30 February / March 2020 Table 4. Peak characterisation data reversed-phase.
Peak [Lys8 [Arg8
]vasopressin
Asymmetry factor (EP) Resolution (EP) 3.3
1.5 ]vasopressin 3.4
The chromatograms from injecting the aqueous sample in HILIC, with and without the column-switch installed, is compared in Figure 2. The results illustrate the problem of injecting an aqueous solution directly on to the HILIC column. With direct injection on to the column, the sample solvent effect causes severe peak splitting and peak broadening. However, when using the column switch approach, the sample solvent is removed before the HILIC column and thereby also the sample solvent effect.
The proposed setup with column switching and a trapping column works like an on-line solid-phase extraction method coupled with HILIC. Theoretically, there is no upper limit for the injection volume of aqueous sample since the sample is concentrated on the trap column and the sample solvent removed.
It should also be noted that the column- switching setup also de-salts the sample
N/A
Peak width (50% height) 11 sec 13 sec
before being transferred to the HILIC column, since any buffer salts in the sample are not retained by the reversed-phase trap column and are sent to waste. Buffer salts are a potential problem in HILIC, since they are typically poorly soluble in high amounts of acetonitrile which could lead to precipitation and column blockage.
In summary, the presented technique provides a fully automated method to eliminate the sample solvent effect when analysing aqueous solutions by HILIC. A suggestion of a potential expansion of the principle can be to test different types of trap columns in combination with different mobile phases to explore selectivity and trapping.
References
1. Snyder LR, Kirkland JJ, Glajch JL. Practical HPLC method development 2nd edition (2012), 7.3.3.2. Silanol effects
2. Nshanian M, Lakshmanan R, Chen H, Ogorzalek Loo RR, Joseph A. Loo J. Enhancing Sensitivity of Liquid Chromatography-Mass Spectrometry of Peptides and Proteins Using Supercharging Agents. Int J Mass Spectrom. 2018 Apr;427:157-164. doi: 10.1016/j. ijms.2017.12.006.
3. Alpert AJ. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds, Journal of Chromatography A, Volume 499, 19 January 1990, Pages 177- 196.
doi.org/10.1016/S0021-9673(00)96972-3
4. Buszewski B, Noga S. Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique. Analytical and Bioanalytical Chemistry, January 2012, Volume 402, Issue 1, pp 231-247
5. Keunchkarian S, Reta M, Romero L, Castells C. Effect of sample solvent on the chromatographic peak shape of analytes eluted under reversed-phase liquid chromatographic condition, Journal of Chromatography A, Volume 1119, Issues 1-2, 30 June 2006, Pages 20-28
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
