Bypass high-performance liquid chromatography for purification of trace analytes

A technique that we have termed “bypass HPLC” is introduced as a noncontaminating way to define the retention time of a trace analyte for purification purposes on an HPLC column when the amount of the analyte in real samples is too low for on-line detection. The technique employs two HPLC columns (“calibration” and “purification”) of the same type that are connected in parallel, with appropriate valving, along with use of two accessory compounds. By injecting ordinary (on-line detectable) amounts of authentic analyte plus the two accessory compounds on the calibration column, and similarly the two accessory compounds onto the purification column, one can predict the retention of the analyte on the latter column without contaminating this column, as follows. The migration times of the first accessory compound provide a reference time on each column; the migration times of the second accessory compound are normalized on each column by subtracting the corresponding reference times; and then the retention time of analyte can be calculated on the second column since the ratio of its normalized retention times on the two columns equals the corresponding ratio for the second accessory compound.