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29


was purchased as lyophilised powder from Sigma-Aldrich and were dissolved in mobile phase B and mixed to contain 20 µg/ml of each component.


The experiment was performed on an Agilent 1260 HPLC with UV-detector, two pumps and one six port – two position switching valve, see Figure 1 for the instrument configuration. The trapping


column was a 2.1x10 mm XTerra MS C18 precolumn (Waters). The HILIC column was an Accucore HILIC, 3.0 x 150 mm, 2.6 µm (Thermo Fischer Scientific). The column temperature was 60°C, and the detection wavelength was 277 nm. The injection volume was 100 µl.


The HILIC gradient is presented in Table 1. The flow of the trapping mobile phase was 1.0 ml/min.


Table 1: The HILIC gradient


HILIC gradient %A %B Flow (ml/min) 0 min 1 min


20 min 21 min 30 min


85 15 85 15 62 38 85 15 85 15


0.8 0.8 0.8 0.8 0.8


For comparison the mixed vasopressin sample was also injected without using the column switching, i.e., the aqueous solution was injected directly on the HILIC column. All other method parameters remained the same.


Experimental (Reversed Phase method)


, 3.0 x 150 mm, 2.6 µm (Phenomenex). All instrument settings were identical to the HILIC run, except the gradient and flow, see Table 2.


The same mobile phases, sample, and equipment were used in the reversed-phase experiment as in the HILIC experiment. The column switch was disconnected and the sample was injected directly on the column. The reversed-phase column was a Kinetex C18


Table 2.


RP gradient %A %B Flow (ml/min) 0 min 10 90 1 min 10 90 20 min 22 78 21 min 10 90 30 min 10 90


0.6 0.6 0.6 0.6 0.6


The reversed-phase method was not optimised for vasopressins and served only as a comparison to HILIC in terms of peak asymmetry and peak width with similar mobile phases.


Figure 3: Reversed-phase chromatogram. Results and Discussion


The HILIC and the reversed-phase chromatograms are presented in Figures 2 and 3, respectively, and the peak data comparison in Tables 3 and 4. The results show that HILIC is superior to reversed- phase chromatography for vasopressins in terms of peak width, asymmetry and resolution. In HILIC, the positively charged peptides elute as Gaussian peaks with an asymmetry factor of 1.0, whilst using MS- friendly mobile phases. In reversed-phase chromatography, the same peptides interact


with the negatively charged residual silanol groups of the reversed phase bonded silica and show a high degree of tailing and peak broadening. Increased tailing and peak broadening decrease the peak capacity and the possibility to separate closely related impurities, which is observed by the decrease in resolution from 9.5 to 1.5 for HILIC and RPLC, respectively. It can be noted that the elution order of the two vasopressins is reversed in HILIC compared with reversed-phase, due to opposite column polarities.


Table 3. Peak characterisation data HILIC using column-switch. Peak


[Arg8 [Lys8


]vasopressin ]vasopressin


Asymmetry factor (EP) 1.0 1.0


Resolution (EP) 9.5


N/A Peak width (50% height)


4 sec 4 sec


Figure 2: Overlay HILIC chromatograms of a) aqueous sample injected with the column switch approach (upper) and b) aqueous sample injected directly on the HILIC column (lower).


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