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 heig
Trang 1876 TROUBLESHOOTING
Table 17.11
(Continued)
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
Trang 2REFERENCES 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.
Trang 4APPENDIX I
PROPERTIES OF HPLC
SOLVENTS
Solvents 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
Trang 5880 PROPERTIES OF HPLC SOLVENTS
Table I.1
Table I 1 Solvent Properties of Interest in HPLC
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
injection of large samples
in prep-LC or trace analysis
drop; low values of viscosity desirable
and safety; higher boiling solvents preferred
organic solvent and sample solvent
accurate) formulation of mobile phases by weight
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
Trang 6I.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)
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
Trang 7882 PROPERTIES OF HPLC SOLVENTS
Table I.3
Miscellaneous Solvent Properties
aWavelength at which solvent absorbs 1.0 AU in a 10-mm cell.
bRefractive index.
cBoiling point.
dSolvent 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 β2/Σ, and dipolarity π∗/Σ The latter parameters are the basis of
Trang 8I.2SOLVENT POLARITY AND SELECTIVITY 883
Table I.4
Solvent Selectivity Characteristics
H-B Acidity H-B Basicity Dipolarity
α H
aValues from [4].
bPolarity index; values from [5].
cDielectric 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
Trang 9884 PROPERTIES OF HPLC SOLVENTS
Table I.5
Viscosity of RPC Mobile Phases as a Function of Composition (%B) and Temperature (T ) a
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].
aThe 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]
Note: Error of 1◦C
Trang 10I.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 LD50value (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
aData from [2].
bMaximum allowable concentration of solvent vapor in the work place air as established by governmental regulation [2].