Methods for separating compounds

Abstract

Methods and systems for analyzing samples using multi-dimensional chromatography are disclosed.

1 . A liquid chromatography system for separating a mixture of compounds, the system comprising:
 a) a first chromatographic analysis system comprising a first chromatographic column and a pump for pumping a first mobile phase through the first chromatographic column;   b) a second chromatographic analysis system comprising a second chromatographic column and a pump for pumping a second mobile phase through the second chromatographic column;   c) means for selecting at least one compound-containing fraction from an effluent stream of the first chromatographic column and means for introducing the at least one compound-containing fraction into the second chromatographic column;   wherein the pH of the first mobile phase and the pH of the second mobile phase are different.   
     
     
         2 . The liquid chromatography system of  claim 1 , wherein the first and second chromatographic analysis systems carry out first and second chromatographic separation modes wherein the first and second chromatographic separation modes are substantially orthogonal to each other. 
     
     
         3 . The liquid chromatography system of  claim 2 , wherein the first and/or second chromatographic separation mode is high performance liquid chromatography (HPLC). 
     
     
         4 . The liquid chromatography system of  claim 2 , wherein the first and/or second chromatographic separation mode is hydrophilic interaction chromatography (HILIC). 
     
     
         5 . The liquid chromatography system of  claim 3 , wherein the HPLC is reversed-phase HPLC separation. 
     
     
         6 . The liquid chromatography system of  claim 2 , wherein the first chromatographic separation mode has a peak capacity of at least 100 peaks. 
     
     
         7 . The liquid chromatography system of  claim 1 , wherein the first pH and the second pH differ by at least about 3 pH units. 
     
     
         8 . The liquid chromatography system of  claim 1 , wherein the at least one compound is a peptide, polypeptide, or protein. 
     
     
         9 . The liquid chromatography system of  claim 1 , wherein the at least one compound is a small organic molecule. 
     
     
         10 . The liquid chromatography system of  claim 1 , wherein the mixture of compounds contains at least 1000 compounds. 
     
     
         11 . The liquid chromatography system of  claim 1 , wherein the total peak capacity of the system is at least 1,000 peaks. 
     
     
         12 . The liquid chromatography system of  claim 3 , in which the HPLC separation is performed using a microbore column, capillary column, or nanocolumn. 
     
     
         13 . The liquid chromatography system of  claim 1 , wherein the at least one compound-containing fraction is concentrated or diluted prior to introducing the at least one compound-containing fraction into the second chromatographic column. 
     
     
         14 . The liquid chromatography system of  claim 13 , wherein the at least one compound-containing fraction is concentrated by evaporation. 
     
     
         15 . The liquid chromatography system of  claim 13 , wherein the at least one compound-containing fraction is diluted on-line prior to introducing the at least one compound-containing fraction into the second chromatographic column. 
     
     
         16 . The liquid chromatography system of  claim 1 , further comprises a detector for detecting the presence or absence of the at least one analyte in the sample 
     
     
         17 . The liquid chromatography system of  claim 16 , wherein the detector is a mass spectrometer. 
     
     
         18 . The liquid chromatography system of  claim 2 , wherein the first and second chromatographic separation modes are the same. 
     
     
         19 . The liquid chromatography system of  claim 1 , wherein the first and/or second mobile phase is substantially free of non-volatile salts. 
     
     
         20 . The liquid chromatography system of  claim 1 , wherein the first and/or second mobile phase comprises less than about 20 mM of non-volatile salts. 
     
     
         21 . The liquid chromatography system of  claim 1 , wherein a plurality fractions from the effluent stream of the first chromatographic column are collected and introduced into the second chromatographic column. 
     
     
         22 . A liquid chromatography system for separating a mixture of compounds, the system comprising:
 a) a first chromatographic analysis subsystem comprising a first chromatographic column and a pump for pumping a first mobile phase through the first chromatographic column;   b) a second chromatographic analysis subsystem comprising a second chromatographic column and a pump for pumping a second mobile phase through the second chromatographic column;   c) means for selecting at least one compound-containing fraction from an effluent stream of the first chromatographic column and means for introducing the at least one compound-containing fraction into the second chromatographic column;   wherein the pH of the first mobile phase and the pH of the second mobile phase are different.   
     
     
         23 . The liquid chromatography system of  claim 22 , wherein the first and second chromatographic analysis subsystems carry out first and second chromatographic separation modes wherein the first and second chromatographic separation modes are substantially orthogonal to each other. 
     
     
         24 . The liquid chromatography system of  claim 23 , wherein the first and/or second chromatographic separation mode is high performance liquid chromatography (HPLC). 
     
     
         25 . The liquid chromatography system of  claim 23 , wherein the first and/or second chromatographic separation mode is hydrophilic interaction chromatography (HILIC). 
     
     
         26 . The liquid chromatography system of  claim 24 , wherein the HPLC is reversed-phase HPLC separation. 
     
     
         27 . The liquid chromatography system of  claim 23 , wherein the first chromatographic separation mode has a peak capacity of at least 100 peaks. 
     
     
         28 . The liquid chromatography system of  claim 22 , wherein the first pH and the second pH differ by at least about 3 pH units. 
     
     
         29 . The liquid chromatography system of  claim 22 , wherein the at least one compound is a peptide, polypeptide, or protein. 
     
     
         30 . The liquid chromatography system of  claim 22 , wherein the at least one compound is a small organic molecule. 
     
     
         31 . The liquid chromatography system of  claim 22 , wherein the mixture of compounds contains at least 1000 compounds. 
     
     
         32 . The liquid chromatography system of  claim 22 , wherein the total peak capacity of the system is at least 1,000 peaks. 
     
     
         33 . The liquid chromatography system of  claim 24 , in which the HPLC separation is performed using a microbore column, capillary column, or nanocolumn. 
     
     
         34 . The liquid chromatography system of  claim 22 , wherein the at least one compound-containing fraction is concentrated or diluted prior to introducing the at least one compound-containing fraction into the second chromatographic column. 
     
     
         35 . The liquid chromatography system of  claim 34 , wherein the at least one compound-containing fraction is concentrated by evaporation. 
     
     
         36 . The liquid chromatography system of  claim 34 , wherein the at least one compound-containing fraction is diluted on-line prior to introducing the at least one compound-containing fraction into the second chromatographic column. 
     
     
         37 . The liquid chromatography system of  claim 22 , further comprises a detector for detecting the presence or absence of the at least one analyte in the sample 
     
     
         38 . The liquid chromatography system of  claim 37 , wherein the detector is a mass spectrometer. 
     
     
         39 . The liquid chromatography system of  claim 23 , wherein the first and second chromatographic separation modes are the same. 
     
     
         40 . The liquid chromatography system of  claim 22 , wherein the first and/or second mobile phase is substantially free of non-volatile salts. 
     
     
         41 . The liquid chromatography system of  claim 22 , wherein the first and/or second mobile phase comprises less than about 20 mM of non-volatile salts. 
     
     
         42 . The liquid chromatography system of  claim 22 , wherein a plurality fractions from the effluent stream of the first chromatographic column are collected and introduced into the second chromatographic column.