21 increased bleeding behaviour of the NUCLEODUR®
of two bulky isobutyl substituents to the biphenylpropyl should minimise hydrolysis of the surface modifi cation. Therefore, NUCLEOSHELL®
p². Following Kirkland [7], an addition Biphenyl does not show any appreciable
spike (Figure 2) and thus can be used under acidic conditions in gradient mode.
For selectivity testing, forty-fi ve organic compounds specifi ed in Table 2 were chromatographed isocratically with acetonitrile / water (50:50, v/v) and methanol / water (60:40, v/v respectively). These compositions were chosen because they have a similar elution power [1]. 1 µL of a methanolic solution (about 1%) of the compounds were injected at a fl owrate of 1 mL/min. The column temperature was 25 °C. A diode array detector (200-300 nm) was used to detect and to identify the substances. The determined retention factors are summarised in Table 2.
The comparison of two sorbents can be visualised by plotting the retention factors in a dot diagram. If two HPLC columns have equal retention factors, all points are on a straight line with a slope of one. This line is marked with a red line in Figures 3-8. Points above this line are for compounds that retain more strongly on the NUCLEOSHELL®
Biphenyl phase than
on the second phase, points below the line for compounds with lower interactions and thus smaller retention factors.
Table 2. Determined retention factors. retention factor
NUCLEOSHELL® Biphenyl
ACN1)
Acenaphthylene Pentylbenzene Anisole
Fluorene
Chlorobenzene Fluoranthene Phenol
Naphthaline Anthracene Toluene
Propylbenzene Butylbenzene Acetophenone Butylbenzoate Biphenyl
4-Nitrotoluene Ethylbenzoate Ethylbenzene
N.N-Dimethylaniline m-Cresol o-Cresol p-Cresol
2.6-Dimethylphenol 2.6-Dimethylaniline Nitrobenzene
Dimethylphthalate Diethylphthalate 1-Phenylethanol Pyrene
Chrysene
Propiophenone Diethyl adipate
Diisobutylphthalate Triphenylene o-Terphenyl Prometryn
Desoxycorticosterone Estrone
Progesterone
Propylparabene p-Terphenyl
Benzofl uoranthene
5-(p-Methylphenyl)-5- phenylhydantoin
Dibutylphthalate Benzopyrene
1) MeOH1)
retention factor NUCLEOSHELL®
ACN1) retention factor
p² NUCLEOSHELL® RP 18plus
MeOH1) ACN1) 7.97 15.02 6.08 13.87 7.43 1.92 1.79 2.58 2.16
MeOH1) 9.06
1.84 retention factor
NUCLEOSHELL® RP 8plus
ACN1) 4.24
1.71
MeOH1) 3.75
15.63 33.47 12.86 38.44 32.83 56.82 14.88 21.80 1.96
8.45 17.14 9.06 31.97 11.25 17.80 5.77 3.03
3.12 2.79 5.79
0.54 4.48
4.04
0.53 8.54
3.87
0.61 5.73
3.77
0.52 6.51
2.66
13.98 33.69 16.59 69.62 19.42 33.96 7.99 0..63 0.50 4.60
2.94 9.53
2.58 3.49 3.92 3.96
0.63 3.53
11.04 23.70 13.20 50.92 14.83 23.68 6.77 2.93 6.74
1.36 1.05 1.89 1.13 0.85 2.70 5.75 11.25 11.07 14.38 6.28 1.00
1.16 6.42 2.06 9.64 0.42 2.96 7.62 2.10 6.55
10.25 17.90 8.60 20.88 19.06 28.63 9.63 11.89 1.17
6.00 12.17 5.01 14.69 9.31 11.89 5.45 7.50 12.88 7.47 20.61 9.63 13.42 5.44 2.72 2.53 4.38 2.89 0.86 0.97 0.86 1.47 1.43 1.81 1.30 2.58 0.63
3.57 3.61 5.14 2.98 0.84 0.92 0.85 1.50 1.36 1.85 1.61 4.15 0.84
2.76 2.20 3.79 2.53 0.73 0.82 0.73 1.25 1.25 1.73 1.15 2.20 0.53
6.85 4.53 6.11 3.99 0.93 1.03 0.96 1.72 1.69 2.99 2.33 5.61 0.89
2.79 3.15 6.39 3.22 0.90 1.02 0.89 1.66 1.47 1.73 1.23 2.89 0.64
2.20 3.15 7.20 2.76 0.92 1.03 0.92 1.70 1.19 1.19 0.83 2.43 0.84
2.15 2.27 4.01 2.26 0.85 0.95 0.84 1.40 1.23 1.49 1.12 2.28 0.62
0.68 6.55 5.65 1.46 2.06 3.60 1.58 0.68 0.74 0.66 1.12 0.81 0.87 0.69 1.82 0.67
14.54 35.36 17.50 71.25 20.98 37.65 8.10 10.10 22.29 77.73 31.41 209.02 32.54 70.54 11.18 16.36 1.99 1.90
2.62 3.72
1.83 1.51
3.76 4.23
2.10 2.19
1.70 2.25
1.65 1.81
1.21 1.83
11.81 35.07 8.84 39.60 20.51 26.22 11.75 16.25 21.14 68.82 27.33 152.00 28.49 58.27 9.98 13.83 21.88 68.69 20.74 104.20 31.00 59.94 15.00 24.29 3.42
8.22 2.56 9.33 4.13
3.83 39.14 4.37 117.48 2.05 2.79 13.11 2.72 25.33 2.45
2.37 1.08 2.73 1.49
6.95 5.32 4.85
2.33
2.76 1.58 1.87
1.25
4.40 4.12 3.23
10.64 110.73 12.42 301.80 6.93 14.58 4.44 11.33 1.29
1.56 27.80 116.86 2.95 32.71 255.89 40.95 103.16 16.14 30.05
33.24 154.51 49.33 444.67 54.50 139.42 15.78 27.49 1.14
0.98 4.09 1.07 1.99 0.98 1.28
12.85 42.94 10.09 51.50 21.50 29.70 11.81 17.00 45.07 232.28 51.38 413.73 54.53 139.39 14.91 26.48
Chromatographic conditions: Column 50 mm x 4 mm, acetonitrile/water 50:50 (v/v) or methanol/water 60:40 (v/v), 1 mL/min, 25°C, DAD 220-300 nm.
50
160 140 120 100 80 60 40 20 0
-20 5,00 6,25 7,50 8,75 10,00 min
1: NUCLEOSHELL® π2 2: Raptor® 3: Kinetex®
4: NUCLEOSHELL® , 2.7 µm
Biphenyl, 2.7 µm Biphenyl, 2.6 µm
Biphenyl, 2.7 µm 1
120 100 80 60 40 20 0
2 3 4
-20 55.0
50.0 40.0 30.0 20.0 10.0 0.0 -5.0
200.0 212.5 225.0 237.5 250.0 UV-spectrum of eluate
256.20
200.0 212.5
225.0
237.5
250.0 200.47 UV-spectrum of allylbiphenyl
262.5
275.0
287.5 nm
255.07
262.5
275.0
287.5 nm
Figure 2. pH stability of different biphenyl sorbents using acidic gradients
Chromatographic conditions: column: 100 mm x 3 mm, eluent A: 1 % H3PO4, Eluent B: ACN, 40 °C, UV, 254 nm, 0.56 mL/min, gradient: equilibration 10 min 10 % B, hold 10 % B for 5 min, from 10 % to 90 % B in 5 min, hold 90 % B for 3 min, in 1.0 min to 10 % B, 0.56 mL/min.
60
40
30
20
10
0 0 10 20 30 k'(NUCLEOSHELL® Figure 3. Comparison of NUCLEOSHELL® π²) p² and NUCLEOSHELL® The comparison of the two aryl phases NUCLEOSHELL® Biphenyl (acetonitrile/water). Biphenyl and NUCLEOSHELL® Figure 3 shows that for compounds with lower retention factors, NUCLEOSHELL®
often exhibits stronger interactions. This reverses at higher values. Here the retention factors and thus the interactions of NUCLEOSHELL®
p² in Biphenyl p² are stronger. This certainly depends
on the choice of the analytes. Most of the mononuclear aromatic compounds studied here are located in the lower part of the retention factors. They show relatively weak interactions with the biphenyl group [5]. The isobutyl groups of NUCLEOSHELL®
Biphenyl
increase the hydrophobic interactions of the phase, so that these molecules retain more. At higher k’ values, compounds like polynuclear aromatics and steroids are located. These substances undergo stronger interactions with biphenyl ligands. Therefore, NUCLEOSHELL® p² compared to NUCLEOSHELL® proportion of p-p interactions.
Biphenyl shows longer retention times due to the higher
The comparisons with octadecyl and octyl ligands are shown in Figures 4 and 5. With a few exceptions, the octadecyl phase shows longer retention times. The exceptions are mainly steroids. The retention factor of progesterone shows a value increased by a factor of 1.53, for desoxycorticosterone even a factor of 1.86 could be determined. On the other hand, alkylaromatics like pentylbenzene are more strongly retained on the RP 18 phase. The lower hydrophobicity of the octyl group means that all compounds tested on NUCLEOSHELL®
two phases can be found for steroids and some polynuclear aromatics.
In Figures 6 - 8, the sorbents are compared in the eluent system methanol / water. Under these conditions, NUCLEOSHELL® NUCLEOSHELL®
for desoxycorticosterone. In contrast to the octadecyl column, NUCLEOSHELL® p² retains all compounds more strongly than Biphenyl (Figure 6). For example, a factor of 3.0 could be determined Biphenyl
shows on average stronger interactions. The retention factors of desoxycorticosterone and progesterone are more than seven times greater on the biphenyl phase. In the case of pentylbenzene the retention factor decreases by a factor of 0.59. A similar behaviour is shown by the comparison with the octyl phase (Figure 8). While the retention factors of desoxycorticosterone, progesterone, and benzopyrene increase by factors of 8.8 to 9.8, phenol for example, is only increased by 19%.
40 50 60
Biphenyl have longer retention times. The largest differences between the
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Absorbance [mAU] k'(NUCLEOSHELL® Biphenyl)
Intensity [cps]
Intensity [cps]
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