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Wako FAQ -Chromatography- Q8

Q8: We are examining mobile phase conditions of HPLC.
We intend to examine by changing solvents.Is there any method to estimate the elution profile in advance?

Polarity, pH, etc. may be given as the mobile phase factors affecting the elution profile of a solute (sample).
Let us examine the effects of polarity here.

In the normal phase liquid chromatography (LC), the polarity of a mobile phase is lower than that of a stationary phase and, therefore, sample components with low polarity have a small capacity ratio, k', and are eluted quickly. In the reverse phase LC such as ODS, on the contrary, the polarity of a mobile phase is higher than that of a stationary phase and, thus, a sample component with a high polarity has a small k'and is eluted quickly (Fig. 1).
Fig.1 Elution Profiles in normal and reverse phase chromatography

Various indicators are used for polarity of the solvents in a mobile phase. The indicators concerning polarity of typical mobile phase solvent are shown in Table 1. You can see that change in a solvent results in a change in polarity of the mobile phase.

The elution profile of the solute after changing the polarity of the mobile phase can be estimated by the following equations that correlated Snyder polarity parameter, P', shown in Table 1 and capacity ratio, k', approximately by an exponential relation equation.
- Normal phase: k'2/k'1 =10 (P1'−P2')/2
- Reverse phase: k'2/k'1 =10 (P2'−P1')/2

For example, when the solvent in the mobile phase is changed from acetonitrile to methanol in the reverse phase chromatography, substitution of P' values for acetonitrile and methanol in the relation equations gives

k'2/k'1 = 10(5.1−5.8)/2 = 10−0.35

showing about 0.45 fold decrease in k'.
Thus, if the k' in certain mobile phase is known, k' in a new mobile phase after changing the solvent can be estimated. In the actual analysis, however, a mixture of solvents is often used rather than a single solvent. In this case, P' is determined by the following equation.

P' = φAP'A + φBP'B + •••
where φA, φB ••• are % volumes of the solvents used in the mixture,
P'A, P'B ••• are P' values of the solvents used in the mixture.

For example, when the composition ratio of a mixture of acetonitrile and water is changed from 40/60 (V/V) (i) to 50/50 (V/V) (ii), P' (P'1) of the mobile phase (i) is
P'1 = 0.4 x 5.8 + 0.6 x 10.2 = 8.44 and
P'(P'2) of the mobile phase (ii) is
    P'2 = 0.5 x 5.8 + 0.5 x 10.2 = 8.00 and
    (P'2 - P'1) becomes (-0.44). Thus, the k' is expected to become approximately 0.6 folds.

Table 1. Indices relating polarities of solvents in the mobile phase
Solvent Solvent strength to alumina (ε°) Hildebrand solubility parameter (δ) Snyder polarity parameter (P') Snyder solvent strength (S)
Hexane 0.01 - 0.1 -
Cyclohexane 0.04 8.2 -0.2 -
Toluene 0.29 8.9 2.4 -
Benzene 0.32 9.2 2.7 -
Ethyl ether 0.38 7.4 2.8 -
Chloroform 0.40 9.3 4.1 -
Methylene chloride (dichloromethane) 0.42 9.7 3.1 -
Tetrahydrofuran 0.45 - 4.0 4.4
Methyl ethyl ketone
(ethyl methyl ketone)
0.51 9.3 4.7 -
Acetone 0.56 9.9 5.1 -
Dioxane 0.56 10.0 5.8 -
Ethyl acetate 0.58 9.6 4.4 -
Methyl acetate 0.60 - - -
Dimethyl sulfoxide 0.62 - 7.2 -
Diethylamine 0.63 - - -
Acetonitrile 0.65 11.7 5.8 3.1
Pyridine 0.71 10.7 5.3 -
Isopropanol 0.82 11.5 3.9 4.2
Propanol 0.82 11.5 4.0 -
Ethanol 0.88 12.7 4.3 3.6
Methanol 0.95 14.4 5.1 3.0
Ethylene glycol 1.11 - 6.9 -
Acetic acid Large - 6.0 -
Water Large 21 10.2 0.0
Solvent strength ε°
Snyder has determined the value corresponding to adsorption energy of a solvent per unit area when alumina was used as the adsorbent and proposed to define the solvent strength, ε℃, taking the value of pentane as reference value, 0.

Solubility parameter δ
Hildebrand has defined the solubility parameter given by δ = (E/V)1/2 of the cohesion energy, E, and molecular volume, V, of a liquid molecule and shown that a large δ shows a strong solvent.

Polarity parameter, P'
Snyder has calculated and proposed polarity parameter, P', of a solvent that contains proton donating, proton accepting or bipolar contribution based on the experimental data of Rohrschneider. P' acts as an indicator for regulation of k' and selectivity.

Snyder's solvent strength, S
Snyder, et al. have determined empirical solvent strength parameter, S, acting as indicator for regulation of k' in the reverse phase partition chromatography.

Q1Which reference standard do you recommend for use to check performance of a column?
Q2Is there any simple method to change the solvent concentrations in the mobile phase once prepared?
Q3How are ODS columns, Wakosil-II HG, AR and RS, used in different situations?
Q4Ion pair reagents for low wavelength are on the market. What effects and differences do they show with actual samples?
Q5We wish to fractionate by HPLC. How can we determine the optimal separating conditions?
Q6We wish to perform fractionation by the reverse phase flash chromatography. How can we determine the optimal separating conditions?
Q7Is column performance improved by narrowing the inside diameter of a column?
Q8We are examining mobile phase conditions of HPLC.We intend to examine by changing solvents.Is there any method to estimate the elution profile in advance?
Q9We are examining the conditions of a mobile phase in the HPLC. We would like to know how to regulate retention of a sample that has a readily dissociating functional group.
Q10I have used a semi-microcolumn (2φ mm) but cannot obtain improvement of sensitivity generally offered.
I wonder what is the cause as I am sure to have used the system applicable to microcolumns.
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