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Application Note: AN04-01

Mobile Phase Selection in LC/API-MS

Luisa Pereira, Thermo Electron Corporation, Runcorn, Cheshire UK

Key Words · Mobile phase · Electrospray · APCI · Buffer pH · Solvent composition · Additives

Introduction

In LC/UV, selectivity is generally altered by changing mobile phase composition or stationary phase functionality. The most successful and convenient option in the past, particularly in the analysis of ionizable compounds, has been to optimize the mobile phase composition by changing solvent type and strength, buffer type and concentration, and pH. In LC/MS with atmospheric pressure ionization (API) techniques, the mobile phase composition needs to balance the chromatographic requirements with the ionization efficiency. Solution and gas phase chemistries need to be optimized in order to maximize ionization. Also, components which may cause ionization suppression, may cause background ions or induce the formation of several molecular weight adducts, need to be removed. This application note discusses the selection of mobile phase when developing a method in RP-LC/API-MS. Parameters discussed are solvent nature, buffer and additive type and concentration, mobile phase pH, and the effects these have not only on the chromatographic resolution but also on the detector response.

API Techniques

The two most widely used API techniques are Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI). These MS inlets or interfaces must transfer the analyte from the liquid phase into the gas phase, remove the mobile phase, and generate sample ions. In electrospray (Figure 1), the LC eluent containing the analytes is sprayed into a chamber at atmospheric pressure by the action of a high potential difference and a concentric flow of nebulizing gas. The resulting spray contains highly charged droplets. The ejection of the ions from the charged droplets occurs by the action of heated drying gas. Essentially, electrospray is a desorption technique as it transfers ions from the liquid phase into the gas phase. Thus, an understanding of solution chemistry is essential as ions are formed in solution. Acid-base equilibrium, redox reactions, and interaction with electrolytes in the mobile phase will all affect the formation of analyte ions and thus detector response. In contrast to electrospray, ion formation in APCI occurs in the gas phase (Figure 2). The LC eluent containing the analyte is sprayed into a heated chamber at atmospheric pressure. The heat evaporates solvents and analytes; once in the gas phase the neutral analyte molecules are ionized by collision with reagent ions produced by interaction of the source gases with a corona discharge. Ionization can occur by proton transfer

To MS

Figure 1: Schematic of the electrospray process

To MS

Figure 2: Schematic of the Atmospheric Pressure Chemical Ionization (APCI) process.

or charge exchange, and thus the proton affinity of all sample and mobile phase components is an important parameter to consider. Also, since neutral molecules are more volatile than ions, selection of mobile phase conditions which will maintain analytes in a neutral form in the liquid phase (acid-base equilibrium) will aid ionization in the gas phase and maximize MS sensitivity. The complimentary nature of ESI and APCI make both valuable as LC interfaces to MS.

Although solution chemistry is not as critical in APCI as in electrospray, the solvent properties still need to be considered for best performance. Protic solvents such as methanol improve positive ionization, whereas in negative APCI solvents which can capture electrons are more beneficial. Low polarity solvents generally used in normal phase chromatography can also be used in APCI, since the solvent evaporation and ionization processes are not simultaneous like in ESI.

Solvents

The most common solvents used in LC/API-MS are water, methanol, acetonitrile and mixtures of the above. The solvent composition (aqueous-to-organic ratio) is particularly important in the electrospray nebulization and ionization process. The efficiency of the electrospray process depends on the conductivity and surface tension of the liquid being nebulized. When the conductivity and/or the surface tension are too high (i.e. highly aqueous) it is difficult to produce a stable spray and to vaporize the droplets formed by the action of the high voltage and nebulizing gas. Because the surface tension of water is much higher than the surface tension of methanol or acetonitrile, the sensitivity is reduced when using more than 70-80% of aqueous mobile phase. If the chromatographic separation requires high aqueous content, then one alternative is to add a sheath liquid prior to ionization. The sheath liquid is highly organic (example isopropanol) to help the spray and vaporization, but of course this involves a more complex setup (Figure 3). The aqueous-to-organic ratio is more significant when working at high flow rates since there is more solvent to be nebulized and vaporized. A very high organic content may also lower the sensitivity, especially if no additive is used, because the conductivity of pure organic solvent is too low. A small percentage of water in the mobile phase aids the droplet formation.

Buffers and Additives

Additives are often used in the mobile phase, either to enhance the LC separation and resolution, or to enhance ionization of the sample by post-column addition. The preferred additives are formic and acetic acids (0.01 to 1% v/v) because they improve protonation of basic analytes in positive ionization. The most compatible buffers are ammonium formate, ammonium acetate, and ammonium hydroxide at concentrations of 10 to 20mM in ESI and up to 50mM in APCI (Table 1). Other additives occasionally used include trifluoroacetic acid (TFA), triethylamine (TEA) and diethylamine (DEA) but these need to be used at low concentrations (<0.1% v/v) since they may cause ionization suppression. TFA is the most commonly used ion-pairing agent in RP-LC of polypeptides. The signal suppression by TFA is caused by ion-pairing with the positive charged analyte and surface tension. The TFA anion masks the positive charge on the analyte molecule and thus prevents ion evaporation of that ion1. Postcolumn addition of propionic acid (10%) in isopropanol (75:25 v/v) has been used to counteract the signal suppression (TFA fix)2. Also, TFA has a high surface tension, thus it enriches the droplet surface during droplet formation, preventing the analytes from migrating to the surface and being evaporated.

Buffer Trifluoracetic acid Formic acid / ammonium formate Acetic acid / ammonium acetate Ammonium hydroxide / ammonia

Table 1: Buffers for use in LC/MS

pK <1 3.8 4.8 9.2

Buffer range 1.5 ­ 2.5 2.8 ­ 4.8 3.8 ­ 5.8 8.2 ­ 10.2

Concentration < 0.1% (v/v) 10 ­ 20 mM 10 ­ 20 mM 10 ­20 mM

DEA and TEA have high proton affinities and thus will compete with analyte molecules for protons, preventing positive ionization of the sample molecules. Traditionally, TEA has been used in the analysis of basic compounds at intermediate pH to ensure deactivation of the acidic silanols on the column. Newer silica-based stationary phases have fewer acidic silanols and thus symmetrical peaks are obtained for basic compounds at neutral pHs, without the need for ion-pairing agents. If the chromatographic method requires an ion-pairing agent then, heptafluorobutyric acid (HFBA) and tetraethylammonium hydroxide (TEAH) can be used in LC/API-MS as anionic and cationic reagents respectively. Involatile buffers are very popular in RP-LC because of their good buffering capacity and wide buffer range. However, the use of high purity silica-based stationary phases with volatile buffers allows for good chromatographic performance to be obtained. Involatile buffers such as phosphates, citrates and borates, ion pairing agents and inorganic acids are generally avoided in LC/API-MS method development, although modern orthogonal (off-axis) sources are more robust, and have been designed to operate with low concentrations (<10 mM) of involatile buffers or additives with minimal sample clean-up. Since blockage of the ion sampling cone can still occur with involatile buffers, a more robust orthogonal source is the M-PathTM triple orthogonal TM sampling system on the FinniganTM SurveyorTM MSQ. This interface includes a self-cleaning feature with patented cone wash that can operate effectively over the range of buffer concentrations commonly used in classical RP-LC. Post-column modification of the mobile phase is the approach taken when the conditions necessary to promote ionization interfere with chromatographic performance. Generally, an additive is introduced into the LC eluent either via a T-piece (Figure 3) or coaxially if the source design allows it.

Mobile Phase pH

The pH of the mobile phase determines the ionization state of the analytes, when working with acids, bases or amphoteric species, and therefore affects the response in LC/API-MS. The mobile phase pH also determines chromatographic selectivity for ionizable compounds: at pH values where the analyte is in its non-ionic form then the hydrophobic interactions with the stationary phase are maximized and so is retention; at pH values where analytes are fully ionized then retention decreases in RP-LC; at pH values within ±2 units of the pK the biggest changes in retention occur (Figure 4). For good pH control of the mobile phase it should be within ±1 pH unit of the buffer pK value (Table 1).

Experimental conditions:

Column: HyPURITYTM C18, 5µm, 50x2.1mm; Mobile phase: Aqueous (50%) 0.1% Formic acid pH 3 - Ammonium formate 20 mM pH 5 - Ammonium acetate 20 mM pH 8.2 - Ammonium acetate 20 mM pH 9 - Ammonium acetate 20 mM Organic (50%) MeOH MeOH MeOH MeOH MeOH

Flow rate: 0.2 mL/min; Temperature: 25°C; Detection: +ESI, 450°C, 4.5kV, 20V (-ESI, 450°C, 3.5kV, 20V); Scan: 120 ­ 480u. Analytes: Nortriptyline, Propranolol, Tetracycline, Caffeine, Paracetamol, Tryptophan, Salicylic acid, Nicotinic acid

Figure 4: Effect of mobile phase pH on the retention factor of several compounds in RP-LC

Figure 3: Sheath liquid addition

Information on the pK of each proton on the analyte will allow prediction of the ionization state of the analyte as a function of pH. With APCI, which requires the sample to be vaporized before ionization, pH should be adjusted to a value where analytes exist in the neutral form: for acids, 2 pH units below pK, for bases 2 pH units above pK. Conversely, for ESI the analyte should be ionized in solution, thus pH needs to be adjusted to 2 pH units below pK for bases and 2 pH units above pK for acids (Figure 5). However, these are general guidelines, often the pK of the analytes is not known, or may be difficult to predict based on the pK values for each proton, particularly if the analytes are complex molecules containing diverse functionalities. In this situation, several regions of pH need to be evaluated for best response both in positive and negative polarity modes. Figure 6 and 7 illustrate the variation of response in positive and negative ESI-MS with pH of the mobile phase for 8 compounds with basic, acidic or both functionalities. The best response in positive ESI (Figure 6) is obtained with 0.1% formic acid, for nortriptyline, propranolol, tryptophan and nicotinic acid. The response of the most basic compounds (nortriptyline and propranolol) decreases as the pH increases, other compounds such as tetracaine and tryptophan show a different trend because of the presence of functional groups with diverse acidity or basicity. Salicylic acid cannot be detected under positive ionization. In negative ESI (Figure 7), the response of salicylic acid and nicotinic acid increases as the pH increases, as the carboxylic group becomes deprotonated. The response for compounds such as tetracycline and tryptophan show a similar trend in positive and negative ESI as the basic and acidic group become protonated or deprotonated respectively.

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Figure 5: Representation of the favored pH regions for APCI and Electrospray: relative abundance of an acid (AH) and its conjugated base (A­), and a base (B) and its conjugated acid (BH+) at various pH values

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Figure 6: Effect of mobile phase pH on the peak area obtained in positive ESI-MS for several compounds. For experimental conditions refer to Figure 4.

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Conclusion

The mobile phase composition is an important parameter to consider when developing a method in RP-LC/API-MS. The nature of the buffer and its concentration, the buffer pH, the organic solvent nature and the aqueous/organic ratio all affect chromatographic selectivity, resolution and also the detector response.

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References

1. W. Niessen, Liquid Chromatography-Mass Spectrometry, Chromatographic Science Series, Volume 79, page 320. 2. A. Apffel, S. Fisher, G. Goldberg, P.C. Goodley, F.E. Kuhlman, J. Chromatogr. A, 712 (1995) 177.

Thermo Hypersil Ltd, Runcorn, UK is ISO Certified. ©2004 Thermo Electron Corporation. All rights reserved worldwide. We make no warranties, expressed or implied, in this product summary, and information is subject to change without notice. All product and company names are property of their respective owners. AN20021_E 04/04C

Figure 7: Effect of mobile phase pH on the peak area obtained in negative ESI-MS for several compounds. For experimental conditions refer to Figure 4.

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