876 TROUBLESHOOTING Table 17.11 (Continued) Failed Test Symptom Symptom/Possible Source Solution Gradient distortion (Fig. 17.28b) Gradient mixing volume too large relative to gradient volume Use shallower gradient; reduce mixing volume Gradient step-test failure Steps in gradient step-test are of uneven height and/or are distorted Bubble, check-valve failure, or leak; Degas mobile phase, clean or replace check valve, fix leak; blocked reservoir frit; bad proportioning valve Gradient proportioning valve (GPV) test failure GPV test steps > 5% Blocked reservoir frit; restricted solvent supply tubing; bad proportioning valve Replace frit; clear or replace tubing; replace proportioning manifold Dwell-volume differences between systems Changes in retention and/or selectivity between systems Normal for differences in dwell-volume Adjust method to compensate for dwell-volume differences Flow-rate check failure Flow rate > ±2% from set value Bubble; bad check valve or pump seal; leak; wrong compressibility set Degas mobile phase; clean or replace check valve, replace pump seal; adjust compressibility (or ignore) Pressure bleed-down failure > 15% pressure loss in 10 min Check valve or pump-seal failure; leak Clean or replace check valve, replace pump seal; fix leak Retention time reproducibility failure > ±0.05 min for standard test; more than normal method performance Bubbles, leaks, check-valve or pump-seal failure; pump or mixing failure Degas solvents, fix leaks, clean or replace check valve, replace pump seal; run gradient performance test to isolate further Peak area reproducibility failure > 1% imprecision in standard test; more than normal method performance Autosampler problem Clean or replace needle; replace seals; see autosampler manual REFERENCES 1. J. W. Dolan and L. R. Snyder, Troubleshooting LC Systems, Humana Press/Springer, Clifton, NJ, 1989. 2. V. R. Meyer, Pitfalls and Errors of HPLC in Pictures, Wiley-VCH, Weinheim, 2006. 3. J. W. Dolan, ‘‘LC Troubleshooting,’’ in LCGC, a monthly column (1983-present). 4. http://www.chromforum.org. REFERENCES 877 5. http://www.lcresources.com/resources/TSWiz/. 6. S. J. Williams, J. Chromatogr. A, 1052 (2004) 1. 7. M. D. Nelson and J. W. Dolan, LCGC, 16 (1998) 992. 8. C. K. Cheung and R. Swaminathan, Clin. Chem., 33 (1987) 202. 9. C. T. Mant and R. S. Hodges, eds., High-Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis, and Conformation, CRC Press, Boca Raton, 1991. 10. J. W. Dolan, J. R. Kern, and T. Culley, LCGC, 14 (1996) 202. 11. D. W. Bristol, J. Chromatogr., 188 (1980) 193. 12. P L. Zhu, L. R. Snyder, and J. W. Dolan, J. Chromatogr. A, 718 (1995) 429. 13. J. W. Dolan, LCGC, 11 (1993) 640. 14. J. W. Dolan, LCGC, 9 (1991) 22. 15. M. L. Ledtje and D. Long, Jr., US Patent 5,002,662 (March 26, 1991). 16. J. W. Dolan, LCGC, 26 (2008) 532. 17. J. W. Dolan, personal communication. 18. L. R. Snyder and J. W. Dolan, High-Performance Gradient Elution, Wiley, Hoboken, NJ, 2007. 19. SPD-10Avp UV-Vis Detector Instruction Manual, Shimadzu, Kyoto, 1997. 20. N. S. Wilson, R. Morrison, and J. W. Dolan, LCGC, 19 (2001) 590. 21. J. W. Dolan, LCGC, 23 (2005) 370. 22. Reviewer Guidance: Validation of Chromatographic Methods, USFDA-CDER, Nov. 1994, http://www.fda.gov/cder/guidance/index.htm. 23. D. V. McCalley, Anal. Chem., 78 (2006) 2532. 24. M. T. W. Hearn, ed., Ion-pair Chromatography. Theory and Biological and Pharma- ceutical Applications, Dekker, New York, 1985. 25. E. Rajakyl ¨ a, J. Chromatogr., 218 (1981) 695. 26. R. D. Morrison and J. W. Dolan, LCGC, 23 (2005) 566. 27. J. W. Dolan, LCGC, 25 (2008) 610. 28. R. G. Wolcott, J. W. Dolan, L. R. Snyder, S. R. Bakalyar, M. A. Arnold, and J. A. Nichols, J. Chromatogr. A, 869 (2000) 211. 29. T L. Ng and S. Ng, J. Chromatogr., 389 (1985) 13. 30. W. R. Melander, H J. Lin, and C. Horv ¨ ath, J. Phys. Chem., 88 (1984) 4527. 31. M. R. Euerby, C. M. Johnson, I. D. Rushin, and D. A. S. Sakunthala Tennekoon, J. Chromatogr. A, 705 (1995) 229. 32. M. R. Euerby, C. M. Johnson, I. D. Rushin, and D. A. S. Sakunthala Tennekoon, J. Chromatogr. A, 705 (1995) 219. 33. J. J. Gilroy and J. W. Dolan, LCGC, 22 (2004) 982. 34. T. Culley and J. W. Dolan, LCGC, 13 (1995) 940. 35. J. W. Dolan, LCGC, 13 (1995) 940. 36. U. D. Neue, personal communication (1995). 37. T. Eidenberger, personal communication (1995). 38. J. W. Dolan and L. R. Snyder, Troubleshooting LC Systems, Humana Press, Totowa, NJ, 1989. 39. G. Hendriks, J. P. Franke, and D. R. A. Uges, J. Chromatogr. A, 1089 (2005) 193. APPENDIX I PROPERTIES OF HPLC SOLVENTS S olvents are used in HPLC for formulating mobile phases, for dissolving the sample, and for carrying out sample preparation. Mobile-phase solvents are of primary concern, because their properties must often fall within narrow limits for acceptable performance. However, these same properties also influence the choice of the sample-injection solvent and solvents used for sample preparation. Table I.1 lists several solvent properties that can be important when selecting solvents for an HPLC application. Some of these properties have been discussed previously in one or more sections of this book (second column of Table I.1). The present appendix contains several tables that list values of one or more solvent properties (third column of Table I.1). A brief comment on each solvent property is given in the last column of Table I.1; this serves as an introduction to following sections that deal with individual solvent properties. I.1 SOLVENT-DETECTOR COMPATIBILITY I.1.1 UV Detection The mobile phase will preferably have an absorbance A < 0.2 AU at the wavelength used for detection of the sample; a lower absorbance may mean improved assay precision and better results with gradient elution, but higher absorbances may be acceptable for some isocratic separations. Table 1.2 summarizes values of solvent absorbance at different wavelengths (200–260 nm) for solvents that are used for RPC (exclusive of NARP). Very rarely, there may be a reason to use UV detection at a wavelength <200 nm, for the detection of solutes with low absorptivity at higher wavelengths. Because water does not absorb at 200 nm or above, the absorbance of aqueous mobile phases that contain these solvents will equal the pure-solvent absorbance times the volume-fraction φ of the B-solvent in the mobile phase. For example, a mobile phase of 25% B would have the following absorbance values A for different B-solvents at 215 nm: ACN, 0.00 AU; MeOH, 0.09 AU; degassed MeOH, 0.05 AU; THF, 0.22 AU; IPA, 0.07 AU. Note that degassing methanol lowers the Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan Copyright © 2010 John Wiley & Sons, Inc. 879 880 PROPERTIES OF HPLC SOLVENTS Table I.1 Table I. 1 Solvent Properties of Interest in HPLC Property Section Reference Table of Values Comment UV cutoff 4.4 I.2 For UV detection; useful solvents depend on wavelength required for sample detection Refractive index 4.11 1.3 For RI detection; low values generally preferred Polarity 2.3.2.1, 6.2.1, 8.2.1 I.4 Determines solvent strength for 1 ≤ k ≤ 10 Selectivity 6.3, 8.3.2 I.4 Determines differences in solvent-type selectivity Sample solubility 15.3.2.3 Can be important for injection of large samples in prep-LC or trace analysis Viscosity 2.4.1 I.3, I.5 Determines column pressure drop; low values of viscosity desirable Boiling point I.3 Affects pump performance and safety; higher boiling solvents preferred Miscibility Important for choice of RPC organic solvent and sample solvent Density I.6 Required for the (more accurate) formulation of mobile phases by weight Stability Not usually an issue Safety I.7 Generally important, but not usually critical concentration of oxygen in the pure solvent, with a resulting decrease in solvent absorbance by about 1/3 over the range 200 to 240 nm. When the mobile phase is degassed, as by helium sparging, the absorbance of other B-solvents will also be lowered, in proportion to the amount of oxygen that is normally present in the solvent. Oxygen is more soluble in less polar solvents such as THF and IPA; the absorbance values of Table 1.2 for these solvents may therefore be higher than found in practice. While water should not absorb light at wavelengths ≥200 nm, this assumes that the water has been properly purified. Specifications for HPLC-grade water are described in ASTM D1193, but water for use with low-wavelength detection may require total organic carbon (TOC) levels below 50 ppb, as well as high resistivity values. Table 1.2 also lists UV absorbance data for some commonly used buffers, and Table 1.3 provides UV cutoff wavelengths for several additional solvents. I.1 SOLVENT-DETECTOR COMPATIBILITY 881 Table I.2 UV Absorbance as a Function of Wavelength of Various Solvents and Buffers Used for RPC Absorbance at Indicated Wavelength (nm) 200 205 210 215 220 230 240 250 260 Solvents Acetonitrile 0.06 0.02 0.02 0.01 0.00 0.00 0.00 0.00 0.00 Methanol 1.0+ 1.0 0.53 0.35 0.23 0.10 0.04 0.02 0.01 Methanol (degassed) 1.0+ 0.76 0.35 0.21 0.15 0.06 0.02 0.00 0.00 Tetrahydrofuran 1.0+ 1.0+ 1.0+ 0.85 0.70 0.49 0.30 0.17 0.09 Isopropanol 1.0+ 0.98 0.46 0.29 0.21 0.11 0.05 0.03 0.02 Buffers Acetate Acetic acid, 1% 1.0+ 1.0+ 1.0+ 1.0+ 1.0+ 0.87 0.14 0.01 0.00 Ammonium salt 10 nM 1.0+ 0.94 0.53 0.29 0.15 0.02 0.00 0.00 0.00 Carbonate (NH 4 )HCO 3 , 10 mM 0.41 0.10 0.01 0.00 0.00 0.00 0.00 0.00 0.00 Formate Sodium salt, 10 mM 1.00 0.73 0.53 0.33 0.20 0.03 0.01 0.01 0.01 Phosphate H 3 PO 4 , 1% 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 KH 2 PO 4 , 10 mM 0.03 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 K 2 HPO 4 , 10 mM 0.53 0.16 0.05 0.01 0.00 0.00 0.00 0.00 0.00 (NH 4 ) 2 HPO 4 , 10 mM 0.37 0.13 0.03 0.00 0.00 0.00 0.00 0.00 0.00 sodium salt, pH-6.8, 10 mM 0.20 0.08 0.02 0.01 0.00 0.00 0.00 0.00 0.00 Trifluoroacetic acid 0.1% in water 1.0+ 0.78 0.54 0.34 0.20 0.06 0.02 0.00 0.00 0.1% in ACN 0.29 0.33 0.37 0.38 0.37 0.25 0.12 0.04 0.01 Source: Data of [1, 2]. It is preferable that the detector response of the mobile phase remains constant during gradient elution. This requires that the A-solvent (water) and the B-solvent each respond similarly. When UV detection is used at low wavelengths, this may not be the case, especially for B-solvents other than acetonitrile. A related problem is a variation in the absorbance of the buffer or other mobile-phase additives as %B changes. Each of these effects is discussed in Section 17.4.5.1. I.1.2 RI Detection For isocratic separation, the choice of mobile phase is usually not limited for RI detection. Detection sensitivity can be increased by selecting a mobile phase whose RI-value is more different than that of sample components (Table 1.3). Detectors based on differential measurement (e.g., RI) cannot be used for gradient elution because of the usual large difference in response for the A- and B-solvents. 882 PROPERTIES OF HPLC SOLVENTS Table I.3 Miscellaneous Solvent Properties Solvent UV Cutoff RI b [3] Viscosity Boiling Point ε (nm) a [2] (cP) [3] ( ◦ C) c [3] (silica) d Acetone 330 1.359 0.36 56 0.53 Acetonitrile 190 1.344 0.38 82 0.52 1-Butanol 215 1.399 2.98 118 0.40 1-Chlorobutane 220 1.402 0.45 78 0.20 Chloroform 245 1.446 0.57 61 0.26 Cycohexane 200 1.424 1.00 81 0.00 Dimethyl formamide 268 1.430 0.92 153 — Dimethylsulfoxide 268 1.478 2.24 189 0.50 1,4-Dioxane 215 1.422 1.37 101 0.51 Ethyl acetate 256 1.372 0.45 77 0.48 Heptane 200 1.388 0.40 98 0.00 Hexane 195 1.375 0.31 69 0.00 Isooctane 215 1.391 0.50 99 0.00 Methanol 205 1.328 0.55 65 0.70 Methyl-t-butyl ether 210 1.369 0.27 55 0.48 Methylethyl ketone 329 1.379 0.43 80 0.40 Methylene chloride 233 1.424 0.44 40 0.30 i-Propanol 205 1.377 2.40 82 0.60 n-Propanol 210 1.386 2.30 97 0.60 Tetrahydrofuran 212 1.407 0.55 66 0.53 Toluene 284 1.497 0.59 111 0.22 Water 190 1.333 1.00 100 a Wavelength at which solvent absorbs 1.0 AU in a 10-mm cell. b Refractive index. c Boiling point. d Solvent strength parameter [4]. I.1.3 MS Detection The MS interface evaporates the mobile phase, so mobile phases comprising water, organic solvent, and volatile additives are used (i.e., no nonvolatile buffers or salts). Because the mobile phase is removed, UV absorbance is of no concern for LC-MS, but the solvents must be free of particulates. I.2 SOLVENT POLARITY AND SELECTIVITY Table 1.4 lists solvent properties that affect solvent strength, selectivity, and solu- bility. These ‘‘normalized selectivity’’ properties recognize three contributions of the solvent to solute–solvent interaction: solvent hydrogen-bond (H-B) acidity α H 2 /Σ H-B basicity β 2 /Σ, and dipolarity π ∗ /Σ. The latter parameters are the basis of I.2 SOLVENT POLARITY AND SELECTIVITY 883 Table I.4 Solvent Selectivity Characteristics Solvent Normalized Selectivity a H-B Acidity H-B Basicity Dipolarity α H 2 / β 2 / π ∗ / P b ε c Acetic acid 0.54 0.15 0.31 6.0 6.2 Acetone 0.06 0.38 0.56 5.1 20.7 Acetonitrile 0.15 0.25 0.60 5.8 37.5 Benzene 0.14 0.86 0.00 2.7 2.3 Chloroform 0.43 0.00 0.57 4.1 4.8 Diethyl ether 0.00 0.64 0.36 2.8 4.3 Dimethylsulfoxide 0.00 0.43 0.57 7.2 4.7 Ethanol 0.39 0.36 0.25 4.3 24.6 Ethylacetate 0.00 0.45 0.55 4.4 6.0 Ethylene chloride 0.00 0.00 1.00 3.5 10.4 Formamide 0.33 0.21 0.46 9.6 182 Glycol 0.38 0.23 0.39 6.9 37.7 Hexane 0.00 0.00 0.00 0.1 1.9 Isopropanol 0.22 0.35 0.43 3.9 19.9 Methanol 0.43 0.29 0.28 5.1 32.7 Methylacetate 0.05 0.40 0.55 ≈ 56.7 Methylene chloride 0.27 0.00 0.73 3.1 8.9 Methylethyl ketone 0.05 0.40 0.55 4.7 18.5 Methyl-t-butylether 0.00 ≈0.6 ≈0.4 ≈2.4 ≈4 N,N-dimethylformamide 0.00 0.44 0.56 6.4 36.7 Nitromethane 0.17 0.19 0.64 6.0 35.9 Pyridine 0.42 0.58 0.00 5.3 12.4 Sulfolane 0.00 0.17 0.83 43.3 Tetrahydrofuran 0.00 0.49 0.51 4.0 7.6 Toluene 0.17 0.83 0.00 2.4 2.4 Triethylamine 0.00 0.84 0.16 1.9 2.4 Trifluoroethanol 0.68 0.00 0.32 Water 0.43 0.18 0.45 10.2 80 a Values from [4]. b Polarity index; values from [5]. c Dielectric constant; values from [3]. the solvent-selectivity triangle (Fig. 2.9). Solvent polarity P is a measure of overall solvent polarity. Sample solubility tends to correlate with values of P —‘‘like dissolves like,’’ so samples tend to be more soluble in solvents of similar P .Less polar compounds, such as hydrocarbons, will be preferentially dissolved by solvents with low values of P , and the reverse will be true for solvents with high values of P . The dielectric constant ε similarly correlates with the ability of the solvent to 884 PROPERTIES OF HPLC SOLVENTS Table I.5 Viscosity of RPC Mobile Phases as a Function of Composition (%B) and Temperature (T) a T %B ◦ C 0 10 20 30 40 50 60 70 80 90 100 15 1.10 1.43 1.72 1.92 2.00 2.02 1.91 1.69 1.40 1.05 0.63 1.10 1.18 1.23 1.30 1.09 0.98 0.89 0.81 0.70 0.54 0.40 20 1.00 1.32 1.57 1.75 1.83 1.83 1.72 1.52 1.25 0.93 0.60 1.00 1.14 1.10 1.13 0.99 0.90 0.81 0.69 0.56 0.50 0.37 25 0.89 1.18 1.40 1.56 1.62 1.62 1.54 1.36 1.12 0.84 0.56 0.89 1.01 0.98 0.98 0.89 0.82 0.72 0.59 0.52 0.46 0.35 30 0.79 1.04 1.23 1.36 1.43 1.43 1.36 1.21 1.01 0.76 0.51 0.79 0.90 0.87 0.86 0.80 0.74 0.65 0.52 0.45 0.43 0.32 35 0.70 0.92 1.07 1.19 1.24 1.26 1.21 1.09 0.91 0.69 0.46 0.70 0.73 0.78 0.76 0.72 0.68 0.59 0.47 0.43 0.39 0.30 40 0.64 0.82 0.96 1.05 1.11 1.12 1.08 0.98 0.83 0.64 0.42 0.64 0.72 0.70 0.68 0.65 0.62 0.54 0.44 0.41 0.36 0.27 45 0.58 0.75 0.87 0.96 1.00 1.02 0.98 0.89 0.76 0.58 0.39 0.58 0.61 0.64 0.61 0.59 0.58 0.50 0.43 0.38 0.33 0.25 50 0.54 0.71 0.82 0.89 0.93 0.94 0.90 0.82 0.70 0.54 0.37 0.54 0.60 0.60 0.57 0.55 0.53 0.46 0.41 0.36 0.31 0.24 55 0.51 0.67 0.77 0.84 0.88 0.88 0.84 0.76 0.65 0.50 0.36 0.51 0.53 0.56 0.53 0.51 0.49 0.43 0.38 0.34 0.29 0.23 60 0.47 0.61 0.70 0.77 0.81 0.81 0.79 0.72 0.61 0.47 0.33 0.47 0.52 0.53 0.50 0.49 0.46 0.41 0.35 0.37 0.27 0.22 Source: Data from [6, 7]. a The composition is given as %B (v/v), where B is either methanol (top) or acetonitrile (bottom); for example, the viscosity of 30% methanol water at 30 ◦ C is 1.36. See [8, 9] for viscosity compared to com- position, temperature, pressure, as well as compressibility data. Table I.6 Density of Solvents for RPC Mobile Phases [3] Solvent Density at Temperature (g/mL) 20 ◦ C22 ◦ C25 ◦ C Acetonitrile 0.7822 0.7800 0.7766 Methanol 0.7913 0.7894 0.7866 2-Propanol 0.7855 0.7838 0.7813 Tetrahydrofuran 0.8892 0.8874 0.8847 Water 0.9982 0.9977 0.9970 Note: Error of 1 ◦ C = 0.1%, error in weight =0.1%, error in %v. I.3 SOLVENT SAFETY 885 dissolve ionized solutes or buffers; high values of ε favor increased solubility for ionized compounds. Table I.5 provides viscosity values for mixtures of MeOH/water and ACN/water as a function of temperature. These data are useful in estimating column pressure drop (Eq. 2.13). Table I.6 provides densities for some common solvents, to facilitate the more accurate formulation of reversed-phase mobile phases by weighing each solvent in the mixture. I.3 SOLVENT SAFETY The solvents commonly used for HPLC are often flammable and moderately toxic. Consequently most of these solvents should be stored in a secure, metal cabinet. Solvent flammability can be roughly assessed by the flash point, values of which are listed in Table 1.7. Common experience suggests that methanol is only moderately flammable, so that solvents with flash points above 12 ◦ C should not normally present a problem in terms of fire safety. However, solvents with lower flash points present a greater danger and should be treated accordingly. Many factors can contribute to solvent toxicity, and solvents other than water should be manipulated in a hood. A very rough measure of immediate toxicity is the solvent LD 50 value (Table 1.7), the administered amount in mg/kg body weight that causes mortality in 50% of the population. However, solvents in the laboratory Table I.7 Flammability and Toxicity Data for Various Solvents Solvent Flash Point ( ◦ C) a Threshold Limit b (ppm) Acetone −18 1000 Acetonitrile 42 40 Carbon tetrachloride None 10 Chloroform None 25 Ethyl acetate 13 400 Ethyl ether −45 400 Heptane −485 Hexane −26 100 Methanol 12 200 Methyl-t-butyl ether −28 — Methylene chloride None 500 n-Propanol 27 200 i-Propanol 12 400 Tetrahydrofuran −20 200 Water None none a Data from [2]. b Maximum allowable concentration of solvent vapor in the work place air as established by governmental regulation [2]. . AU; THF, 0.22 AU; IPA, 0.07 AU. Note that degassing methanol lowers the Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan Copyright. (1996) 202. 11. D. W. Bristol, J. Chromatogr., 188 (1980) 193. 12. P L. Zhu, L. R. Snyder, and J. W. Dolan, J. Chromatogr. A, 718 (1995) 429. 13. J. W. Dolan, LCGC, 11 (1 993) 640. 14. J. W. Dolan,. TROUBLESHOOTING Table 17.11 (Continued) Failed Test Symptom Symptom/Possible Source Solution Gradient distortion (Fig. 17.28b) Gradient mixing volume too large relative to gradient volume Use shallower gradient; reduce