Third, new column technologies using charged surface and other approaches provide a better solution to improve peak shape of basic analytes. These three solvents have distinctively different properties in terms of proton acceptor ability, proton donor ability, and dipole interactions (1,11). (28) H. Hahne, F. Pachl, B. Ruprecht, S.K. Tetrahydrofuran is rarely used in reversed-phase LC, despite its strong solubilizing power and eluotropic strength. Paradoxically, innovation in reversed-phase LC centers on new instruments and columns while scant research efforts are placed on the development of newer reagents for mobile phases. Dong, D. Guillarme, S. Fekete, R. Rangelova, J. Richards, D. Prudhomme, and N.P. Prior to the 1990s, the primary complaint against silica-based HPLC columns was the difficulty in method transfer for quality control applications caused by batch-to-batch differences of raw silica support or bonded phase chemistry. Reversed-phase LC uses a hydrophobic stationary phase and a polar mobile phase, and analytes are retained primarily by hydrophobic interaction. Modern trends in gradient methods include the use of simple binary mobile phases (such as 0.1% formic acid or 0.1% ammonia) with high-efficiency columns packed with superficially porous particles and low-silanophilic bonded phases (8). Dong, LCGC North Am. All rights reserved. The use of 0.1% phosphoric acid appears to be under-utilized because there is a traditional preference for phosphate buffer. 2, C. Horvth, Ed. Historically, the most common buffer used in HPLC is phosphate. Many analysts prepare buffers by dipping the pH electrode directly in mobile phase A and titrating the solution until the designated pH is reached. Adapted with permission from reference 20. Separation of proteins and peptides is a challenging application for HPLC requiring bonded phases with low silanophilic activity, specific mobile-phase additives to allow detection at low UV wavelengths (210220 nm), and higher column temperatures (>60 C) to improve peak shapes, and mass recovery (2, 3). Column: 150 mm x 2.1 mm HALO Peptide ES-C18; flow rate: 0.3 mL/min; temperature: 60 C, gradient: 247% acetonitrile in 40 min; detection: MS,(+)TIC 3001800 m/z, 4 kV, with a Shimadzu MS-2020 SQ. (15) M.W. Rev.16(4), 36-43 (2013). Here, we discuss those and other trends and best practices, as well as the fundamentals behind them. (19) M.W. Snyder and J.J. Kirkland, Introduction to Modern Liquid Chromatography (Wiley, Hoboken, New Jersey, 3rd Ed., 2010, Chapters 2, 6 and 7. In 2009, Barry returned to developing separations technologies at AMT, including the refined superficially porous particles pioneered by Jack Kirkland. Finally, the rapid emergence of HPLCMS as a standard technology in high-throughput screening, in-process control, bioscience research, and clinical diagnostics also favors the use of simpler HPLC methods and mobile phases (10). Basic analytes are ionized at low pH, though most basic drugs have sufficient hydrophobicity for adequate reversed-phase LC retention. Ion-pairing reagents are detergent-like molecules added to mobile phase A to provide retention of acidic or basic analytes (1). There is also a common practice to use the same levels of additives in both mobile phases A and B, such as 0.1% trifluoroacetic acid in acetonitrile when using 0.1% trifluoroacetic acid (TFA) in water as mobile phase A. Technically, there are no perceivable differences between 100% acetonitrile or 0.1% trifluoroacetic acid in acetonitrile for separation reproducibility or elution order in reversed-phase LC except for reducing gradient shifts with UV detection at low wavelengths (to be covered in a later section). Note the excellent peak shapes of all peaks under high-pH conditions without any peak splitting. Ion chromatography sometimes employs a mobile phase additive (such as 3 mM sodium phthalate) to perform ion chromatography by conventional HPLCUV equipment with indirect photometric detection of the displaced complex (21). Iraneta, and D. Morrison, Waters Corporation, Milford, Massachusetts, 720003720EN, Sep. 2010. The low pH suppresses the ionization of weakly acidic analytes, leading to higher retention (3). For instance, a mobile phase of 44% methanol:water was found to have equivalent elution strength of 35% acetonitrile:water or 28% tetrahydrofuran:water for a reference application (11). Other Mobile Phase Additives for Chiral Separations and Ion Chromatography. First, improved column technologies have reduced the need for mobile-phase additives or buffers to improve peak shapes or column batch-to-batch reproducibility (8). During the method development process, separation of coeluted peaks can be fine-tuned with infinite combinations of mobile phase factors, including solvent type, pH, additives, and operating conditions (such as temperature, flow rate, and gradient time). 31(6), 472-479 (2013). In pharmaceutical analysis, most drugs are ionizable, that is, acidic, basic, or zwitterionic. Kind, J. Anal. It also suffers from poor solubility in acetonitrile, particularly at high concentration (such as 50 mM), causing precipitation problems during pump blending. Adapted with permission from reference 3. An acidic pH of 24 is used for most pharmaceutical applications. In addition to supplying a strongly acidic modifier for reversed-phase LC separations, trifluoroacetic acid shows a favorable increment in retention of acidic peptides, because of the formation of an N-terminal amino ion pair, which is notably absent in formic acid mobile phases. Dong, G. Miller, and R. Paul, J. Chromatogr.987, 283290, (2003). Trifluoroacetic acid is an excellent ion pair reagent with primary -amino lysine group, as well as the N-terminal -amino group. The example shown is for a mixture of 5 synthetic peptides, resolved in 10 mM of each acid or acid plus salt (in the case of ammonium formate). It is possible to use a chiral additive as a pairing reagent to form a diastereomer, enabling chiral separations to be achieved on conventional reversed-phase columns (1). Buffers are required to tightly control the pH of mobile phase A for critical assays. Modern trends in LC mobile-phase selection and preparation include using simpler mobile phases, increased use of MS-compatible mobile phases, and eliminating filtration and certain mobile-phase additives. Dong,. Pharm. (16) J.J. Kirkland, M.A.van Straten, and H.A.Claessens, J. Chromatogr. A universal HPLC method using a sub-3-m charged surface hybrid (CSH) column in the analysis of 12 new chemical entities (NCEs). (3) M.W. Many publications have appeared in recent years using acetonitrile mixed with n- or i-propanol, or even n-butanol, which seems to be of benefit to improve mass recovery of some monoclonal antibodies (mAbs). Snyder and J.W. (b) Comparative chromatogram of the same mixture using identical mobile-phase conditions on a C18 column showing peak tailing for four of the more basic NCEs marked with arrows. Gradient: 2565% B in 15 min; baseline shift: (a) 0.1 AU, (b) 0.002 AU; detection: UV absorbance at 230 nm. The main cause of this non-reproducibility was the presence of active silanols, which are activated by metallic impurities (1,3). Before the 1990s, silica-based columns could not be used with high pH mobile phases, because of the dissolution of silica support at pH values >8. Column: 50 mm x 3.0 mm, 1.7-m Waters BEH C18. In any case, labeling of the preparation date is encouraged and required in many regulated laboratories. With the improvements of modern pumps and on-line mixers, this additional step appears to be less beneficial. This elimination of the filtration step can reduce potential mobile phase contaminations from the filtration process. Commonly used acids are trifluoroacetic acid, formic acid, and acetic acid at concentrations of 0.05 to 0.1% v/v. Figure 5: A case study illustrating the potential issue of a ghost peak caused by dipping the pH electrode into mobile-phase A during pH adjustment. Many laboratories have eliminated the filtration process with 0.2 or 0.5 m membrane filters by using high-purity reagents (such as 99.995% ammonium formate from Aldrich, Cat# 516961), and HPLC-grade solvents and water (22). Anecdotal evidence suggests that the advantages of difluoroacetic acid as a replacement are more apparent for ESI signal intensities of peptides and smaller proteins compared to larger proteins up to the size of immunoglobins. Buffers of ammonium salts of volatile acids are used for the development of MS-compatible HPLC methods. Figure 2 further illustrates the effect of mobile-phase pH to dramatically alter the retention and elution orders of acidic and basic drugs (17). LCGC North Am.31(8), 612621 (2013). Higher concentrations can be used effectively to increase retention in reversed-phase LC by the "salting out" effect or the Le Chatelier's Principle to drive solutes into the hydrophobic stationary phase (1,11). Acetonitrile is an aprotic solvent and is a proton acceptor with capability for interaction (3). Recent research by Boyes and associates has uncovered two promising additives that deliver a good compromise of performance for peak shape and MS sensitivity: difluoroacetic acid and 3-fluoropropionic acid (26). Barry has published more than 85 peer-reviewed papers, reviews, patents, and applications. Again, the magnitude of such baseline disturbances is a complex function of the on-line mixer design, volume, and apparent absorbance differences between pure mobile phase A and mobile phase B. Figure 8: A comparison of a separation of an intact monoclonal antibody (Sigma/Millipore SILu Lite SigmaMAb) using difluoroacetic acid compared to trifluoroacetic acid and formic acid as acidic mobile phase modifiers in mobile phases A and B. The highlighted trends focus on the stability-indicating analysis of pharmaceuticals, including both small molecule drugs and biotherapeutics, increased UV and mass spectrometer (MS) detection sensitivity, and the attainment of symmetrical peak shapes for basic analytes. The disadvantages are the instability of some drugs in basic mobile phases and potential method robustness issues if the mobile phase pH is close to the pKa values of the basic analytes (for example, most amines have pKa values in the range of 8-10) (3,15). Historically, the three most common reversed-phase LC organic solvent choices are acetonitrile, methanol, and tetrahydrofuran. One often sees HPLC methods with a stipulated mobile phase B such as 95% acetonitrile in water. For this mAb, mobile phases using formic acid exhibit poor retention and peak shape, compared to either trifluoroacetic acid or difluoroacetic acid mobile phases. Reduced Usage of Silanol-Masking, Ion-Pairing and Chaotropic Reagents. Another approach that has emerged is the use of additives to mobile phase A to increase, or rescue, ionization efficiency lost to trifluoroacetic acid ionization suppression.