Buffers and Other Mobile Phase Additives

Another way to avoid unwanted phase separation is to force the solvent system to be saturated in one component. For example, a distinct water layer is put in contact with water-saturated hexane and the solution is continuously stirred to ensure that the hexane will remain water-saturated. Unfortunately, the solubility is still temperature dependent, so that the solvent/colunnn system must be temperature regulated to guarantee stable and reproducible chromatographic results. 

As described earlier, mobile phase modifiers (MPMs) are present at low levels— typically 2% or less. MPMs are typically considered as additives or buffers. To distinguish the role of additives and buffets, the following definitions are used. Additives are present at a defined level and the resulting equilibrium establishes the solvent characteristics. Buffers are MPMs that are added in order to hold a particular aspect of the mobile phase constant a common case being pH. Exanples of MPMs that are additives include 0.1 % triethylamine (TEA), 0.2% trifluoroacetic acid (TFA), 1% phosphoric acid, and 50 mM sodium dodecyl sulfate. 

Buffers are typically mixtures of solids and or a liquid and a solid. Examples of buffers include acetate buffer (50 mM at pH 4.0), phosphate buffer (lOOmM at pH 7.0), and 0.1 % TEA/0.1 % TFA. Since the use of buffers in RP separations is commonplace, basic knowledge of the solubility of each buffer component in the mobile phase is critical. Consider the case of phosphoric acid and its series of conjugate acid/base pairs as the buffer for acetonitrile/water mobile phase. Phosphate has negligible UV absorbance down to 200 nm (see Fig. 1.5 f), does not denature proteins, and has three effective buffer regions around the pH values of 2, 7, and 12. These properties make phosphate buffers very attractive for many HPLC separations. 

The use of buffers is common and is critical enough to many separations to warrant a detailed discussion here. As mentioned above, HPLC buffers are commonly used to control solution hydronium ion, H+. The H concentration [H+] is represented by the pH scale where pH = - log[H+]. 

Buffers are chosen on the basis of their dissociation constants. For example, acetic acid has a dissociation constant, K, of 1.76 x 10 (also expressed as a p/T value of 4.76). The chemical equilibrium for acetic acid is represented as 

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Solvent systems encompass a dizzying array of permutations of organic solvents, buffers, and other mobile-phase additives. However, the most commonly employed solvent systems involve acetonitrile, methanol, and/or tetrahydrofuran. Buffers are typically acetate (pKa 4.8) or phosphate (pKa 1.3 and 6.7) at approximately 100 mM. For the analysis of a small number of free amino acids, isocratic elution is often possible. For the determination of an overall amino acid profile from a hydrolysate sample, complicated ternary gradients are often necessary. 

However, despite the great advantages it offers, LC-MS coupling has some limitations, apart from the interfacing need Incompatibility with some of the nonvolatile buffers and other mobile phase additives. Hence, phosphates, ion-pairing agents, and amine modifiers are replaced by ammonium acetate, ammonium formate, and so forth. 

BUFFERS AND OTHER MOBILE PHASE ADDITIVES 27 TABLE U Common Components Used for BnfTers... 

Reverse-phase and ion-exchange columns have been used for the separation of acesulfame-K. Veerabhadrarao et al. (27) and Hannisdal (62) separated acesulfame-K from other sweeteners and additives on reverse-phase Cl 8 columns using methanol acetic acid and methanol-.phosphate buffer mobile phase, respectively. However, most of the reverse-phase methods for the separation of acesulfame-K use acetonitrile phosphate buffer as the mobile phase (14,16,33,44,51,63). According to Prodolliet and Bruelhart (33), the use of acetonitrile in the mobile phase provides a better resolution for sweeteners than methanol. 

Mobile-phase additives are used in HPLC to control the pH and ensure efficient and reliable separations. They also have to be compatible with ESI or APCI conditions. If the pH of the mobile phase needs to be reduced for better LC separations, the most suitable additives in LC/MS are acetic acid and formic acid with typical concentrations ranging from 0.1% to 1%. Note that addition of acids will suppress ionization in negative ion mode. Weakly acidic compounds may not form deprotonated ions under acidic conditions. If the pH of the mobile phase needs to be increased to enhance LC separations, ammonium hydroxide (0.1% to 1%) is suitable. Weakly acidic compounds can be ionized effectively in negative ion mode. Triethylamine is another additive that may be useful to enhance ionization of other compounds in negative ion mode because it is basic. It should be cautioned that the presence of triethylamine might suppress ionization of other compounds in the positive ion mode. A commonly used volatile salt in LC/MS to buffer mobile phases is ammonium acetate (<0.1 M). It is used to replace nonvolatile salts such as phosphates because these nonvolatile salts tend to crystalUze in the ion source and block the source, suppressing ionization of analytes. 

Sometimes other variables must be investigated such as the pH and/or the ionic strength of the buffer in the mobile phase or the concentration of additives in the mobile phase such as for instance tensio-active substances in micellar chromatography. In such a case the first step in an optimization is to screen these factors and to identify the most important ones for the subsequent optimization. The screening (Section 6.4.2) leads to a definition of the experimental domain in which the optimum is probably situated. This is somewhat similar to the retention optimization step. It is followed by an optimization step (Sections 6.4 and 6.7), in which the most important variables are changed, often according to an experimental design. Similar methods are used in capillary zone electrophoresis. 

For compounds with groups that can be protonated or deprotonated, i.e., compounds that show liquid-phase acid-base behaviour, a buffer must be added to the RPLC mobile phase in order to avoid problems with poor retention, poor resolution, and/or poor repeatability in retention time. Phosphate buffers are applied for this purpose in RPLC with UV or fluoresoence detectors, because of the low UV eut-ofif (<200 nm). In LC-MS, the use of the nonvolatile phosphate buffers is not reeommended. Although most of the modem ESI somee will no longer show elogging due to phosphate buffers or other nonvolatile additives, their nse may lead to signal suppression, the formation of addnet ions, and backgronnd noise. Volatile mobile-phase additives are preferred in LC-MS. 

The effectiveness of various protein precipitation additives in terms of protein removal and matric effects was investigated [86]. Acetorritrile, trichloroacetic acid (TCA), and zinc sulfate were formd most effective in removing proteirrs (applied in a 2 1 additive-to-plasma ratio). By a post-colunm infusion setup (Figme 11.6), these three methods were further evaluated with respect to matrix effect for five different mobile-phase compositions. As both buffered, acidified, and pure methanol-water mobile phases were compared, actual conclusions are difficult. In the pure methanol-water mobile phases, the use of TCA enhances the response, probably by generating acidic conditions more favourable in ESI-MS. With buffered or acidified mobile phases, the difference in matrix effects between acetonitrile or TCA as protein precipitation additive was less pronounced. A similar comparison between acetonitrile, perchloric acid, and TCA as protein precipitation additives was reported by others [100]. With acid precipitation, lower analyte recoveiy and higher %RSD was observed. Therefore, precipitation by acetonitrile was preferred. 

RPIP chromatography uses a hydrocarbonaceous stationary phase and either an aqueous or aqueous-organic mobile phase which also contains the counter-ion. The stationary phase is usually an octadecyl bonded phase and the mobile phase is usually an aqueous buffer with either methanol or acetonitrile as an organic modifier. The choice of counter-ions depends on the solutes to be separated, but generally for the separation of acids a hydrophobic organic base is added to the mobile phase, while for the separation of bases a hydrophobic organic acid is added. Separations of other compounds are similarly obtained by the addition of an appropriate counter-ion. 

Several variables should be considered in the development of an MLC procedure the nature of surfactmit and modifier, their concentrations, and pH. When a surfactant solution is used as mobile phase, the retention of solutes can be adequately controlled through the addition of a small amount of alcohol, and through variation of pH. The alcohol usually also improves the efficiency of the chromatographic peaks. Other variables that affect the retention and efficiency are temperature and ionic strength. However, most procedures are performed at room temperature, and the ionic strengfti is given by the combination of the surfactant and buffer in the mobile phase, and is not studied as a separate variable. 

In a similar study with ESI the influence of different buffers was studied [12]. In the presence of acetic acid (HAc) only in the MeOH/H20 mobile-phase, a mass spectrum resulted with ion adducts of Na and K appearing as the most abundant ones. However, minor peaks could also be observed in the mass spectrum resulting from ammonium adducts (Fig. 4.3.2(A)). The respective ions could be suppressed or enhanced by changing the nature of the buffer used in the mobile-phase. For example, when a potassium buffer was used, sodium and ammonium adducts were suppressed, and the spectrum became less complicated with primarily the potassium adduct ion being visible (Fig. 4.3.2(C)). In addition, the signal-to-noise ratio improved by about a factor of 1.5-2. Similarly, sodium or ammonium acetate buffers enhanced the sodium and ammonium adduct ions, meanwhile suppressing other adducts (Fig. 4.3.2(B) and (D), respectively). 

The addition of organic solvent thus suppresses the ionisation of the acid reducing the [H+] in solution and the overall effect is an increase in pH. The same effect can be observed for other buffers such as phosphate and citrate and with 50% organic solvent the effective pH of the mobile phase may be 1-1.5 units higher than the measured pH of the buffer before mixing. 

FAB ionization has been used in combination with LC/MS in a technique called continuous-flow FAB LC/MS (Schmitz et al., 1992 van Breemen et al., 1993). Although any standard HPLC solvent can be used, including methyl-ferf-butyl ether and methanol, the mobile phase should not contain nonvolatile additives such as phosphate or Tris buffers. Volatile buffers such as ammonium acetate are compatible at low concentrations (i.e., <10 mM). Continuous-flow FAB has also been used in combination with MS/MS (van Breemen et al., 1993). The main limitationsof continuous-flow FAB compared to other LC/MS techniques for carotenoids, such as ESI and APCI, are the low flow rates and the high maintenance requirements. During use, the 3-nitrobenzyl alcohol matrix polymerizes on the continuous-flow probe tip causing loss of sample signal. As a result, the continuous-flow probe must be removed and cleaned approximately every 3 hr. 

The first and most often encountered separation mechanism in CE is based on mobility differences of the analytes in an electric field these differences are dependent on the size and charge-to-mass ratio of the analyte ion. Analyte ions are separated into distinct zones when the mobility of one analyte differs sufficiently from the mobility of the next. This mechanism is exemplified by capillary zone electrophoresis (CZE) which is the simplest CE mode. A number of other recognized CE modes are variations of CZE. These are micellar electrokinetic capillary chromatography (MECC), capillary gel electrophoresis (CGE), capillary electrochromatography (CEC), and chiral CE. In MECC the separation is similar to CZE, but an additional mechanism is in effect that is based on differences in the partition coefficients of the solutes between the buffer and micelles present in the buffer. In CGE the additional mechanism is based on solute size, as the capillary is filled with a gel or a polymer network that inhibits the passage of larger molecules. In chiral CE the additional separation mechanism is based on chiral selectivity. Finally, in CEC the capillary is packed with a stationary phase that can retain solutes on basis of the same distribution equilibria found in chromatography. 

Other common practices include the use of well-degassed solvents, low concentrations of electrolytes, a relatively large amount of the organic component in the mobile phase, working at reduced temperatures (e.g., 15°C) when possible, and the use of low conductivity electrolytes (i.e., zwitterionic buffers). The addition of sodium dodecyl sulfate (SDS) into the mobile phase at low concentrations has also been used to minimize bubble formation [67]. 

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