Practical HPLC Method Development, Second Edition

by Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. Glajch

Copyright 1997 John Wiley & Sons, Inc.

APPENDIX II
PROPERTIES OF SOLVENTS USED
IN HPLC

Chromatographers have a choice among hundreds of solvents for use as

mobile-phase components, sample solvents, or in sample pretreatment. A
particular selection is usually affected by solvent characteristics that relate to
detection, separation, flow resistance (column pressure drop or mobile phase
viscosity), and miscibility. Commercial availability in adequate purity and at
a reasonable price are also important factors. The solvent properties described
in this appendix will be useful to chromatographers when it comes to selecting
one or more solvents.
II.1 DETECTION
The choice of mobile-phase solvent can have a profound effect on the ease
and sensitivity of HPLC detection. The lowest usable (cutoff) wavelength is
important for UV detectors (Tables 3.2 and 3.3), solvent refractive index (RI)
affects the sensitivity of RI detection for a particular sample, and solvent
volatility (boiling point) is important for evaporative light-scattering detectors.
Table II. 1 summarizes these and other properties that may affect detection
for a number of common solvents. Additional data are provided for most
HPLC-grade solvents in Refs. 2 and 3 (e.g., complete UV spectra for most
HPLC-grade solvents, blank gradients for some A- and B-solvent combinations, etc.).
II.2 SEPARATION
Mobile-phase solvents can affect separation by their polarity and selectivity.
More polar solvents cause increased retention in RPC and reduced retention
721

330
190
254
215
287
220
245
200
200
200
295
268
268
268
215
256
228
215
220
200
190
195
215
220
205
205

UV
Cutoff (nM)

Viscosity
(cP)
0.36
0.38
0.734
2.98
0.80
0.45
0.57
1.0
0.44
2.42
1.32
0.84
0.92
2.24
1.37
0.45
0.79
0.24
0.46
0.40

0.31
0.50

2.40
0.55

Refractive
Index (20C)
1.3587
1.3441
1.3942
1.3993
1.5249
1.4021
1.4458
1.4242
1.4064
1.4758
1.5514
1.4384
1.4305
1.4783
1.4224
1.3724
1.4448
1.3524
1.3796
1.3876
1.4340
1.3749
1.3914
1.3959
1.3772
1.3284
56.29
81.60
126.11
117.5
131.69
78.44
61.15
80.72
49.26
191.7
180.48
166.1
153.0
189.0
101.32
77.11
83.48
34.55
83.5
98.43
287.0
68.7
99.24
107.7
82.26
64.7

Boiling Point
(C)

Some Solvent Properties of Interest in HPLC Method Development" '

Acetone
Acetonitrile
n-Butyl acetate
1-Butanol
Chlorobenzene
1-Chlorobutane
Chloroform
Cyclohexane
Cyclopentane
Decahydronaphthalene
o-Dichlorobenzene
Dimethyl acetamide
Dimethyl formamide
Dimethyl sulfoxide
1,4-Dioxane
Ethyl acetate
Ethylene dichloride
Ethyl ether
Glyme
Heptane
Hexadecane
Hexane
Isooctane
Isobutyl alcohol
Isopropyl alcohol
Methanol

Solvent

TABLE n.l

29
29
15
15
12

29

12
9
17
19

23

19
28

15,17
11,17
22
15
21

Miscibility
Number (M)b

0.1
0.5
0.1
0.1
4.0
3.9
5.1

2.7
6.5
6.4
7.2
4.8
4.4
3.5
2.8

5.1
5.8
4.0
3.9
2.7
1.0
4.1
0.2
0.1

Polarity

0.01
0.01

0.82
0.95

0.01

0.62
0.56
0.58
0.49
0.38

8.3
1.0

7.6

11.7

0.40
0.04
0.05

8.8
3.1

c,8

0.56
0.65

Alumina

0.00

0.6
0.7

0.00

0.51
0.48

0.43

0.26

0.53
0.52

Silica

Eluotropic Values

210
210
329
330
334
331
233
285
190
210
280

212
284
308
273
231
210
190
288

1.4020
1.3689
1.3788
1.4072
1.3957
1.3901
1.4241
1.4680
1.3575
1.3856
1.4210
1.5102
1.4072
1.4969
1.5717
1.4767
1.3557
1.2850
1.3330
1.5054
1.72
0.27
0.43
0.80
0.506
0.51
0.44
1.67
0.23
2.3

0.95
0.55
0.59
0.566
0.567
0.711
0.926
1.00
0.81
124.6
55.2
79.64
144.9
116.5
102.4
39.75
202.00
36.07
97.2
241.7
115.25
66.0
110.62
213.5
87.19
47.57
71.8
100.0
144.41

17

20

16
17
23

25

5.5
2.5
4.7
4.0
4.2
4.5
3.1
6.7
0.0
4.0
6.1
5.3
4.0
2.4

1.0
0.0

10.2
2.5

0.35
0.51

0.43

0.42

0.00
0.82

0.71
0.45
0.29

0.26

3.7

0.48

0.30

0.00

0.53
0.22

0.02

Source: Ref. 1; Original data were obtained from Ref. 2 with the exceptions noted in b.
* Missing values indicate that data are unavailable.
* All pairs whose M numbers differ by 15 units or less are miscible in all proportions at 15C. Each pair whose M number difference is 16 has a critical solution
temperature between 25 and 75C, approximately 50C preferably. A difference of 17 or more corresponds to immiscibility or to a critical solution temperature
above 75C. Miscibility data were obtained from Ref. 4.

2-Methoxyethanol
Methyl terf-butyl ether
Methyl ethyl ketone
Methyl isoamyl ketone
Methyl isobutyl ketone
Methyl n-propyl ketone
Mthylne chloride
n-Methyl-2-pyrrolidone
Pentane
Propyl alcohol
Propylene carbonate
Pyridine
Tetrahydrofuran
Toluene
1 ^,4-Trichlorobenzene
Trichloroethylene
Trichlorotrifluoroethane
Trifluoroacetic acid
Water
o-Xylene

n*/2

0.86
0.83
0.81
0.90
0.91
0.93
1.00
0.84
0.77
0.72
0.69
0.66
0.65
0.58
0.58
0.51
0.45

Solvent

Aromatics
Benzene
Toluene
p-Xylene
Fluorobenzene
Chlorobenzene
Bromobenzene
Iodobenzene
Phenyl oxide
Anisole
Nitrobenzene
Benzonitrile
Dibenzylether
Acetophenone
Quinoline
Pyridine
2,6-Lutidine
Benzyl alcohol
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.32

all

Normalized Selectivity
Factors

0.14
0.17
0.19
0.10
0.09
0.07
0.00
0.16
0.23
0.28
0.31
0.34
0.35
0.42
0.42
0.49
0.22

/2

TABLE n.2 Classification of Solvents According to Normalized Selectivity"

0.16
0.20

Amines
Triethylamine
Tributylamine

0.36
0.36
0.34
0.51
0.54
0.60

Ethers
Diethyl
Diisopropyl
Dibutyl
Tetrahydrofuran
1,2-Dimethoxyethane
p-Dioxane

0.64
0.64
0.66
0.49
0.46
0.40

0.40
0.45
0.36
0.40
0.05
0.00
0.00
0.00
0.55
0.55
0.64
0.60

0.00
0.00
0.00
0.00
0.00
0.00

0.15
0.54

0.84
0.80

/2

0.31

0.00
0.00

a/2

Acetic acid
Esters
Methyl acetate
Ethyl acetate
-y-Butyrolactone
Ethylacetoacetate

Carboxylic acids

it*l2.

Solvent

Normalized Selectivity
Factors

0.46
0.56
0.54
0.46
0.51
0.57

Amides
Formamide
N, iV-Dimethylformamide
N,N-Dimethylacetamide
Hexamethylphosphoramide
Tetramethylurea
N-Methylpyrrolidinone
0.33
0.00
0.00
0.00
0.00
0.00

0.43
0.39
0.36
0.37
0.35
0.33
0.38
0.75
0.68
0.21
0.44
0.46
0.54
0.49
0.43

0.29
0.36
0.40
0.41
0.43
0.48
0.23
0.00
0.00

Source: Ref. 5.
" See Fig. 2.5 for a plot of these values.
6
The value used for water was 0.48, which is based on more recent estimates.

0.28
0.25
0.24
0.22
0.22
0.19
0.39
0.25
0.32

Methanol
Ethanol
Propanol
Butanol
Isopropanol
f-Butanol
Glycol
Hexachloro-2-propanol
Trifluorethanol

Alcohols

0.64

Nitro compounds
Nitromethane
0.73
0.57
1.00
0.57
0.83
0.45

0.60

Nitrites
Acetonitrile

X-miscellaneous
Mthylne chloride
Chloroform
Ethylene chloride
Dimethyl sulfoxide
Sulfolane
Water1'

0.56
0.55
0.59

Ketones
Acetone
2-Butanone
Cyclohexanone

0.27
0.43
0.00
0.00
0.00
0.43

0.17

0.15

0.06
0.05
0.00

0.00
0.00
0.00
0.43
0.17
0.18

0.19

0.25

0.38
0.40
0.41

TABLE IL3 Viscosity of RPC Mobile Phases as a Function of Composition

and Temperature
(a) Mobile-phase viscosity at 25C (ifcs) for reversed-phase systems
Vis (cP)'
Mobile Phase(%v organic/water)

MeOH

ACN

THF

0
10
20
30
40
50
60
70
80
90
100

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

0.89
1.06
1.22
1.34
1.38
1.43
1.21
1.04
0.85
0.75
0.46

MeOH, methanol; ACN, acetonitrile; THF, tetrahydrofuran (THF values approximate).

(fe) Variation of the viscosity (cP) of methanol-water and acetonitrile-water mixtures
with temperature"
Temperature
(Q
15
20
25
30

35
40
45
50
55
60
65

Water Content (%, v/v)

0

10

20

30

40

50

60

70

80

90

100

0.63
0.40
0.60
0.37
0.56
0.35
0.51

1.05
0.54
0.93
0.50
0.84
0.46
0.76

1.40
0.70
1.25
0.56
1.12
0.52
1.01

1.69
0.81
1.52
0.69
1.36
0.59
1.21

1.91
0.89
1.72
0.81
1.54
0.72
1.36

2.02
0.98
1.83
0.90
1.62
0.82
1.43

2.00
1.09
1.83
0.99
1.62
0.89
1.43

1.92
1.30
1.75
1.13
1.56
0.98
1.36

1.72
1.23
1.57
1.10
1.40
0.98
1.23

1.43
1.18
1.32
1.14
1.18
1.01
1.04

1.10
1.10
1.00
1.00
0.89
0.89
0.79

0.32
0.46
0.30
0.42
0.27
0.39
0.25
0.37
0.24
0.36
0.23
0.33
0.22
0.28

0.43
0.69
0.39
0.64
0.36
0.58
0.33
0.54
0.31
0.50
0.29
0.47
0.27
0.45

0.45
0.91
0.43
0.83
0.41
0.76
0.38
0.70
0.36
0.65
0.34
0.61
0.31
0.59

0.52
1.09
0.47
0.98
0.44
0.89
0.43
0.82
0.41
0.76
0.38
0.72
0.35
0.68

0.65
1.21
0.59
1.08
0.54
0.98
0.50
0.90
0.46
0.84
0.43
0.79
0.41
0.72

0.74
1.26
0.68
1.12
0.62
1.02
0.58
0.94
0.53
0.88
0.49
0.81
0.46
0.72

0.80
1.24
0.72
1.11
0.65
1.00
0.59
0.93
0.55
0.88
0.51
0.81
0.49
0.69

0.86
1.19
0.76
1.05
0.68
0.%
0.61
0.89
0.57
0.84
0.53
0.77
0.50
0.64

0.87
1.07
0.78
0.%
0.70
0.87
0.64
0.82
0.60
0.77
0.56
0.70
0.53
0.58

0.90
0.92
0.73
0.82
0.72
0.75
0.61
0.71
0.60
0.67
0.53
0.61
0.52
0.51

0.79
0.70
0.70
0.64
0.64
0.58
0.58
0.54
0.54
0.51
0.51
0.47
0.47
0.40

" The composition is given in % (v/v) of water at 20.5C. Upper figures, methanol-water mixture;
lower figures, acetonitrile-water mixture.
Source: Refs. 6-8.
726

727

APPENDIX II

in NPC. Table II.l lists polarity values P' for 46 solvents. These polarity values
range from P' = 0 for a non-polar solvent like pentane to P' = 10.2 for the
very polar solvent water. Relative solvent strength values for some of these
solvents are also listed in Table II.l for use in normal-phase (alumina, silica)
chromatography. See also Figs. 6.4 (reversed-phase), 6.23, and 6.24 (normal
phase).
Selectivity depends both on solvent polarity and the position of a solvent
in the solvent-selectivity triangle (Fig. 2.7). The latter classification of solvent
selectivity is based on the interaction of the solvent with sample molecules
as a dipole, an acid, or a base. Table II.2 summarizes these selectivity interactions (x = n*f,, a/S, fS,) for a number of common solvents. The x values of
Table II.2 are normalized in terms of solvent polarity, so if different strong
solvents B are diluted with a weaker solvent A to give mobile phases A/B of
the same polarity and solvent strength, solvent selectivity will be determined
mainly by the selectivity factors x of Table II.2. By choosing solvents with
very different selectivity factors from Table II.2, mobile-phase selectivity can
be varied significantly.
The first column of values in Table II.2 (JC = ir*fX) gives the fractional
polarity of the solvent due to dipole interactions, the second column (x = a/
2) gives the fractional polarity due to the acidity of the solvent, and the third
column (x = fZ) gives the fractional polarity due to solvent basicity. For
example, among the alcohol solvents, methanol receives 28% of its polarity
from its dipole, 43% from its acidity, and 29% from its basicity. Similarly,
among the amine solvents, triethylamine has 16% of its polarity from its dipole,
and 84% from its basicity; it has no acidity for Chromatographie separation.

II3 SOLVENT VISCOSITY

To maintain an acceptable pressure drop (< 2500 psi or < 160 bar) with a
reasonable flow rate through the column, the mobile-phase viscosity should
be as low as possible. Pressure drop is proportional to mobile-phase viscosity
(Eq. 2.9). Viscosity values TJ at 20C for several pure solvents are listed in
Table II.l. For non-aqueous mobile phases (NPC or NARP), the viscosity TJ
can be estimated as a function of the viscosities of the A- and B-solvents (ja
and -qb) and their mole fractions (Xa and Xb):
log(T,) = Xa log(77(I) + Xb log(r,fc)

(II.l)

The volume fractions of each solvent can be substituted for Xa and Xb in

Eq. II.l with little error. Equation II.l is not reliable for mobile phases that
contain water, due to their non-ideal behavior as a result of the very strong
interactions between water molecules. Viscosities at 25C for RPC mobilephase mixtures are listed in Table II.3a. Viscosity decreases with temperature,

728

APPENDIX II

as illustrated in Table 11.36 for mobile-phase mixtures that were recommended

in Chapter 9 (acetonitrile-water and methanol-water).
II.4 SOLVENT MISCIBILITY
Mobile phases formulated for HPLC separations must be miscible, preferably
in all proportions. Whether any two solvents from Table II. 1 will be miscible
in all proportions can be estimated by calculating the difference in their
miscibility numbers M, shown in Table ILL For example, hexane has M =
29 and acetonitrile has M = 11, so the difference is 29 - 11 = 18. If this
difference is >17, the two solvents will not be miscible in all proportions,
which is the case for hexane and acetonitrile. Water is miscible with a limited
range of organic solvents, mainly those with polarity values P' equal to 5 or
greater and including ethanol and propanol.
REFERENCES
1. C. Seaver and J. Przbytek, LC/GC, 13 (1995) 220.
2. Burdick & Jackson Solvent Guide, 3rd ed., Burdick & Jackson Laboratories, Muskegon, MI, 1990.
3. HPLC Solvent Reference Manual, J. T. Baker Chemical Co., Phillipsburg, NJ, 1985.
4.
5.
6.
7.

B. A. Bidlingmeyer, Practical HPLC Methodology, Wiley, New York, 1992, p. 245.

L. R. Snyder, P. W. Carr, and S. C. Rutan, J. Chromatogr., 656 (1993). 537.
L. R. Snyder and P. E. Antle, Liq. Chromatogr., 3 (1985) 99.
H. Colin, J. C. Diez-Masa, G. Guiochon, T. Czajkowska, and I. Miedziak, /. Chromatogr., 167 (1978) 41.
8. M. A. Quarry, R. L. Grob, and L. R. Snyder, J. Chromatogr., 285 (1984) 1.