Pretreatment for the purpose of biomass component separation and solid substrate retention was investigated using percolation methods on a milled hardwood substrate. A two-stage dilute acid percolation (DA) was investigated using an extremely low acid concentration (0.05 wt% sulfuric acid) at moderate temperature rages (150~190 ℃). Under these conditions hemicellulose was completely, solubilized, 90~100% of the cellulose was retained in the solid, and nearly 60% of the Klason Lignin was removed in the hydrolyzate. A ammonia recycled percolation (ARP) was optimized with a 17.5% ammonia solution, and run for 1 h at a constant temperature of 185 ℃. The ARP process yielded a primarily glucan residue, with almost 50% of the hemicellulose and 60% of the Klason Lignin having been removed in the hydrolyzate. In both cases the enzymatic digestibility after pretreatment was significantly higher than that or the untreated substrate, yet it remained below 85% of the theoretical value. In an attempt to increase the separation and thereby the percent digestibility, combined pretreatment methods were investigated. A sequential DA then ARP treatment exhibited little advantage over a DA treatment alone, whereas an ARP then DA process showed a continued delignification and hemicellulose removal as the temperature of the second DA stage increased. The combined pretreatment essentially fractionated the biomass feedstock into three major components. The remaining solid contained about 80% of glucan and 10% of lignin in the ARP-DA process.