Buffer kit and method of generating a linear pH gradient

A method of separating at least one analyte from matrix components in a sample with a chromatographic separation device using a gradient eluent flow having a linear pH gradient from a first pH value to a second pH value as a function of time, the method comprising:

• injecting the sample into an injection valve, the injection valve being in fluidic communication with the chromatographic separation device;
• pumping a first eluent solution that has the first pH value into the chromatographic separation device, the first eluent solution comprising:
• a first buffer salt, a second buffer salt, a third buffer salt, and a fourth buffer salt, where at least three of the four buffer salts for the first eluent solution are a monovalent buffer salt, have a net negative charge or a net neutral zwitterionic charge over a pH range of about 6 to about 10, and include a sulfonate group and an amine;
• pumping a second eluent solution that has the second pH value into the chromatographic separation device, the second eluent solution comprising:
• a first buffer salt, a second buffer salt, a third buffer salt, and a fourth buffer salt, where at least three of the four buffer salts for the second eluent solution are a monovalent buffer salt, have a net negative charge or a net neutral zwitterionic charge over a pH range of about 6 to about 10, and include a sulfonate group and an amine,
• for both the first and second eluent solutions, the first buffer salt has a first pKa, the second buffer salt has a second pKa, the third buffer salt has a third pKa, and the fourth buffer salt has a fourth pKa, where the first pKa is the smallest of the four pKa values and the fourth pKa is the largest of the four pKa values, and that the first pKa is about the same as the first pH value and that the fourth pKa is about the same as the second pH value,
• a first difference between the second pKa and the first pKa is less than about 1.5, a second difference between the third pKa and the second pKa is less than about 1.5, and a third difference between the third pKa and the fourth pKa is less than about 1.5;
• varying a proportion of the pumped first eluent solution and the pumped second eluent solution as a function of time;
• generating a linear pH gradient based on a function of time and pH values, that forms an approximately straight line from about the first pH value to the second pH value;
• eluting the sample through the chromatographic separation device;
• separating the analyte from matrix components in the sample;
• and
• detecting the analyte at a detector.

A buffer kit comprising:

• a) a first eluent solution consisting of a first buffer salt, a second buffer salt, a third buffer salt, a fourth buffer salt, and a monovalent non-buffer ionic salt, where the first eluent solution has a first pH of about 6 and a total buffer salt concentration of greater than about 25 millimolar;
• and
• b) a second eluent solution consisting of a first buffer salt, a second buffer salt, a third buffer salt, a fourth buffer salt, and a monovalent non-buffer ionic salt, where the second eluent solution has a second pH of about 10 and a total buffer salt concentration of greater than about 25 millimolar;
• in which each of the buffer salts are a monovalent buffer salt, have a net negative charge or a net neutral zwitterionic charge, and include a sulfonate group and an amine.