Liquid chromatography (LC) is an analytical technique in which a column or tube is packed with a stationary phase material that typically is a finely divided solid or gel. FIG. 1A depicts an LC system 10 in which a source of mobile phase liquid 20 typically contains species to be examined with system 10. The liquid containing species is passed via tubing or the like 30 to a primary pump 40 for input to a column 50 that contains a finely divided solid or gel. Regions within column 50 not occupied by the packing becomes filled with the mobile phase liquid that typically is continuously pumped through the column. A porous plug or filter (not shown) at the lower end of the column in FIG. 1A supports the packing. Those skilled in the art will recognize that in some applications, a pre-column flow splitting configuration is used, indicated in phantom as element 55.
Eluent fluid exiting the column through the porous plug passes through tubing 60 (or the like) and is input or otherwise subjected to a detector 70, which seeks to detect species present in the liquid pumped through the column. As shown in FIG. 1B, concentration of different species (e.g., species A, species B) within the eluent fluid typically will peak at different times, as some species can pass through the column and through the plug more rapidly than other species. Thus in FIG. 1B, detector 70 notes a peak in concentration for species A at time t1, and notes a peak (here a smaller amplitude peak) in concentration for species B at time t2. The earlier peak for species A in FIG. 1B could result from many factors, including a relatively rapid mobility for species A, a smaller molecular size, etc. Typically the liquid and species 80 that has been analyzed with detector 70 is collected in a container 80 and is discarded.
One shortcoming with many LC systems such as system 10 is that it can be difficult for detector 70 to adequately sense when the various species within liquid 20 pass through the system. While FIG. 1B depicts two rather definitive concentration peaks, in practice the various peaks may be difficult to discern. The various peaks may exhibit very close concentration peaks and/or occur very close in time.
In many LC analysis systems, liquid 20 may be a biological sample that is difficult to obtain and that is analyzed with a so-called nano-sized capillary column. Understandably it is desired that such valuable liquid samples not be discarded until all possible information as to species within can be obtained. However conventional LC systems such as shown in FIG. 1A are somewhat limited in the analysis that can be carried out with conventional detectors. Where the fluid under analysis includes cellular extracts and/or biological fluids, numerous amounts of proteins will be present. In applications such as proteomics, it is desired to identify these proteins, for example to help elucidate complex medical and biological problems. Identification of proteins that are lacking or over-expressed in abnormal cells is of particular interest.
A general strategy for the identification of proteins is to perform a digestion with an enzyme, e.g. trypsin, and then measure the peptide masses using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). In applications where this method does not allow protein identification, the peptides must be sequenced using fragmentation mass spectrometry. The measured amino acid sequence of a peptide is then compared with theoretical sequences from protein databases. Mass spectrometric (MS) detection requires that compounds be ionized and in gas phase. Electrospray ionization (ESI) is a commonly applied technique when dealing with biomolecules such as peptides and proteins. In ESI, a potential is applied to the outlet of a conducting needle to spray sample and solvent within. The solvent droplets disintegrate and the solvent evaporates. In so-called nanospray, the sample is sprayed from a needle with a tip diameter less than about 5 μm, using a sample flow rate between 5 nl/min. and 50 nl/min. Nanospray MS is used for the analysis of low fmol amounts of peptides in small sample volumes. However, most samples are too complex for direct nanospray MS analysis and require some physical separation.
High performance liquid chromatography (HPLC) can be used for high resolution peptide separation, and may readily be used on-line with ESI-MS analysis. In addition, HPLC on-line connected to MS offers the possibility for pre-concentration of dilute samples, desalting and removal of detergents. Available sample amounts in proteomic research are often limited and relatively precious. Therefore it can be very important to carry out analysis using a minimal amount of biological molecules.
In many applications, and especially where relatively small volumes of sample are under analysis, improving detection sensitivity can become especially important. Improvement of detection sensitivity using concentration sensitive detectors such as UV/Vis absorbance and ESI mass spectrometers can be achieved by employing HPLC columns with smaller internal diameters (I.D.). For example, increased sensitivity during peptide analysis can result from using nano- and capillary LC with column I.D.s of 50 μm and 300 μm, respectively. Flow rate of the mobile phase through such columns is from several nl/min, to several μl/min, and the mobile phase can be sprayed directly without splitting.
For maximum LC separation resolution, peak widths should be minimal, but narrow peaks limit the acquisition time for mass spectral data. Preferably, an analytical system should enable separation of all compounds within an HPLC system (narrow peaks and high peak capacity), while enabling essentially unlimited time to acquire mass spectrometric data for each separated compound. For example, peak width at half height of compounds eluting from capillary- or nano-LC columns is generally in the range of 5 seconds to 30 seconds. During this period, the peptide must be sequenced, which requires multiple stages of MS detection. The peptide must be identified, isolated, and dissociated to produce fragment ions. Sequencing of the peptides is performed by comparing measured and calculated fragment spectra. These scan cycles take approximately a few seconds, which allows the experiment to perform only limited cycles during the elution of a chromatographic peak.
Reducing the flow rate during an HPLC experiment can increase the elution time window. Preferably, a reduction of the mobile phase flow rate should instantaneously be effective when the mass spectrometer detects a compound, for example as shown by Lee et al., “A Microscale Electrospray Interface for On-Line, Capillary Liquid Chromatography Tandem Mass Spectrometry of Complex Peptide Mixtures”, Anal. Chem. 1995, 67, 4549-4556. Lee disclosed storing a preformed gradient in a loop, and using a programmable syringe pump to slow the mobile phase flow rate. To compensate the delay time before the low flow rate was achieved, the direction of the pump was reversed to try to quickly lower the column head pressure. While this experimental system seemed to work for Lee et al., it is typically not possible to reverse the flow rate with most widely used HPLC pumps, which are reciprocating pumps. Further, flow reversal in a Lee type system that decreases the column inlet pressure can result in severe damage to the column bed and can reduce column lifetime. Further, such flow reversal can cause severe band broadening of the components that are still in the separation column.
U.S. Pat. No. 6,139,734 to Settlage et al. describes a system to perform variable flow rate chromatography using two different lengths of fused silica capillaries that functioned as restrictors. However in practice, one cannot easily control or adjust flow rate through a column, especially if a restrictor becomes partially clogged. Further, Settlage's system splits the injected sample, a procedure that is not acceptable for sample limited applications in proteomics. An additional drawback of a Settlage type system is a loss of separation efficiency due to flow reduction. In practice, a certain time is required to actually reduce the flow rate through Settlage's column due to the relaxation of the column pressure. Unfortunately, during this time a significant part of an eluting peak could already have been detected by the MS, thereby minimizing the advantage of flow reduction on sensitivity.
Thus there is a need for an improved LC analysis system that provides better resolution or granularity of detection information as to species within a liquid sample under examination. Further, such system should provide additional analytical tools to extract as much information as possible from the sample before it is discarded, preferably by broadening elution peaks without loss of chromatographic resolution, while enabling good control of the eluent flow rate. In biotechnology applications, not only such enhanced and further data be provided, but such data should be provided while using relatively small amounts of sample.
The present invention provides such a system and a method for implementing same.
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