Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 8F) docx

8.6 EFFECT OF pH ON UV ABSORBANCE The extent to which an organic molecule absorbs electromagnetic radiation in the ultraviolet region (UV light) depends on the structure of the molecule. Generally, molecules that contain one single double bond absorb weakly in the UV region. However, if multiple double bonds are present in a molecule and they are conjugated, the molecule absorbs more strongly and the absorbance is shifted to longer wavelengths. The pH of the mobile-phase effects the ionization of ionogenic solutes and also the analyte UV response. The change in pH can change the electronic structure of the molecule and result in changes in the molar absorptivity and the absorption maximum of the molecule [25]. Ionization of aromatic com- pounds containing a pyridinal nitrogen, amino, carboxylic acid, and phenolic group can lead to significant changes of their UV response. Understanding the effects of charge delocalization and conjugation on the UV response and detection wavelength will allow the chromatographer to choose the proper pH and wavelength of detection to obtain a method with higher sensitivity. Sil- verstein et al. [26] and Shenk [27] provide a good overview for predicting how the structure of a molecule and its environment will affect its molar absorp- tivity and the wavelength of the absorption maximum. Most applications of absorption spectroscopy to organic compounds are based upon transitions for n or π electrons to the π* excited state. Energies required for these processes bring absorption peaks into the spectral region (200–700nm). π-electrons are further delocalized by conjugation. The effect of this delocalization is to lower the energy level of the π* orbital and give it less antibonding character and as a result absorption maxima are shifted to longer wavelengths [28]. UV spectra of aromatic hydrocarbons are characterized by three sets of bands (E1, E2, and B bands) that originate from π→π* transitions. Gener- ally the E2 and B bands are of most interest to chromatographers, since the solvent cutoff for most mobile phases is <200nm. For example, benzene has strong absorption peaks at E1: 184nm, ε max ~ 60,000 E2: 204nm, ε max = 7,900 B: 256nm, ε max = 200 Table 8-9 shows E2 and B bands for some organic molecules. Auxochromes are a functional group that does not itself absorb in the UV region but have the effect of shifting chromophore peaks to longer wavelengths and increas- ing their intensity. The —OH and —NH 2 groups have an auxochromic effect on benzene chromophore. These substituents have at least one pair of n elec- trons capable of interacting with π electrons of the ring. This stabilizes the π* state and lowers its energy. The phenolate anion auxochromic effect is more pronounced than for phenol because the anion has an additional pair of EFFECT OF pH ON UV ABSORBANCE 429 unshared electrons. Aniline has a pair of n electrons capable of interacting with the π electrons of ring. This stabilizes the π* state by the relationship shown in Equation (8-3), thereby lowering its energy [28]. With a decrease in protonation, the absorption maxima would be shifted to longer wavelengths and increasing intensities and a red shift occurs. However, upon protonation the nonbonding electrons are lost by formation of the anilinium cation, and the auxochromic effect disappears as a consequence. (8-3) The change in the mobile-phase s s pH at a constant organic composition may have an effect on an ionizable analyte’s UV response (Figure 8-55A). Also, at constant w w pH as the organic concentration is increased, this may also lead to a change in the analytes absorbance at a particular wavelength. Increasing concentration of the organic shifts the pH of the mobile phase upward (for an acidic modifier), and changes in UV absorbance may be observed (Figure 8-55B). At 232nm there is a decrease in aniline’s absorbance as this analyte becomes progressively more ionized. A plot of the UV absorbance at a par- ticular wavelength versus the w w pH of the aqueous phase will lead to a sig- moidal dependence (Figure 8-56). The inflection point corresponds to the analyte pK a (not corrected for pH shift of the mobile phase). When perform- ing method development experiments a judicious choice for the wavelength of the detection should be carefully considered because this can lead to desired/undesired effects (change in sensitivity at particular wavelength as a function of pH) on the resulting chromatography. Figure 8-57 demonstrates that a greater response for aniline is observed at w w pHs where the analyte is in Ehv hc == l 430 METHOD DEVELOPMENT TABLE 8-9. Molar Absorptivity Values for Neutral, Acid, and Basic Species a Molecular E 2 Band B Band Compound Formula λ max (nm) ε max λ max (nm) ε max Benzene C 6 H 6 204 7,900 256 200 Naphthalene C 10 H 8 286 9,300 312 289 Toluene C 6 H 5 CH 3 207 7,000 261 300 Chlorobenzene C 6 H 5 Cl 210 7,600 265 240 Phenol C 6 H 5 OH 211 6,200 270 1,450 Phenolate ion C 6 H 5 O − 235 9,400 287 2,600 Thiophenol C 6 H 5 SH 236 10,000 269 700 Aniline C 6 H 5 NH 2 230 8,600 280 1,430 Anilinium ion C 6 H 5 NH 3 + 203 7,500 254 160 a Values from reference 28. EFFECT OF pH ON UV ABSORBANCE 431 Figure 8-55. Effect of pH on UV absorbance for aniline (obtained from diode array). (A) 10 v/v% acetonitrile and pH of 15 mM K 2 HPO 4 ·7H 2 O adjusted to w w pH 1–9 with H 3 PO 4 . (B) w w pH2.0 and acetonitrile concentration changed from 10 to 50 v/v%. Figure 8-56. Absorbance at 232 nm versus the w w pH of the aqueous phase. Mobile phase contains 10 v/v% acetonitrile. its neutral state.As the analyte ionization state varies with pH so does the con- jugation. In some cases, the wavelength in a specific region does not vary with the pH and the sensitivity of the analysis will not change as a function of pH as seen for 2,4-dihydroxybenzoic acid, at 280 nm in Figure 8-58. 8.7 ANALYTE pK a —FROM AN ANALYTICAL CHEMIST’S PERSPECTIVE In order to avoid any secondary equilibrium effects on the retention of iono- genic analytes, it is preferable to use a mobile-phase pH either two units greater or less than the analyte pK a . Therefore knowledge of the analyte pK a is very important. A basic understanding of how functional group substitution on a molecule affects the pK a of the ionizable group on the substrate is given. An exhaustive description of all the nuances of analyte substitution on analyte pK a is not included in this section. However, further details can be found in the references 29–31. 8.7.1 Aromatic Acids Effect of Analyte Substitution on Analyte pK a . The acidity of substituted phenols or carboxylic acids depend upon the substituent attached to the cor- 432 METHOD DEVELOPMENT Figure 8-57. Effect of pH on UV absorbance for aniline. Conditions: Column: 15-cm × 0.46-cm Luna C18(2). Eluent: 90% aqueous:10% MeCN. Aqueous: 15 mM K 2 HPO 4 ·7H 2 O adjusted to w w pH 1.5–9 with H 3 PO 4 , Flow rate, 1 mL/min; temperature, 25°C; detection, PDA. responding substrate, phenol, or carboxylic acid Aromatic acids with an electron-withdrawing substituent are more acidic because these substituents stabilize the ion by delocalizing the negative charge. Aromatic acids with elec- tron-donating groups are less acidic because the substituents destabilize the ion by localizing the charge. 8.7.1.1 Electron-Withdrawing Effects—Aromatic Acids. Electron-withdrawing groups in the nucleus of the substrate increases the acidity. Inductive effect usually falls off with distance: ortho (o) > meta (m) > para (p). However, electron-withdrawing mesomeric effects also play a role when the electron- withdrawing substituent is in the o- or p-position (see Figure 8-59). This promotes ionization by stabilization (through delocalization) of resultant anion. 8.7.1.2 Electron-Donating Groups—Aromatic Acids. The effect of elec- tron-donating groups such as alkyl groups attached to the benzene nucleus are small. These substituents destablize the phenoxide anion and disturb the interaction of the negative charge with delocalized p orbitals of the aromatic nucleus, shown in the following table. ANALYTE pK a —FROM AN ANALYTICAL CHEMIST’S PERSPECTIVE 433 Figure 8-58. Effect of pH on UV absorbance for 2,4-dihydroxybenzoic acid. Condi- tions: Column: 15-cm × 0.46-cm Luna C18(2). Eluent: 90% aqueous:10% MeCN. Aqueous: 15 mM K 2 HPO 4 ·7H 2 O adjusted to w w pH 1–7 with H 3 PO 4 . Flow rate, 1 mL/min; temperature, 25°C; detection, PDA. 8.7.2 Amines 8.7.2.1 Arylamines. Arylamines like aliphatic amines are basic. A lone pair of nonbonding electrons on nitrogen can bond to acids, yielding an arylam- monium salt. Base strength of arylamines are lower than aliphatic amines. A stronger base corresponds to a less acidic ammonium ion (higher pK a ). A weaker base corresponds to a more acidic ammonium ion (lower pK a ). 8.7.2.2 Aromatic Amines—Electron-Donating Groups. Electron-donating groups tend to disperse the positive charge of the anilinium ion, and this stabilizes the ion relative to the amine. Electron-donating groups increase the basicity. Electrons are being pushed toward nitrogen and makes the fourth pair more available for sharing with acid. These activating substituents make the aromatic ring electron-rich. Some examples are shown in Figure 8-60A. Electron donors (–CH 3 , –NH 2 , –OCH 3 ) increase the basicity of arylamines. 8.7.2.3 Aromatic Amines—Electron-Withdrawing Groups. Electron-withdrawing groups tend to intensify the positive charge of the anilinium ion, and this desta- bilizes the ion relative to the amine. This increase the reactivity of an aromatic 434 METHOD DEVELOPMENT Figure 8-59. pK a values of aromatic acid (phenols) species with electron-withdrawing groups. Figure 8-60A. pK a values of aromatic amine species with electron-donating groups. Compound pK a phenol 9.95 o-MePhenol 10.28 m-MePhenol 10.08 p-MePhenol 10.19 REVERSED-PHASE VERSUS NORMAL-PHASE SEPARATIONS 435 Figure 8-60B. pK a values of aromatic amine species with electron-withdrawing groups. Figure 8-61. pK a values of secondary amine species of two pharmaceutical compounds. ring toward electrophilic substitution. Electron-withdrawing groups decrease the basicity and pull electrons away from nitrogen and make the fourth pair less available for sharing with acid. These deactivating substituents make the aromatic ring electron-poor. Some examples are shown in Figure 8-60B. Electron-withdrawing groups (–C1, –NO 2 , –CN) decrease arylamine basicity. 8.7.2.4 Alkyl Amines and Amides. Nonaromatic secondary and tertiary amines have pK a values greater than 8, as shown for two beta blockers in Figure 8-61. Amides are nonbasic, poor nucleophiles and do not protonate in aqueous acids. As with carboxylic acids, the resonance stabilization of the neg- ative charge of the CH 3 CONH– rationalizes the higher acidity of the amide. pK a values of amides are typically greater than 15. 8.8 REVERSED-PHASE VERSUS NORMAL-PHASE SEPARATIONS Reversed-phase HPLC is the dominant method used for the majority of pharmaceutical applications (>95%). Normal-phase chromatography may be required for separations that are not compatible with reversed-phase mode. Solutes that are labile (i.e., reacts with protic solvents) or exhibit poor solu- bility in aqueous media are prime candidates for normal-phase chro- matography. Normal phase is well-suited for the separation of isomers and diastereomers, as well as for separating compounds with saturated and unsaturated side chains. Generally, the greater is the amount of unsat- uration the greater the retention due to increased polarizability of double bond. Diol phases are a good starting point for normal-phase application. Silica, amino, and cyano are alternative phases. Silica tends to strongly retain solutes that can interact with its highly active sites. Hexane or heptane modified with a polar organic solvent is generally utilized as the mobile phase. The polar organic solvent can be chosen based on it physicochemical properties (dipole, hydrogen bond acceptor/donor). Generally, small changes of the polar organic solvent can cause large changes in retention, and this should be investigated during method development. Common solvents include ethanol, isopropanol, tetrahydrofuran, ethyl acetate, and dichloromethane. The level of water in the solvents needs to be controlled as well, since differences in retention may be observed. Additives such as trifluoroacetic acid or triethylamine can be used to reduce interactions with the highly active sites of silica, allowing for reduced retention and improved peak shape. A further description of normal-phase chromatography can be found in Chapter 5. Normal-Phase Chromatography Example. Vitamin E, an antioxidant, is a complex made up of tocopherols and tocotrienols (Figure 8-62), which are sometimes used to stabilize formulations. Tocopherols are a series of related benzopyranols with a C16 saturated side chain. Tocotrienols contain three double bonds on the C16 side chain [32]. Could you predict the elution order of the alpha, beta, gamma, and delta isomers in the normal-phase mode? Note that in the normal phase, the less hydrophobic the compound and the more substituents that could potentially hydrogen bond to the stationary phase, the greater the affinity for the sta- tionary phase and the longer the retention. The order of elution for the alpha, beta, gamma, and delta isomers for both the tocopherols and the tocotrienols series is the same (Figure 8-63) [32]. The order of elution for beta and gamma would be hard to predict because they have very similar hydrophobicity and same number of potential hydrogen bonding moieties. Their differences in elution order depend on the planarity of the molecule and its interaction with the stationary phase. In normal-phase chromatography, the more unsaturated molecules, tocotrienols, elute later compared to the tocopherols, which have a saturated side chain, and this could be attributed to the increased polarizabil- ity of the double bond [33]. Comparing the separation to reversed mode, the elution order is reversed, where the retention is as follows: delta tocopherol < gamma tocopherol < beta tocopherol < alpha tocopherol < alpha tocopheryl acetate (Figure 8-64). 436 METHOD DEVELOPMENT REVERSED-PHASE VERSUS NORMAL-PHASE SEPARATIONS 437 Figure 8-62. Structures of substituted tocopherol and tocotrienols. Figure 8-63. HPLC conditions: Genesis silica column (250 × 4.6 mm, 4 µm). Flow rate, 1.5 mL/min. Mobile phase: Hexane-1,4-dioxane (96 : 4). Fluorimetric detection: Fluor LC 304 (excitation @ 294 nm and emission @ 326 nm). (Reprinted from reference 32, with permission.) 8.9 INSTRUMENT/SYSTEM CONSIDERATIONS The four common causes for high-performance liquid chromatography (HPLC) column failure include column clogging at the inlet frit (from samples/mobile phase), voids generated in the column, strongly adsorbed impurities from solvent/sample, and chemical attack of the stationary phase from the mobile phase or analytes. Procedure for removal of strongly adsorbed impurities from sample/mobile phase was discussed in Chapter 3, Section 3.9.2. 8.9.1 Column/System Backpressure Column backpressure gives a good indication of how the column and/or system are operating. The initial backpressure of the column should be 438 METHOD DEVELOPMENT Figure 8-64. Separation of synthetic tocopherols by reversed-phase HPLC (280 nm). (1) δ-tocopherol, (2) γ-tocopherol, (3) β-tocopherol, (4) α-tocopherol, (5) α-tocopheryl acetate. (Reprinted from reference 33, with permission.) [...]... of salt crystals that will damage the seal over time Therefore it is recommended to wash the HPLC system with acetonitrile/water (20 : 80) for at least 30 min before the system is shut down to remove any potential buffer residues A shut-down method with this wash method is recommended at the end of the sequence Pump piston seals usually last for at least 6 months (if system is continually used throughout... coupled with reverse phase HPLC, for monitoring the formation of an enolate intermediate, J Liq Chrom Relat Technol 25 (2002), 1049–1062 14 A N Heyman and R Henry, Importance of Controlling Mobile Phase pH, Keystone Technical bulletin, 99-06 (page 2, Figure 2), http://www.hplcsupply.com/ pdf/App_9.pdf#search=‘Biobasic%20and%209906%20and%20henry’ 15 M L Mayer, Selecting filters for chromatographic applications,... at system/column performance and method transfer considerations for pharmaceutical analytes, HPLC 2005 Conference, Stockholm, Sweden, 2005 41 NIOSH Manual of Analytical Methods, 4th ed., Issue 2, Method 7601: Silica, crystalline, August 15, 1994, pp 1–5 42 H A Claessens, M A van Straten, and J J Kirkland, Effect of buffers on silica based column stability in reversed phase high performance liquid chromatography,... often used and recommended for analysis of proteins, lipids, and other highmolecular-weight species, if the sample is biological in origin (urine, plasma, etc.), to prevent contamination of the column with matrix components The need for replacing the guard column depends on the matrix, the number of injections, and whether decreased performance is observed (change in efficiency for isocratic separations... modifier, especially if the probe analytes are ionizable For a given set of probes the selectivity may be high on a specific column; however, for another set of analytes (“your pharmaceutical compounds”) the selectivity may be poor There is no universal selectivity test that can ensure that a particular column will give the desired selectivity for a set of compounds However, if enough knowledge is gained... retention may be observed for all components (neutral and basic) However, if loss of bonded phase and end-capping reagent and/or change to stationary-phase surface occurs and greater exposure of the residual silanols is prevalent, then the increase in retention and peak tailing for protonated basic components may be observed while the retention for neutral compounds may decrease Columns for a particular laboratory... peak tailing for protonated basic components may be observed (secondary interactions with residual silanols) while the retention for neutral compounds may decrease (due to increased hydrophilicity of the surface) 8.11 CONCLUDING REMARKS A well-defined method development plan with clear aim of analysis is critical to the success for fast and effective method development The general approach for the method... amine, J Pharm Sci 87 (1998), 31–39 5 ICH, Guidance for the Industry: Q3B(R): Impurities in new drug products, November 2003 6 J D Higgins II and W L Rocco, Pharma preformulation, Today’s Chemist (2003), 22–26 7 R LoBrutto and Y V Kazakevich, Retention of ionizible components in reversedphase HPLC, in S Kromidas (ed.), Practical Problem Solving in HPLC, WileyVCH, New York, 2000, pp 122–158 8 E Loeser... readily available, and an analyst should know how to replace them to avoid waiting for a metrologist or vendor engineer or contract engineer to change them Also, a proper maintenance log for each HPLC must be maintained in a regulated environment, and any type of maintenance should be properly recorded according to the pharmaceutical departments standard operating procedures 8.9.2 Column Inlet and Outlet... (10) (1996), 902–905 16 M L Mayer, Filtration: Preventive maintenance for HPLC, Am Lab 29 (1997), 34–37 REFERENCES 453 17 J A Dean, Analytical Chemistry Handbook, McGraw-Hill, New York, 1995 18 J C Merrill, Avoiding problems in HPLC through filtration, Am Lab 19 (1987), 74–81 19 L R Synder, J J Kirkland, and J L Glach, Practical HPLC Method Development, 2nd ed., John Wiley & Sons, New York, 1997 20 . Reversed-phase HPLC is the dominant method used for the majority of pharmaceutical applications (>95%). Normal-phase chromatography may be required for separations. the seal over time. Therefore it is recommended to wash the HPLC system with acetonitrile/water (20:80) for at least 30min before the system is shut down