Automated capillary liquid chromatography small volume analysis system

• US 20020146349A1
• Filed: 04/04/2002
• Published: 10/10/2002
• Est. Priority Date: 04/05/2001
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Gygi, Steven P., Peng, Junmin, Licklider, Lawrence J.
• Granted Patent: US 6,989,129 B2
• Time in Patent Office: Days
• US Class Current: 422/70
• CPC Class Codes: G01N 2030/085 using absorbing precolumn
• CPC Class Codes: G01N 2030/162 electromigration
• CPC Class Codes: G01N 2030/201 multiport valves, i.e. having more than two ports
• CPC Class Codes: G01N 2030/202 rotary valves
• CPC Class Codes: G01N 2030/285 electrically driven carrier
• CPC Class Codes: G01N 30/08 using an enricher
• CPC Class Codes: G01N 30/16 Injection G01N30/24 takes precedence
• CPC Class Codes: G01N 30/24 Automatic injection systems
• CPC Class Codes: G01N 30/466 with separation columns in parallel
• CPC Class Codes: G01N 30/7266 by electric field, e.g. electrospray
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

A system for automatically performing liquid chromatography analysis of low volume liquid chemical samples at nanosecond flow rates using an analysis column that integrates a pre-concentration trapping column and a chromatography separation column terminating at an electrospray nozzle of an online mass spectrometer. The analysis column consists of a capillary having an inside diameter of between 75 and 125 microns packed throughout with a porous bed of micron particles. A branch outlet positioned 10 to 16 centimeters upstream from the nozzle divides the analysis column into an upstream pre-concentration trap and a downstream separation column. An autosampler delivers low volume liquid samples to the upstream inlet via a two-position valve. Feed connections couple the autosampler to upstream inlet when the valve is open to inject a liquid sample into the pre-concentration trap at a maximum loading flow rate in the range from 0.5 to 50 microliters/minute. Thereafter, when the valve closes, it terminates the further injection the sample, and a concentrated portion of the sample then passes though the chromatography separation column at a much slower flow rate between 10 and 1,000 nanoliters per minute. Throughput can be doubled by coupling two such analysis columns to a single autosampler using a ten-port, two position valve. A single column can be supplied through a six port two-position valve.

Claims

1. An automatic capillary liquid chromatography chemical analysis system comprising, in combination,

a capillary defining a fluid passageway having an inlet end, and outlet end, and an intermediate branch outlet, said capillary being filled with a porous material to form a trapping pre-concentration column between said inlet end and said intermediate branch outlet and a separation column between said branch outlet and said outlet end,

loading flow control means coupled to said inlet end and to said branch outlet for passing a liquid sample through said trapping pre-concentration column,

analysis flow control means for terminating the further introduction of said liquid sample into said pre-concentration column via said inlet end and for thereafter passing a concentrated liquid sample from said pre-concentration column through said separation column at a separation flow rate between 10 and 1,000 nanoliters per minute to deliver chromatography eluent through said outlet end, and

an analyzer positioned at said outlet end for receiving and analyzing said chromatography eluent delivered from said separation column via said outlet end.

2. The system according to claim 1 wherein said loading flow control means limits the maximum loading flow rate of said liquid sample through said trapping pre-concentration column to between 0.5 and 50 microliters/minute.

3. The system according to claim 1 wherein said separation flow rate is between 100 and 300 nanoliters per minute.

4. The system according to claim 2 wherein said separation flow rate is between 100 and 300 nanoliters per minute.

5. The system according to claim 1 wherein said analyzer is a mass spectrometer.

6. The system according to claim 4 wherein said analyzer is a mass spectrometer.

7. The system as set forth in claim 5 including electrical contact means coupled to said capillary and to a voltage source for applying an electrical potential to create an electrospray of said chromatography eluent delivered via said outlet end to said mass spectrometer.

8. The system as set forth in claim 1 wherein said porous material is a bed of C₁₈ bonded phase particles having a 5 micron particle size and a 200 Å

pore size.

9. The system as set forth in claim 5 wherein said porous material is a bed of C₁₈ bonded phase particles having a 5 micron particle size and a 200 Å

pore size.

10. The system as set forth in claim 1 wherein said loading flow control means comprises an automatic sample loader for delivering a succession of liquid samples through said inlet end to said trapping pre-concentration column.

11. The system as set forth in claim 10 wherein said loading flow control means includes a second capillary having an inside diameter between 50 and 500 microns connected to said branch outlet to carry a flow of said liquid sample from said trapping pre-concentration column.

12. The system as set forth in claim 10 further including a frit positioned at said branch outlet to prevent said porous material from passing from said capillary through said branch outlet.

13. The system as set forth in claim 1 wherein said capillary has an inside diameter between 50 and 150 microns.

15. The system according to claim 1 wherein said maximum loading flow rate is between 5 and 50 microliters/minute and said lower flow rate is between 100 and 300 nanoliters per minute.

16. The system as set forth in claim 15 including a voltage source for applying an electrical potential to said downstream outlet produce an electrospray of said chromatography eluent directed to said mass spectrometer.

17. The system as set forth in claim 1 wherein said porous bed of particles comprises a bed of bonded phase particles having a particle size of about 5 microns.

18. The system as set forth in claim 10 wherein said feed connections include a second capillary having an inside diameter between 50 and 500 microns connected to said branch outlet to carry a flow of said liquid sample from said pre-concentration trap.

20. A system as set forth in claim 19 wherein said two position valve is a ten port two position valve.

14. A system for automatically analyzing a plurality of liquid chemical samples comprising, in combination,

a first capillary having an inside diameter of between 50 and 150 microns packed with a porous bed of particles to form an analysis column having an upstream inlet, a downstream outlet, and a branch outlet positioned between said inlet and said outlet at a distance between 10 and 16 centimeters from said outlet, said analysis column forming a pre-concentration trap between said upstream inlet and said branch outlet and forming a chromatography separation column between said branch outlet and said downstream outlet,

a mass spectrometer positioned to receive chromatography eluent from said downstream outlet,

an autosampler for delivering said plurality of liquid samples,

a two-position valve having an open position and a closed position, and

feed connections coupling said autosampler to said upstream inlet via said two-position valve for injecting one of said liquid samples into said pre-concentration trap via said upstream inlet at a maximum loading flow rate in the range from 0.5 to 50 microliters/minute when said two-position valve is in said open position, for thereafter terminating the further injection of said one of said liquid samples via said upstream inlet when said two-position valve moves to said closed position, and for permitting the passage of a concentrated portion of said one of said liquid samples though said chromatography separation column at a lower flow rate between 10 and 1,000 nanoliters per minute to thereby deliver chromatography eluent through said downstream outlet to said mass spectrometer when said two position valve is in said closed position.

19. A system for automatically analyzing a plurality of liquid chemical samples comprising, in combination,

first and second analysis columns each of which comprises capillary having an inside diameter of between 50 and 150 microns packed with a porous bed of particles to form an analysis column having an upstream inlet, a downstream outlet, and a branch outlet positioned between said inlet and said outlet at a distance between 10 and 16 centimeters from said outlet, said analysis column forming a pre-concentration trap between said upstream inlet and said branch outlet and forming a chromatography separation column between said branch outlet and said downstream outlet, and a mass spectrometer positioned to receive chromatography eluent from said downstream outlet of each of said analysis columns

an autosampler for delivering said plurality of liquid samples,

a valve having first and second positions,

a first set of feed connections coupling said autosampler to said upstream inlet of first analysis column via said two-position valve for injecting a second one of said liquid samples into the pre-concentration trap of said first analysis column via said upstream inlet of said first analysis column at a maximum loading flow rate in the range from 0.5 to 50 microliters/minute when said two-position valve is in said first position, for thereafter terminating the further injection of said first one of said liquid samples via said upstream inlet of said first analysis column when said two-position valve moves from said first to said second position, and for permitting the passage of a concentrated portion of said first one of said liquid samples though said chromatography separation column of said first analysis column at a lower flow rate between 10 and 1,000 nanoliters per minute to thereby deliver chromatography eluent derived from said first one of said liquid samples to said mass spectrometer positioned to receive chromatography eluent from said downstream outlet of said first analysis column when said two position valve is in said second position.