22
60
60
50
50
40
40
30
30
20
20
10
10
0 0 10 20 30 k'(NUCLEOSHELL® Figure 4. Comparison of NUCLEOSHELL® 40 RP18plus) RP 18plus and NUCLEOSHELL® Biphenyl (acetonitrile/water).
The different behaviour of acetonitrile and methanol can be explained by the triple bond of the nitrile function. This allows acetonitrile to compete with the analytes for the p-p interactions of the stationary phase. This explains the moderate differences between the phases when using acetonitrile as organic modifier. Methanol, on the other hand, does not reduce the p-p interactions between stationary phase and analyte. The influence of the biphenyl group is therefore much more pronounced.
k’rel(i, c)= formula k’rel k’(i, c) k’(i, ref )
Table 3. Description of the formulas used. k’(i, c)
k’rel(i, c)= k’rel(i, c)=
(c) k’rel
RSD= ∑[k’rel(i, c)–k’rel(c)]2 k’rel(c)
k’rel
(c) (c)
√
1 n
For quantitative evaluation, the quotient of the retention factor of the respective column and that of the biphenyl column is formed for each compound (Table 3). The mean of the values of a column is a measure of the average interaction strength of this phase compared to NUCLEOSHELL®
quotient describes the selectivity differences of the two stationary phases. The larger this value, the more the phases differ in their interactions and thus in their selectivity. When acetonitrile is used as the organic modifier, NUCLEOSHELL® interaction strength of 95% compared to NUCLEOSHELL®
RSD= ∑[k’rel(i, c)–k’rel(c)]2 k’rel(c)
RSD= ∑[k’rel(i, c)–k’rel(c)]2 k’rel(c)
√ √
1 n
1 n
Biphenyl. The relative standard deviation (RSD) of the p² shows an average Biphenyl (Table 4). This is
surprising to the extent that due to the bulky isobutyl groups a lower occupation density and thus smaller retention factors were expected. The higher average interaction strength is mainly caused by the increase in retention of mononuclear aromatics. These compounds are only weakly retained by biphenyl ligands. For the retention of NUCLEOSHELL®
the interactions with the additional isobutyl groups are responsible. Compared to the alkyl modified silica, the interaction strength of NUCLEOSHELL®
octadecyl (123%) and octyl phases (78%). The relative standard deviation is 0.24 in both cases, but a value of 0.18 could be determined between NUCLEOSHELL® Thus, the two biphenyl phases also show different selectivities.
Biphenyl is between the Biphenyl and p².
In contrast to methanol, acetonitrile has a nitrile group with a triple bond. The solvent competes with the analytes for the p-p-interactions of the stationary phase. This explains the moderate differences between the phases when using acetonitrile as organic modifier. With methanol the mean interaction strength of NUCLEOSHELL®
with 154%. In contrast, the interaction strengths of the pure alkyl phases decrease to 91% (octadecyl) and 48% (octyl) compared to NUCLEOSHELL®
p² increases significantly Biphenyl. The relative
standard deviations and thus the selectivity differences between the phases also increase significantly if the organic modifier is switched to methanol.
Biphenyl Figure 6. Comparison of NUCLEOSHELL® p² and NUCLEOSHELL® Biphenyl (methanol/water)
Figures 9 - 11 show the great difference for the chromatograms of the investigated sorbents. NUCLEOSHELL®
RP 8plus is compared to NUCLEOSHELL® 0 0
50 100 150 200 250 300 350 400 450 500 k'(NUCLEOSHELL®
π2 )
k’(i, ref ) k’(i, c)
k’(i, ref ) description
Relative retention factor of compound i on column c in comparison to the reference column (NUCLEOSHELL® Biphenyl)
Averaged relative retention factor over all compounds on column c as a measure of the mean interaction strength
Relative standard deviation as a measure of the dispersion of the relative capacity factors
50 150 50 60
0 0 2 4 6 8 10 k'(NUCLEOSHELL® Figure 5. Comparison of NUCLEOSHELL® 250 12 RP8plus) RP 8plus and NUCLEOSHELL® Biphenyl (acetonitrile/water) 14 16 18 20
200
100
biphenyl in Figure 9.
The higher interaction strength leads to the longer retention times of the aryl phase. This and the stronger interactions between biphenyl and steroidal structures lead to the better resolution of the first three compounds. The next chromatogram (Figure 10) shows a comparison of NUCLEOSHELL®
RP 18plus and NUCLEOSHELL® Biphenyl for the separation
of a further test mixture. Desoxycorticosterone is clearly separated from butyl paraben and propiophenone due to its steroidal structure on the biphenyl phase. The separation of the diisobutyl phthalate and pyrene peak pairs is probably based on stronger p-p interactions with the conjugated aromatic system. This is also the reason for the elution reversal in pentylbenzene and chrysene.
In Figure 11 the aryl phases NUCLEOSHELL® Biphenyl and NUCLEOSHELL® compared. Under the same conditions NUCLEOSHELL® p² are Biphenyl needs more time for
the separation. This is because the sorbents in the lower k’-region often show stronger retention (Figure 3).
LABMATE UK & IRELAND - SEPTEMBER 2020
k'(NUCLEOSHELL®
Biphenyl)
k'(NUCLEOSHELL®
Biphenyl)
k'(NUCLEOSHELL®
Biphenyl)
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