In RPLC mode, the typical column balance required for reproducible retention is approximately 10 column volumes. This is clearly reflected in Figure 14, but is different for HILIC separation. Typically, HILIC columns have a longer equilibration time than RPLC columns. This is a feature of the technology and is necessary for reproducible retention. The time spent also varies by phase (stationary phase) characteristics, eluent and analyte, and therefore depends on the specific application. The following guidelines can be used as an approximation (approximate reference). Importantly, in the development of the HILIC method, the balance will be studied as a parameter for each separation (for each mature separation method, the equilibrium time will be studied as a parameter during the development of the HILIC method). Figure 14 Once the water layer is established, reproducible (reproducible) and consistent retention and chromatography (reproducibility of chromatographic methods, consistent retention times) can be achieved. For the second and subsequent runs of the column, it is recommended to use only 20 column volumes to achieve a stable number of retentions (time), see Figure 15. Figure 15 Gradient HILIC balance At the same time, the ACE HILIC phase can also use the reproducible gradient HILIC method (the ACEHILIC column can also be used to reproduce the gradient elution of the HILIC method). It was determined that approximately 10 column volumes were required between injections. The equilibration time between injections is lower than the time required for the new column because only the initial conditions need to be re-established. The new column needs to be equilibrated to form a hydrated layer on the stationary phase particles (while the new column needs to be equilibrated for a long time because of the need to form a hydrated layer on the stationary phase particles). Figure 16
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The establishment and formation of a stable hydration layer around the stationary phase particles is important for reproducible HILIC chromatography (reproducibility of HILIC chromatography is critical).
For new, unused columns, it is recommended to use 60-80 column volumes to initially establish the water layer required for HILIC.
Comparison of the number of column volumes required to reach a new, unused column in RPLC and HILIC modes 
After the run is complete, the column should be cleaned and stored according to the relevant instructions in Section 9. 
The number of column volumes required for equilibrium in the second and subsequent analyses in the HILIC mode is compared to the column: ACE 5 HILIC-A,
100 x 3.0 mm
Mobile phase: 10 mM ammonium formate, pH 4.7 (dissolved in MeCN/H2O) (90:10 v/v)
Flow rate: 0.43 mL/min
Temperature: 25 °C
Detection: UV, 214 nm
sample:
1) 2'-deoxyguanosine
2) 4-hydroxybenzoic acid
3) Salbutamol
Excellent retention reproducibility of two consecutive injection gradient HILIC analyses with 20 column volume balances between injections is shown. 
HILIC method balance time