By applying a“hydrophobic”HPLC system, five of peptidic substances in the middle molecule range were separated from the hemofiltrate fluid of a dialysis patient with chronic uremia. This system was based on the hydrophobicity value of peptide hormones having molecular weight up to 5,000 daltons by using gradient elution with increasing acetonitrile concentration in the eluent. Many ultraviolet-absorbing solutes of the hemofiltrate detected on this system were classified as hydrophilics, low-hydrophobics and hydrophobics. Numerous peaks of the hydrophobic solutes could be detected only in the hemofiltrate but not in healthy subject urines. Both middle molecule crude fractions of low-hydrophobicity and hydrophobicity were separated by step-wise gradient elution and further separation of the middle molecule subfractions was achieved by isocratic elution. Each of five subfractions corresponded to a single middle molecule and was found to be peptidic substances.
