicilling G ESI relative intensity versus flow rate.
The ideal in terms of capillary tip droplet charging is to have a small drop that forms over a relatively long period of time. This will ensure that the droplet contains many charged species and the surface spacing of the charges will cause the initial coloumbic fissions at a point within the source that will allow optimal sampling of gas phase analyte ions.
Pneumatically Assisted ESI
The use of high pressure, high flow rate nitrogen from a concentric tube at the capillary tip means that this Pneumatically Assisted ESI optimizes at slightly higher flow rates than the non-assisted technique.
Figure 30. The use of an axially sprayed gas limits the droplet size and ensures more efficient droplet charging at high eluent flow rates.
In general, pneumatically assisted ESI optimizes at flow rates of around 0.2 mL/min, but flow rates of up to 1.0 mL/min can be endured by the source with a moderate reduction in sensitivity.
Figure 31 shows a sensitivity reduction of around 40% for Penicillin G [M+H]+ when the eluent flow rate is increased from 0.2 to 1.0 mL/min, this significant reduction can be tolerated in some cases.
Figure 31. Penicilling G pneumatically assisted ESI relative intensity versus flow rate.
Essentially, for a given eluent flow rate (flowing through the capillary) the nebulising gas flow rate has to be optimized. Figure 32 illustrates the procedure.
Figure 32. Nebuliser gas flow rate effect on signal response.
For applications where sample amounts are limited or for high sensitivity work, micro- and nano-flow ESI interface devices are available. Flow rates of between 100 and 10,000 nL/min are usual with these devices and the samples may be loaded directly into the needle, infused from pressurised vials or introduced via packed silica capillaries when using capillary LC systems. Sensitivities in the range of fempto-moles on column have been reported.
Table 7. Column diameter versus flow rate.
Essentially APCI is a high flow rate technique optimizing at flow rates of over 1.0 mL/min, above 0.4 mL/min an unstable analyte signal is usually observed due to the instability of the gas plasma caused by non-reproducible discharge processes. At high flow rates (2-4 mL/min), the heating and drying gas should be adjusted to ensure that all species are in the gas phase as they encounter the discharge region, allowing maximum ion generation.
Figure 33 illustrates the relative intensity of the protonated [M+H]+ ion of penicillin G for APCI applications over the flow rate range of 0.1 - 2.0 mL/min. The mobile phase was 50:50 acetonitrile:0.1% formic acid (aq).
Figure 33. Penicilling G pneumatically APCI relative intensity versus flow rate.
The signal intensity increases as the flow rate is raised corresponding to the formation of a stable and reproducible solvent gas plasma that is effective in ionising the analyte molecules present in the discharge region of the source.
The actual value of the optimal flow rate for a particular application will depend upon the analyte, the nature of the eluent system and the instrumentation used. To this effect, flow rate must be optimised when analysing compounds not previously studied using LC-MS. Once the eluent flow rate was set, then the nebulizer gas flow rate must be optimized as with pneumatically assisted ESI.
The flow rate incompatibility can be solved through the use of LC columns with reduced internal diameters as these columns generally have flow rates that can be directly introduced into both APCI and pneumatically assisted ESI sources.
Table 8. Typical flow rates used with narrow and micro-bore LC columns.
A very important instrumental parameter common to both ESI and APCI is the capillary (or ionization) voltage. While in ESI a high capillary voltage is typically required (4 to 5kV), APCI applications usually require low voltages (in the range of 2.5 to 3.0 kV).
The formation of a stable nebulization aerosol is dependent 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.
Figure 34. 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.
Figure 35 illustrates the effect of capillary voltage on signal response.
Figure 35. Capillary voltage effect on signal response.
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. A great advice on ESI, which pertains to many of its experimental parameters is ‘If a little bit works, a little bit less probably works better’. This particularly pertains to concentrations of mobile phase additives, flow-rates and spray voltage.
Figure 36 illustrates the capillary voltage effect when performing the analysis of mitoguazone.
Figure 36. Flow injection analysis of mitoguazone. Courtesy of Agilent Technologies.
A less well documented mechanism for ionization is derived via the triboelectric effect. As the mobile phase and analyte species exit the nebuliser, the sheer forces generated by the nebulising gas tear the liquid stream into droplets. The ‘friction’ generated by this process generates an electric charge (the triboelectric effect) at the liquid-gas interface. This can cause ion formation in some non-volatile analytes. Triboelectric charging is most common for analyte molecules that are moderately polar and/or non-volatile.
Figure 37. Triboelectric charging process.
Triboelectric charging depends neither upon the electrons generated by the corona discharge electrode, nor upon the applied capillary potential. Ions may be generated (with their associated signals in the mass spectrum) even when the corona discharge and/or capillary voltage values are kept very low or even turned off.
Corona Electrode Pin Potential (APCI)
An efficient APCI process only occurs when the corona is kept at the right potential.
Figure 38. Corona electrode pin voltage effect on signal response.
Cone Voltage and Ion Declustering
The cone voltage, also known as orifice voltage, is used to extract ions from the atmospheric pressure region of the ion source into the high vacuum region of the mass analyzer.
In addition, this parameter can be used to induce some in-source fragmentation for structural determination. In fact, when the cone voltage is increased, ions are accelerated, undergoing collisions with gas species (solvent vapor and desolvation gas) and resulting in some fragmentation. Bear in mind that cone voltage is not selective but it can be used to generate certain ions, in particular, it can be adjusted to obtain either molecular ions (of a compound) or structural information (fragmentation).
Typical cone voltage parameters for both ESI and APCI are in the region of 10 to 60 V.
Figure 39. Cone voltage effect on signal response.
The cone voltage provide a means to reduce the incidence of ion clusters (declustering potential). Note that your voltage settings may not be transferable to another instrument.
Figure 40. ESI +ve mass spectrum of sulfamethazine acquired with no collision induced dissociation – little fragmentation.
Figure 41. ESI +ve mass spectrum acquired with CID.
The effects of declustering analyte species require more attention with APCI than with ESI, as many of the gas phase analyte ions produced are clustered with water molecules and reagent ions derived from the eluent system. Declustering of analyte ions may be achieved using one or a combination of the following approaches:
Using a counter current gas flow at the nozzle plate, also known as a curtain gas
Using a heated transfer capillary between the API region and the nozzle-skimmer region
Using a drift voltage between the nozzle and skimmer plates, to promote intermolecular collisions between the ion clusters and background gas molecules (collision induced dissociation CID)
Figure 42. Ion Declustering in ESI/APCI.
Solvent clusters formed without the analyte of interest have to be removed in order to prevent noisy baselines obscuring of the analyte signal. Increasing the drying gas flow rate and temperature in the main interface can reduce the formation of these clusters, however, too high drying gas flow rates will attenuate the signal from the analyte.
Gas Flow Rates and Temperature Settings
Interface Gas Flow Rates
In LC/MS the eluent flow rate affects not only the chromatographic separation but the performance of the mass spectrometer.
In LC-MS, both instrument response and sensitivity will decrease with the size of the droplets formed at the capillary tip. By introducing an axially sprayed gas around the forming droplet (nebulizing gas) the droplet size is restricted and the droplets are charged more efficiently. The ion source temperature is usually set to 100oC. A desolvation gas (usually nitrogen at high temperature) is also delivered through the ion source to help evaporation and solvent removal. The table below lists common settings for the aforementioned parameters.
Table 9. Typical LC-MS flow rates and temperatures.
The basic approach consists in setting your initial experimental values as indicated on table 9, then altering the other parameters until signal response is optimized. This could be a time consuming process and therefore, the experience and expertise of the instrument operator should be complemented with proper experimental design, so LC-MS parameter selection will be optimized in minimum time.
In general APCI-MS interfaces are considered very simple to operate. Choosing the optimum settings for the more important interface parameters is in general less critical than for electrospray interfaces but choosing the wrong parameters could have a serious impact on your application. The case study presents an unrecognizable spectrum for the drug ibuprofen when the APCI parameters (such as probe temperature, reagent gas type, eluent flow rate and composition, etc.) are not optimized.
Vaporizing Probe Temperature (APCI)
Of all the parameters that may be optimized within the APCI, the vaporizing probe temperature has perhaps the most profound effect on signal intensity.
Figure 43. Vaporizing probe temperature effect on peak shape (the remaining parameters were kept constant).
Figure 44. Vaporizing probe temperature effect on signal response.
Figure 45 illustrates the effect of the vaporizing probe temperature on APCI signal response.
Figure 45. APCI Vaporizing Probe Temperature. Courtesy of Agilent Technologies.
In general the flow rates of gas used as nebulising, auxiliary, make-up and countercurrent flows in the APCI interface vary widely between instrument manufacturers. The flow rates of gas used will also vary depending upon the chemical nature of the eluent system. However, it should be noted that the required nitrogen supply could be as high as 600 L/h in some systems with a line pressure of 0.7MPa, this will almost always require the Nitrogen supply to be directly pumped from a liquid nitrogen pressurized vessel or from a nitrogen generating unit supplied with high-pressure compressed air.
Figure 46. Optimized vs. non-optimized APCI spectrum parameters (cone voltage, probe temperature and flow rates of eluent, nebulizer gas and desolvation gas).
Due to the milder ionization conditions of ESI/APCI compared with EI, there are less ion source contamination problems in LC-MS than in GC-MS. However, for optimum results, the LC-MS ion source should be cleaned on a periodic basis.
Contamination of the sampling orifice or tube can prove to be detrimental to the performance of the instrument, in some cases leading to very frequent source cleaning when dealing with samples in dirty matrices or when using non-volatile solvents and buffers.
The layer of contamination inside an orifice or tube will attract a buildup of charge that can effectively stop the passage of ions, while the flow of neutrals is not affected, thus significantly decreasing the instrument response.
Cleaning regimes will differ for instruments from different manufacturers but may include some physical abrasion (aluminium powder is a popular choice) and / or wipe cleaning of the sampling cone and other source components followed by sonication in a range of solvents matched to the polarity of the contaminants – (hexane, acetone and methanol are all popular choices for source cleaning).
Common Background and Contaminant Ions in LC-MS
Some peaks in your spectrum may not be from your compound of interest (contaminants, impurities, etc.). Tables 10 to 13 list and identify background and cluster ion signals typically encounter with ESI and APCI.
Table 10.Background and cluster ions typically encounter with ESI positive ion mode.
Table 11. Background and cluster ions typically encounter with ESI negative ion mode.
Table 12. Background and cluster ions typically encounter with APCI positive ion mode.
Table 13. Background and cluster ions typically encounter with APCI negative ion mode.
The process of tuning and calibration is crucial to guarantee analysis repeatability across LCMS instruments and labs. By calibrating the spectrometer, confidence that the detected mass of an ion will agree with its true mass. Likewise, tuning the MS will give confidence on signal response (intensity).
Mass spectrometry involves the separation of charged species which are produced by a variety of ionization methods in LC-MS. These include:
Electrospray Ionisation (ESI) – this ionization mode uses condensed phase (liquid) charge separation and ion evaporation techniques to produce vapour phase analyte ions
Atmospheric Pressure Chemical Ionization (APCI) – this ionization mode uses analyte desolvation and charge transfer reactions in the vapour phase to produce vapor phase analyte ion
The process of tuning and calibration is critical to achieve optimum LC-MS results, as this process sets:
voltages on the source elements
amu gain and offset for correct peak width
set the electron multiplier voltage
the mass axis for proper mass assignment
Tuning and calibration is important as it:
calibrates the m/z scale of the spectrometer versus standards of known m/z ratio
provide the means (if performed on a periodic basis) to realize that spectrometer contamination or degraded electronic components have not changed the selected mass positions
ensures repeatable analysis from instrument to instrument or lab. to lab
checks that spectrometer gives expected relative ratios of ion fragment intensities for a target compound
acts as a diagnostic tool to indicate the service / cleaning requirements of the spectrometer
provides results that act as a chronicle of system performance
Unfortunately, manufacturers do not provide m/z calibrated instruments. So this process must be accomplished by infusing a standard of well known mass spectrum into the LC-MS instrument and adjusting the response of the MS component accordingly. Broadly speaking, the user should provide the known m/z values for the peaks in the mass spectrum of the standard, so the system can make appropriate adjustments.
Modern GC-MS and LC-MS have very stable m/z scales, so strictly speaking, mass calibration may not need to be repeated for several days; however, it is a good practise to check the calibration on a daily basis.
Most instruments are calibrated using infused tune compounds that contain spectral lines due to ions whose mass is known to the level of accuracy required i.e. unit mass values for quadrupole mass analyzers or accurate mass values for double focusing magnetic/electric mass analyzers.
Mass axis calibration should be independent of the API technique used for ion generation so long as the calibrant is capable of efficient ionization under a given set of interface conditions.
Calibration of the mass axis requires series of ions with known mass that cover the required operating mass range, the ions being as evenly spaced as possible.
Calibration of the mass axis is usually a discrete process that is carried out at regular intervals to ensure correct mass assignment i.e. at the beginning of each working day or prior to a campaign of analysis -depending upon the laboratory and its applications. However, when working at high resolution where high mass accuracy is required a lock mass compound may need to be constantly infused alongside the analyte. This is particularly prevalent with time-of-flight mass analyzers where there may be a tendency for the mass axis calibration to drift due to the highly sensitive nature of the mass analyzing device.
The voltages applied to the various ion source components must be ‘tuned’ to achieve target ion abundances for various analyte ion masses. This ensures optimal instrument sensitivity and a predictable response across a range of masses, which allows for subsequent library searching of spectra.
During tuning (sometimes called ‘autotuning’), the relative and absolute abundances of fragments of a known tune compound are established and the mass assignment, resolution and spectral peak width generated by the mass analyser are also adjusted and set.
The tuning process involves adjusting a number of mass spectrometer parameters. Some are purely electronic and only affect the way the electronics process the signal. Other parameters affect the voltage settings or current to ion source components, the mass analyser and detector. In general terms, the tuning process will set:
voltages on the source elements
gain and offset of the mass analyser for correct spectral peak width (resolution)
the electron multiplier voltage
the mass axis for proper mass assignment
The importance of tuning cannot be overemphasized and is performed to check the mass spectrometer is working correctly and/or to ensure that spectra (mass assignment and relative abundance of spectral signals) resemble a previously determined standard. The process of tuning will:
check to see that spectrometer contamination or degraded electronic components have not changed assigned mass positions (calibration of the mass axis)
ensure repeatable analysis from instrument to instrument (or lab to lab)
check the spectrometer gives expected relative ratios of ion fragment intensities for a target compound
act as a diagnostic tool to indicate the service/cleaning requirements of the spectrometer
act as a chronicle of system performance
match fragments from a known calibration compound and adjust mass axis so it agrees with the expected mass assignments
API Suitable Compounds for Tuning and Calibration
As API-MS produces little fragmentation from a single ion, LC-MS calibration is usually achieved by using a mixture of compounds. Mass axis calibration should be independent of the API technique used for ion generation so long as the calibrant is capable of efficient ionisation under a given set of interface conditions.
Calibration of the mass axis requires series of ions with known mass that cover the required operating mass range, the ions being as evenly spaced as possible. Nowadays, a wide range of substances is available for calibrating the m/z scale of an API-MS instrument, including:
Poly ethylene glycol (PEG)
Poly ethylene glycol dimethyl ether (PDE)
Poly propylene glycol (PPG)
Perfluorinated phosphoazides (similar to a commercial calibration material known as Ultra Mark)
Other alternatives such as mixtures of sugars and solvent clusters such as methanol clusters) are also available for calibrating the m/z scale of an API-MS instrument. Of course, series of proprietary solutions have been developed by different instrument manufacturers.
For many years, poly ethylene glycol (PEG) and polypropylene glycol (PPG) were the most widely used calibrants for LC-MS; however, these compounds are very sticky and tend to remain in the system for long periods. As a consequence, their use is falling out of favor. Table 14 lists selected API calibration standards and their applicability range.
The selection of mass analyzer for a particular application will depend upon the analytical needs and budget. Table 16 will help with your mass analyzer selection.
Table 16. Figures of merit of selected mass analyzers.
MS-MS experiments can be performed using either tandem-in-time or tandem-in-space mass spectrometry:
Tandem in Time: The term tandem-in-time refers to the MS/MS operation mode in which a given mass analyser is used to select, store, isolate and exposed the precursor to some dissociation process (during a third time interval). Typical examples of tandem in time mass analysers include the quadrupole ion traps and ion cyclotron resonance (FTMS) mass spectrometers.
Tandem in Space: The term tandem-in-space refers to the MS/MS operation mode in which a given mass analyser is used to select the precursor ion whilst a second mass analyser is used to analyze the products resulting from fragmenting such precursor ion. Typical examples of tandem in space mass analysers include the triple quadrupole and the Q-TOF.
In practice, tandem in time is easier to achieve than tandem in space, and this is because, tandem in space requires more points of ion focus that must be optimized simultaneously to achieve a successful experiment.
Broadly speaking, tandem in space spectrometers are characterized by their high collision energy (larger than with their tandem in time counterparts).
Table 17. MS/MS analyzers.
* Such as Q-TOF, TOF-TOF and double focusing instruments.
Triple Quadrupole Efficient Operation
Tandem MS/MS with Triple Quadrupoles
Tandem mass spectrometry (or MS/MS analysis) is used to regain structural information by fragmenting ions (that in this case were produced in the API interface). The hardware used to perform MS/MS experiments usually includes a combination of two mass analysers and a collision cell:
First mass analyser (Q1), used to select the initial ion(s) of interest
Collision cell (Q2), where the ion(s) of interest receive enough collisional energy to fragment
Second mass analyser (Q3), used to collect and measure the fragment ions of interest
The design of a triple quadrupole instrument can be found in figure 52.
Figure 48. Triple quadrupole representation.
Triple quadrupole instruments are well suited for the study of low energy ion-molecule reactions. These devices are in general outperformed by magnetic instruments; however, triple quadrupole instruments have many advantages that have made them the analyzers of choice in many situations, such as:
Relatively low cost
Simple to operate
Excellent sensitivity in the SRM mode
Capability of MS3 experiments
Modern instruments are capable of achieving high MS/MS efficiency
Multiple Reaction Monitoring (MRM)
In Multiple Reaction Monitoring (MRM) selected ions only are allowed to pass through the first mass analyzer (Q1) to be fragmented in the collision cell (Q2), selected fragments are permitted to pass through the second mass analyser (Q3)
Q1 operated in SIM Mode, allowing transmission of specific ions
Q2 is operated as a collision cell, where ions coming from Q1 are fragmented
Q3 is operated in SIM Mode, allowing transmission of specific fragments
Figure 49. The MRM process.
MS/MS Parameter Optimization
There is a large number of parameters that have real incidence on the overall LC-MS/MS process (in terms of separation, ionization and fragmentation). Therefore, if looking for optimum system performance, a systematic approach that permits to adjust every major parameter is needed, otherwise, optimum response would not be achieved easily or at all. Nowadays, the use of experimental design is becoming mandatory as it helps to find the correct LC-MS/MS parameters in minimum time.
In LC-MS/MS the choice of optimization criteria is not evident, for in order to achieve optimum system performance, both LC separation and MS/MS conditions must be optimized. From an HPLC point of view, you can use the traditional criterion descriptors currently used for LC optimization (selectivity, resolution, peak symmetry, etc.) which depend upon the chromatographic conditions. Likewise, from an MS perspective, all parameters that optimize signal response should be considered (capillary voltage, source temperature, etc.), so abundant ions of interest (or high signal to noise ratio) are obtained.
Basically the whole process should commence by having a set of HPLC conditions that provide levels of selectivity and resolution that are sufficient to undertake MS/MS analysis. Once this is done, the optimization of the MS/MS parameters can take place. Interestingly, the optimization of the LC separation can take place only after the MS/MS conditions were optimized.
Parameter optimization is usually performed in one out of two ways:
Sequential Optimization: a few experiments are initially conducted, then the optimum conditions are found by using a search routine
Simultaneous Optimization: a large number of experiments are initially conducted according to an experimental design, then the optimum conditions are found by using a mathematic model that relates the response to the LC-MS/MS parameters
Developing a robust LC-MS/MS method is a multidimensional task that requires both HPLC and MS conditions being optimized. Note that we are not going to explain how to optimize LC separations; however, interested readers are invited to visit the links below:
Interface voltage optimization (capillary and cone)
Interface gas flows (nebulizing and desolvation)
Sample information is crucial to start the optimization process. Note that even an experienced operator will need, to some extent, trial and error. Due to the large number of parameters to consider, experimental design should be used as it will help to find the correct MS parameters in minimum time.
The most important parameters required in order to optimize the API ionization process (ion source conditions) include:
Eluent flow rate
Position of the spray
Nebulizer gas flow
Curtain gas (instrument dependent)
Ion source temperature
Pin electrode potential (APCI)
Warning: it is not recommended to change the positioning of the spray during a chromatographic run, as this in general will compromise reproducibility.
The most important parameters required in order to optimize the MS/MS process (which is instrument dependent) include:
Collision gas (type and pressure)
Filling time (ion trap mass analysers)
Last but not least, design of experiments provides not only a means for achieving LC and MS optimization but it is also applicable to sample preparation. In fact experimental design has been successfully used to optimize sample purification and extraction protocols in a number of different LC-MS and GC-MS application scenarios.
MRM Parameter Optimization General Strategy
Typical MS/MS (MRM) parameters for optimization:
Identify unique and /or abundant pre-cursor ion and optimize instrument parameters to gain maximum sensitivity
Identify product ion(s) (fragments) giving highest signal abundance - this may be achieved using a ‘scan’ function on Q3
Pay attention to uniqueness of the fragment ions (larger mass difference between parent and precursor tend to be more specific)
Dwell time – effects the abundance of each ion / transition so if multiple transitions monitored issues can arise where dwell times differ
Whether you work in a lab or manage a lab, you will benefit from being a member of CHROMacademy.
As a member of CHROMacademy, you will get access to our vast e-Learning archive full of great interactive content and animations.
All our Essential Guide Webcasts and tutorials and LCGCs archive of magazine articles and webcasts from your favourite authors - John Dolan, John Hinshaw, Mike Balough, and Ron Majors. Plus vendor application notes, electronic laboratory tools and calculators and with our 'Ask the Expert' function - help is always at hand.
I feel empowered to fix things
I can troubleshoot effectively
I know where to go for help
I understand my analyses
I know where to get applications
I’m up to date
I’m more employable
My career is progressing
Improved equipment utilization
Faster method development/problem solving
Flexible workforce with a common standard
Better quality data
Get up to speed quicker
Less reliant on me
I spend less time on training
Subscribe for $399 per/year and access:
The entire e-Learning archive
All Essential Guide Webcasts and Tutorials
LCGCs archive of articles and webcasts
Expert troubleshooting advice when needed
Like CHROMacademy on Facebook and keep up-to-date with the latest webcast, tutorial and eLearning release schedules.
In this session, Dr. Kevin Schug (Associate Professor-Chemistry & Biochemistry, University of Texas at Arlington) and Tony Taylor (Technical Director, Crawford Scientific) consider the major instrument and method variables in LC-MS analysis as well as some of the more minor ones which can sometimes make a big difference to your analysis. Eluent chemistry, interface parameters and analyzer settings are all considered in a lively discussion format where you will have the chance to ask questions and get answers.
Dr. Kevin Schug
Associate Professor-Chemistry & Biochemistry, University of Texas at Arlington
Key Learning Objectives:
Effects of eluent modifier, pH and additives on sensitivity of Atmospheric Pressure Ionization LC-MS equipment
What interface parameters make a difference to sensitivity or specificity – and why
Interface chemistry issues such as ion suppression or enhancement – just exactly what can be done?
Can you make a difference to the analyzer performance? What performance can you expect from the wide range of analyzer types currently available
MS/MS experiments – critical parameters and how to optimize instrument response with various analyzer combinations