Please check for possible typos, the method is there and this example should work fine:

// Red separator with 50% opacity placed at the current day with width equal to CellWidth, shown above events
DayPilotScheduler1.Separators.Add(DateTime.Today, Color.Red, SeparatorLayer.AboveEvents, DayPilotScheduler1.CellWidth, 50);

You can also create the separator instance manually and then add it to the collection:

Separator s = new Separator();
s.Location = DateTime.Now;
s.Color = Color.Red;
s.Layer = SeparatorLayer.AboveEvents;
s.Opacity = 50;
s.Width = 20;
DayPilotScheduler1.Separators.Add(s);