7 Chromatographic probes
they were also able to observe that the water-rich layer increased in thickness as the aqueous content in the mobile phase increased up to 30%. The authors provided indirect evidence that hydrophilic partitioning is the main mechanism experienced at higher water content but other interactions (such as hydrogen bonding) might become more relevant as the water content decreases.
Subsequently, McCalley demonstrated the existence of a very complex mechanism, consisting of a combination of hydrophilic partitioning, adsorption, ionic interactions and even hydrophobic interactions [4]. Other authors also support this view, Liang et al. proposed a HILIC retention model, where the predominant mechanism depends on the analyte characteristics, the mobile phase composition and the nature of the stationary phase [5].
In HILIC an increase in the percentage of organic solvent leads to an increase in the retention times for polar analytes [1]; this phenomenon was investigated in our laboratory, particularly in order to assess whether partitioning is the main retention mechanism. Electrostatic interactions are secondary forces which can have important contributions to the retention in HILIC, since some polar compounds can be charged at the mobile phase pH conditions typically used [6]. We therefore investigated the electrostatic interaction contribution, by assessing the effect of mobile phase pH and salt concentration/salt type on the retention of polar acids and polar bases.
Column temperature is an important parameter that can also affect retention of polar analytes in HILIC [6]. The equation that is often used is derived from chemical thermodynamics, where the equilibrium point is related to the temperature, and is referred to as the van’t Hoff equation. In a chromatography sense the relationship between column temperature and retention factor is often described by the following:
ln k’= - ΔH°/RT + ΔS°/R + ln Φ where:
• ΔH° = enthalpy of interaction between stationary/mobile phase and analyte
• ΔS° = entropy of interaction between stationary/mobile phase and analyte
• R= universal gas constant • T= column temperature in Kelvin • Φ= phase ratio The van’t Hoff equation should also apply to
Theophylline Toluene Molecular Structure Variable t0 marker Uridine
Hydrophilic/ hydrophilic interaction
pKa LogD Test Mixture 41 2.72 all
12.6
-1.58
1+2
5-Methyluridine
Hydrophobic interaction
12.0
-1.02
1
2’-Deoxyuridine
Hydrophilic interaction
Adenosine
Configurational isomers selectivity
Vidarabine
Configurational isomers selectivity
2’ -Deoxyguanosine
Positional isomers selectivity
3’-Deoxyguanosine
Positional isomers selectivity
Anion Sodium p-toluenesulfonate
exchange selectivity
N,N,N-
trimethylphenylammonium chloride
Cation
exchange selectivity
Uracil
Hydrophilic interaction
Theobromine
Acidic-basic nature of stationary phase
Acidic-basic nature of stationary phase
-2.31 6 -2.8 0.88 5
13.9
-1.26
2
13.9
-1.03
3
13.9
-1.02
3
13.5
-1.14
4
13.5
-1.14
4
13.8
-1.08
5,6
10
-1.06
7
8.6
-0.5
7
Table 2: Structures of characterisation test solutes and their physiochemical properties (pKa and Log D values obtained from
www.chemspider.com)
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60
