JP2005241637A - Mass spectrometer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve a technique for reducing the velocity in identifying a chemical species by a mass spectrometer (variable flow rate chromatography), whereas, in a combined use of liquid chromatography and electronic spray ionization mass spectrometer (LCMS), the quality of data of a low-abundance chemical species has a limitation by its short actual resident time in an ion source, and the length of this time is determined by a peak elution profile. <P>SOLUTION: In a peak parking mode of operation, solvent from A and B of solvent pumps 9 and 10 is immediately diverted to waste to reduce the back pressure on an analytical column 21. An analyte of interest is then allowed to be released from the column 21 at a slower rate by passing a fluid from a separate pump 1 through the column 21. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a liquid chromatography system, a mass spectrometer, a liquid chromatography method, and a mass spectrometry method.

  Liquid chromatography is a method by which various species derived from complex mixtures can be separated into their individual components. The individual species or components elute from liquid chromatography at substantially different times.

  Known liquid chromatography systems include high performance liquid chromatography (HPLC) systems that incorporate a pumping system that includes two solvent channels A and B. Traditionally, solvent channel A comprises an aqueous solvent or solution (eg, HPLC grade water containing 0.1% acid), and solvent channel B is an organic solvent (eg, acetonitrile containing 0.1% acid or Methanol). The aqueous solvent or solution A and the organic solvent B are mixed to provide an isocratic flow. Subsequently, a sample or an analyte to be analyzed is introduced into the mixed solvent flow. The sample may be introduced manually into the mixed solvent flow or may be introduced by an autosampler.

  The sample or analyte combined with the solvent mixture is then passed through an analytical column. The analytical column is typically packed with a stationary phase (eg, 5 μm silicon beads). Initially, the composition of the mixed solvent is set to contain most of the aqueous solvent or solution from solvent channel A. However, the ratio of the organic solvent B to the aqueous solvent or solution A gradually increases with a linear property over a certain time. The components in the liquid initially trapped in the analytical column become movable again as the gradient of the organic solvent increases, i.e. as the proportion of organic solvent B in the solvent mixture increases. start. For example, the relative ratio of flow rates derived from the two solvent channels may vary linearly, so that, for example, the concentration of the solvent mixture initially containing 1% or less of organic solvent is set to a certain time, for example, It may be progressively increased after 60 minutes, at which time the solvent mixture may contain 60% organic solvent B. As the relative composition in the mixture of the two solvents A and B changes, different species begin to be released from the stationary phase of the column and are subsequently detected by various means located at the outlet of the analytical column.

  The inner or inner diameter of analytical columns used in liquid chromatography applications can vary to a considerable extent. For example, the inner or inner diameter of the analytical column may be less than 50 μm in some applications, and in other applications the inner or inner diameter may be greater than 4.6 mm. The transport flow rate required for the pumping system increases as the inner or internal diameter of the analytical column increases, and the transport flow rate varies, for example, from a few nanoliters / minute to several milliliters / minute. Also good.

  If the transport stream passes directly through the analytical column and then passes through an analytical instrument (eg a mass spectrometer) without being split, it is common to use a direct flow arrangement. However, there are circumstances where direct flow arrangement is not suitable.

  In order to provide an accurate gradient at low flow rates (eg, a few nanoliters / minute), it is often necessary to split the transport stream from the liquid chromatograph before the analytical instrument. For example, there are two relatively common situations where the transport flow may need to be split. The first situation is when a large diameter HPLC analytical column is used. Traditional standard large diameter HPLC analytical columns have an internal diameter of 4.6 mm. Columns with an internal diameter of 4.6 mm are industry standard, reliable and rugged. Such a column can be further processed in large quantities, and can be used effectively in purification steps such as fraction collection. However, such large diameter columns generally require relatively high flow rates of a few milliliters / minute. Providing such a flow rate to the analytical column is not a problem, for example, as a flow rate directly processed by an electrospray ionization ion source that may be arranged to receive and ionize a stream eluting from the column. The flow rate of several milliliters / minute may be too high. A relatively high flow rate may be particularly unsuitable for electrospray ionized ion sources, especially if the solvent mixture used to push the sample through the analytical column contains a relatively high percentage or proportion of water. . Therefore, it is subsequently necessary to split the stream upstream or downstream of the HPLC column, after which only a portion of the stream passes directly through the electrospray ionization ion source. The remainder of the stream may simply be discarded, or alternatively, certain components that are important in a process known as fraction collection may be collected in a vial.

  As a second situation, when a nano-flow HPLC system is used, it may be necessary to divide the transport flow. Nanoflow HPLC systems generally use a small internal diameter column, which typically has an internal diameter of less than 360 μm. Nanoflow HPLC systems, therefore, due to their nature typically have a relatively low flow rate in the range of 100 nl / min to 1000 nl / min, ie 3 than the typical flow rate used with a 4.6 mm internal diameter column. Operate at a flow rate of ~ 4 grades lower. For example, if only a very small sample can be prepared, a column having a small internal diameter may be used. For example, the nanoflow HPLC system can be used to analyze samples of less than 100 femtomole in protein digests extracted from human cells. However, HPLC pumps are relatively poor in providing accurate, stable, and reproducible solvent gradients at such relatively low flow rates. Therefore, the pump from the solvent channels A, B is operated at a relatively high flow rate, but after that, the transport flow is divided in front of the nanoflow column, and only the flow rate extremely lower than the transport fluid is reduced to the nanoflow HPLC column. It is known to pass through.

Electrospray ionization is a technique commonly used in mass spectrometry, and species present in a flowing solution are ionized by applying a high voltage to the electrospray probe.
Electrospray ionization is sometimes referred to as a mild ionization technique. This is because the resulting ions generated from the ion source typically contain relatively large molecular weight species (eg, peptides) that can then be detected as intact ions by a mass analyzer. Electrospray ionization can be achieved at a number of different flow rates in the range of several nl / min (ie nanoflow rate) to several ml / min.

  The number of ions observed during electrospray ionization on a mass spectrometer is not flow rate dependent, at least in the first approximation, and therefore at the same signal to noise ratio at relatively low flow rates due to very low sample consumption. Suitable high sensitivity can be achieved.

  Liquid chromatography systems used with electrospray ionization ion source mass spectrometer (LCMS) or tandem mass spectrometer (LCMS / MS) represent powerful analytical instruments widely used in many laboratories around the world . However, when a liquid chromatography system is used in combination with a mass spectrometer, the limit of data quality that can be achieved with low abundance species is that any particular analyte species is actually present in the electrospray ionization ion source. The time is relatively short. This further results in the limited number of different MS / MS product ion mass spectra that can be performed and recorded by the length of time any species of the parent ion is present in the ion source. Bring. This length of time is determined by the peak elution profile of the particular column used.

  In order to effectively extend the peak elution time, attempts have been made to reduce the flow rate when important species are identified by a mass spectrometer. This technique is known as peak parking or variable flow chromatography. Decreasing the flow rate, at least theoretically, allows important species to be present in the ion source for a longer time without losing the number of ions per scan.

  US Pat. No. 6,139,734 (US-6139734) describes a variable flow chromatography method in which the flow rate varies based on different restrictor split ratios. The method described here relies on the use of two different transport diversion ratios to determine one standard flow rate and one decreasing flow rate. However, this approach has the disadvantage that pressure balancing is not immediate. Furthermore, the restrictor may be clogged, causing a change in flow rate. Yet another problem with the disclosed variable flow approach is that the narrow peak elution time, e.g., less than 0.5 minutes for a column with an internal diameter of 75 μm, the elution is before the reduced flow rate is actually fully established. The analyte corresponding to the peak may already have passed completely through the ion source.

  US 2002/0072126 (US 2002/0072126) describes another approach. Here, the post column in which the valve is placed is switched and the species (chemical species) are eluted into the mass spectrometer at a low flow rate using a syringe pump. The post column valve switches when an important species is detected. During the park, stop the gradient transport pump flow rate and block the output from the column. The syringe pump then continues to elute the species into the ion source at a reduced flow rate. However, the use of a post-column valve leads to the introduction of dead volume, which is detrimental to both chromatographic performance and chromatographic separation. The known methods using post-column valves to enable variable flow chromatography are therefore particularly disadvantageous.

  Accordingly, there is a need to provide an improved liquid chromatography system that has disadvantages due to some or all of the problems associated with known liquid chromatography employing variable flow rates. Preferably not.

According to the present invention,
The first column,
A first fluid transport system for transporting a first fluid to the first column; and
A liquid chromatography system comprising a second fluid transport system for transporting a second different fluid to the first column;
In a first mode of operation, the first fluid transport system passes the first fluid through the first column at a first flow rate; and
In a second mode of operation, the first fluid is substantially diverted from the first column and the second fluid transport system diverts the second different fluid to a second different flow rate. Pass through the first column,
A liquid chromatography system is provided.

  Preferably, the first column includes a high performance liquid chromatography (HPLC) column. According to a less preferred embodiment, the column may comprise a normal phase column. (I) <50 μm; (ii) 50-100 μm; (iii) 100-200 μm; (iv) 200-300 μm; (v) 300-400 μm; (vi) 400- 500 μm; (vii) 500-600 μm; (viii) 600-700 μm; (ix) 700-800 μm; (x) 800-900 μm; (xi) 900-1000 μm; (xii) 1.0-1.5 mm (xiii) 1.5-2.0 mm; (xiv) 2.0-2.5 mm; (xv) 2.5-3.0 mm; (xvi) 3.0-3.5 mm; (xvii) 3.5-4.0 mm; (xviii) 4.0-4.5 mm; ( xix) 4.5-5.0 mm; (xx) 5.0-5.5 mm; (xxi) 5.5-6.0 mm; (xxii) 6.0-6.5 mm; (xxiii) 6.5-7.0 mm; (xxiv) 7.0-7.5 mm; (xxv) 7.5-8.0 mm; (xxvi) 8.0-8.5 mm; (xxvii) 8.5-9.0 mm; (xxviii) 9.0-9.5 mm; (xxix) 9.5-10.0 mm; and (xxx)> 10.0 mm It may have an internal diameter.

  (I) <10 mm; (ii) 10-20 mm; (iii) 20-30 mm; (iv) 30-40 mm; (v) 40-50 mm; (vi) 50- 60 mm; (vii) 60-70 mm; (viii) 70-80 mm; (ix) 80-90 mm; (x) 90-100 mm; (xi) 100-110 mm; (xii) 110-120 mm (xiii) 120-130 mm; (xiv) 130-140 mm; (xv) 140-150 mm; (xvi) 150-160 mm; (xvii) 160-170 mm; (xviii) 170-180 mm; ( (xxix) 180-190 mm; (xx) 190-200 mm; (xxi) 200-210 mm; (xxii) 210-220 mm; (xxiii) 220-230 mm; (xxiv) 230-240 mm; (xxv) 240-250 mm; (xxvi) 250-260 mm; (xxvii) 260-270 mm; (xxviii) 270-280 mm; (xxix) 280-290 mm; (xxx) 290-300 mm; and (xxxi)> Preferably it has a length selected from the group consisting of 300 mm.

  The first column preferably comprises a C4, C8 or C18 stationary phase.

  (I) <1 μm; (ii) 1-2 μm; (iii) 2-3 μm; (iv) 3-4 μm; (v) 4-5 μm; (vi) 5- 6 μm; (vii) 6-7 μm; (viii) 7-8 μm; (ix) 8-9 μm; (x) 9-10 μm; (xi) 10-15 μm; (xii) 15-20 μm (xiii) 20-25 μm; (xiv) 25-30 μm; (xv) 30-35 μm; (xvi) 35-40 μm; (xvii) 40-45 μm; (xviii) 45-50 μm; ( xix) preferably comprising particles having a size selected from the group consisting of> 50 μm.

  The first column comprises: (i) <100 angstroms; (ii) 100-200 angstroms; (iii) 200-300 angstroms; (iv) 300-400 angstroms; (v) 400-500 angstroms; (vi) 500- (Vii) 600-700 angstrom; (viii) 700-800 angstrom; (ix) 800-900 angstrom; (x) 900-1000 angstrom; and (xi)> 1000 angstrom It is preferable that the particle | grains which have are included.

  Preferably, the first fluid transport system includes one, two, or more than two pumps. The pump may include one or more piston pumps, syringe pumps, or peristaltic pumps.

  The first fluid transport system (ie, solvent channel A) preferably includes an aqueous solvent or solution transport device. The aqueous solvent or solution transport device A preferably distributes the aqueous solvent or solution A at the time of use. The aqueous solvent or solution A preferably contains HPLC grade water, which may optionally contain a small amount of acid, for example 1% formic acid. Preferably, the first fluid transport system includes an organic solvent transport device (ie, solvent channel B). The organic solvent transport device B preferably distributes the organic solvent B at the time of use. The organic solvent B preferably contains an alcohol such as methanol or propanol, or acetonitrile or tetrahydrofuran (THF).

  The streams from the aqueous solvent or solution transport device A and the organic solvent transport device B are preferably mixed at the time of use, so that the flow of isocratic fluid (e.g., solvent A, B) is the first ( Analytical) column is preferably provided.

  Preferably, the second fluid transport system includes one, two, or more than two pumps. Preferably, the pump comprises one or more piston pumps, syringe pumps or peristaltic pumps.

  The second fluid transport system B-C preferably includes a sample transport device. The second fluid transport system C preferably provides an isocratic fluid flow to the first column in use. Said second fluid transport system C preferably provides an aqueous solvent or solution, said aqueous solvent or solution preferably comprising HPLC grade water, said HPLC grade water optionally comprising a small amount of acid (eg 1% Of formic acid).

  (I) <10 nl / min; (ii) 10-20 nl / min; (iii) 20-30 nl / min; (iv) 30-40 nl / min; (v) 40- (Vi) 50-60 nl / min; (vii) 60-70 nl / min; (viii) 70-80 nl / min; (ix) 80-90 nl / min; (x) 90- (Xi) 100-200 nl / min; (xii) 200-300 nl / min; (xiii) 300-400 nl / min; (xiv) 400-500 nl / min; (xv) 500- 600 nl / min; (xvi) 600-700 nl / min; (xvii) 700-800 nl / min; (xviii) 800-900 nl / min; (xix) 900-1000 nl / min; (xx) 1- 100 μl / min; (xxi) 100-200 μl / min; (xxii) 200-300 μl / min; (xxiii) 300-400 μl / min; (xxiv) 400-500 μl / min; (xxv) 500- 600 μl / min; (xxvi) 600-700 μl / min; (xxvii) 700-800 μl / min; (xxviii) 800-900 μl / min; (xxix) 900-1000 μl / min; (xxx) 1.0- 2.0 ml / min; (xxxi) 2.0-3.0 ml / min; (xxxii) 3.0-4.0 ml / min; (xxxiii) 4.0-5.0 ml / min; (xxxiv) 5.0-6.0 ml / min; (xxxv) 6.0- 7.0 ml / min; (xxxvi) 7.0-8.0 ml / min; (xxxvii) 8.0-9.0 ml / min; (xxxviii) 9.0-10.0 ml / min; and (xxxix)> 10.0 ml / min It is preferable.

  The second flow rate is: (i) <10 nl / min; (ii) 10-20 nl / min; (iii) 20-30 nl / min; (iv) 30-40 nl / min; (v) 40- (Vi) 50-60 nl / min; (vii) 60-70 nl / min; (viii) 70-80 nl / min; (ix) 80-90 nl / min; (x) 90- (Xi) 100-200 nl / min; (xii) 200-300 nl / min; (xiii) 300-400 nl / min; (xiv) 400-500 nl / min; (xv) 500- 600 nl / min; (xvi) 600-700 nl / min; (xvii) 700-800 nl / min; (xviii) 800-900 nl / min; (xix) 900-1000 nl / min; (xx) 1- 100 μl / min; (xxi) 100-200 μl / min; (xxii) 200-300 μl / min; (xxiii) 300-400 μl / min; (xxiv) 400-500 μl / min; (xxv) 500- 600 μl / min; (xxvi) 600-700 μl / min; (xxvii) 700-800 μl / min; (xxviii) 800-900 μl / min; (xxix) 900-1000 μl / min; (xxx) 1.0- 2.0 ml / min; (xxxi) 2.0-3.0 ml / min; (xxxii) 3.0-4.0 ml / min; (xxxiii) 4.0-5.0 ml / min; (xxxiv) 5.0-6.0 ml / min; (xxxv) 6.0- 7.0 ml / min; (xxxvi) 7.0-8.0 ml / min; (xxxvii) 8.0-9.0 ml / min; (xxxviii) 9.0-10.0 ml / min; and (xxxix)> 10.0 ml / min It is preferable.

  The second flow rate is preferably substantially lower than the first flow rate.

  The ratio of the second flow rate to the first flow rate is: (i) <1; (ii) 0.1-1; (iii) 0.01-0.1; (iv) 0.001-0.01; (v) 0.0001-0.001; (vi) Preferably selected from the group consisting of 0.00001-0.0001; (vii) 0.000001-0.00001; (viii) 0.0000001-0.000001; (ix) <0.0000001. The second flow rate in the second mode of operation is preferably non-zero or substantially non-zero.

  The liquid chromatography system determines, analyzes, measures, detects, and predicts that, in use, one or more important analytes or one or more components have emerged, eluted or transmitted from the first column. Alternatively, when evaluating, it is preferable to switch from the first mode of operation to the second mode of operation.

  Preferably, the liquid chromatography system switches from the second mode of operation to the first mode of operation after a predetermined time in use.

  The predetermined time is: (i) <1 second; (ii) 1-10 seconds; (iii) 10-20 seconds; (iv) 20-30 seconds; (v) 30-40 seconds; (vi) (Vii) 50-60 seconds; (viii) 60-70 seconds; (ix) 70-80 seconds; (x) 80-90 seconds; (xi) 90-100 seconds; (xii) 100- 110 seconds; (xiii) 110-120 seconds; (xiv) 120-130 seconds; (xv) 130-140 seconds; (xvi) 140-150 seconds; (xvii) 150-160 seconds; (xviii) 160-170 seconds ; (xix) 170-180 seconds; (xx) 180-190 seconds; (xxi) 190-200 seconds; (xxii) 200-210 seconds; (xxiii) 210-220 seconds; (xxiv) 220-230 seconds; ( (xxv) 230-240 seconds; (xxvi) 240-250 seconds; (xxvii) 250-260 seconds; (xxviii) 260-270 seconds; (xxix) 270-280 seconds; (xxx) 280-290 seconds; (xxxi) Preferably selected from the group consisting of 290-300 seconds; and (xxxii)> 300 seconds.

In use, the liquid chromatography system preferably switches from the first mode of operation to the second mode of operation at time t ₁ , where t ₁ is (i) ≦ 10 seconds; (ii) ≦ 9 seconds (iii) ≤ 8 seconds; (iv) ≤ 7 seconds; (v) ≤ 6 seconds; (vi) ≤ 5 seconds; (vii) ≤ 4 seconds; (viii) ≤ 3 seconds; (ix) ≤ 2 seconds; (x) ≤ 1 second; (xi) ≤ 0.75 seconds; (xii) ≤ 0.5 seconds; (xiii) ≤ 0.25 seconds; (xiv) ≤ 0.1 seconds; and (xv) substantially instantaneous, The

  In the second mode of operation, the fluid dispensed from the first fluid transport systems A, B is preferably diverted from the first (analytical) column and discarded.

  In the second mode of operation, at least 50%, 60%, 70%, 80%, 90%, 95% of the first fluids A, B distributed from the first fluid transport systems A, B, Preferably 99% or 99.9% is not substantially transferred to the first (analytical) column.

  In the second mode of operation, substantially 100% of the fluid dispensed from the first fluid transport devices A, B is diverted from the first (analysis) column, or substantially the first (Analysis) It is preferable not to transmit to the column.

When switching to the second mode of operation from the first mode of the liquid chromatography system operations, or column head pressure for the first (analytical) column is substantially reduced at time t _2, or removed T ₂ is preferably (i) ≤ 10 seconds; (ii) ≤ 9 seconds; (iii) ≤ 8 seconds; (iv) ≤ 7 seconds; (v) ≤ 6 seconds; (vi) ≤ 5 (Vii) ≤ 4 seconds; (viii) ≤ 3 seconds; (ix) ≤ 2 seconds; (x) ≤ 1 second; (xi) ≤ 0.75 seconds; (xii) ≤ 0.5 seconds; (xiii) ≤ 0.25 seconds (xiv) ≦ 0.1 seconds; and (xv) substantially instantaneous, selected from the group consisting of:

  In the first mode of operation, the fluid dispensed by the first fluid transport system A, B causes the analyte to pass from a second column (eg, pre-column) to the first (analytical) column. Is preferred.

  In the first mode of operation, the first fluid transport system A, B serves to keep the flow of fluid (eg, solvent) through the first (analytical) column substantially constant or regular. Is preferred.

  In the first mode of operation, the first fluid (eg, solvent) passes through the first (analytical) column at a flow rate of x ml / min, and in the second mode of operation, the first fluid Preferably, the fluid passes through the first column at a flow rate of y ml / min. y is (i) ≤ 0.2 x; (ii) 0.15-0.20 x; (iii) 0.10-0.15 x; (iv) 0.05-0.10 x; (v) 0.01-0.05 x; (vi) ≤ 0.01 x; ( vii) substantially zero; and (viii) preferably selected from the group consisting of 0.

  An analyte with a specific mass to charge ratio is in said first mode of operation: (i) <1 second; (ii) 1-2 seconds; (iii) 2-3 seconds; (iv) 3-4 seconds (v) 4-5 seconds; (vi) 5-6 seconds; (vii) 6-7 seconds; (viii) 7-8 seconds; (ix) 8-9 seconds; (x) 9-10 seconds; ( xi) 10-15 seconds; (xii) 15-20 seconds; (xiii) 20-25 seconds; (xiv) 25-30 seconds; (xv) 30-35 seconds; (xvi) 35-40 seconds; (xvii) It is preferred to have a peak elution time selected from the group consisting of 40-45 seconds; (xviii) 45-50 seconds; and (xix)> 50 seconds.

  An analyte with a specific mass to charge ratio is in said second mode of operation: (i) <1 second; (ii) 1-10 seconds; (iii) 10-20 seconds; (iv) 20-30 seconds (v) 30-40 seconds; (vi) 40-50 seconds; (vii) 50-60 seconds; (viii) 60-70 seconds; (ix) 70-80 seconds; (x) 80-90 seconds; ( xi) 90-100 seconds; (xii) 100-110 seconds; (xiii) 110-120 seconds; (xiv) 120-130 seconds; (xv) 130-140 seconds; (xvi) 140-150 seconds; (xvii) 150-160 seconds; (xviii) 160-170 seconds; (xix) 170-180 seconds; (xx) 180-190 seconds; (xxi) 190-200 seconds; (xxii) 200-210 seconds; (xxiii) 210- 220 seconds; (xxiv) 220-230 seconds; (xxv) 230-240 seconds; (xxvi) 240-250 seconds; (xxvii) 250-260 seconds; (xxviii) 260-270 seconds; (xxix) 270-280 seconds preferably having a peak elution time selected from the group consisting of: (xxx) 280-290 seconds; (xxxi) 290-300 seconds; and (xxxii)> 300 seconds.

  The analyte having the specific mass to charge ratio is: (i) <100; (ii) 100-200; (iii) 200-300; (iv) 300-400; (v) 400-500; (vi ) 500-600; (vii) 600-700; (viii) 700-800; (ix) 800-900; (x) 900-1000; (xi) 1000-1100; (xii) 1100-1200; (xiii) 1200-1300; (xiv) 1300-1400; (xv) 1400-1500; (xvi) 1500-1600; (xvii) 1600-1700; (xviii) 1700-1800; (xix) 1800-1900; (xx) 1900 Preferably having a mass to charge ratio selected from the group consisting of: -2000; and (xxi)> 2000.

  In the third (preloading) mode of operation, a sample mixture containing the analyte is preferably dispensed from the second fluid transport device C.

  In the third mode of operation, the analyte is stored in the second column (ie, pre-column), stored by the second column, or otherwise held by the second column, or Preferably, it is retained in the second column.

  The second column (ie, pre-column) preferably includes a high performance liquid chromatography (HPLC) column. Although not preferred, the second column (ie, pre-column) may include a normal phase column.

  The second column (ie, pre-column) is (i) <50 μm; (ii) 50-100 μm; (iii) 100-200 μm; (iv) 200-300 μm; (v) 300-400 μm; (vi) 400-500 μm; (vii) 500-600 μm; (viii) 600-700 μm; (ix) 700-800 μm; (x) 800-900 μm; (xi) 900-1000 μm; (xii ) 1.0-1.5 mm; (xiii) 1.5-2.0 mm; (xiv) 2.0-2.5 mm; (xv) 2.5-3.0 mm; (xvi) 3.0-3.5 mm; (xvii) 3.5-4.0 mm; (xviii) 4.0 -4.5 mm; (xix) 4.5-5.0 mm; (xx) 5.0-5.5 mm; (xxi) 5.5-6.0 mm; (xxii) 6.0-6.5 mm; (xxiii) 6.5-7.0 mm; (xxiv) 7.0-7.5 mm; (xxv) 7.5-8.0 mm; (xxvi) 8.0-8.5 mm; (xxvii) 8.5-9.0 mm; (xxviii) 9.0-9.5 mm; (xxix) 9.5-10.0 mm; and (xxx)> 10.0 mm Preferably it has an internal diameter selected from the group consisting of:

  The second column (precolumn) is (i) <10 mm; (ii) 10-20 mm; (iii) 20-30 mm; (iv) 30-40 mm; (v) 40-50 mm; (vi ) 50-60 mm; (vii) 60-70 mm; (viii) 70-80 mm; (ix) 80-90 mm; (x) 90-100 mm; (xi) 100-110 mm; (xii) 110 -120 mm; (xiii) 120-130 mm; (xiv) 130-140 mm; (xv) 140-150 mm; (xvi) 150-160 mm; (xvii) 160-170 mm; (xviii) 170-180 (xix) 180-190 mm; (xx) 190-200 mm; (xxi) 200-210 mm; (xxii) 210-220 mm; (xxiii) 220-230 mm; (xxiv) 230-240 mm; (xxv) 240-250 mm; (xxvi) 250-260 mm; (xxvii) 260-270 mm; (xxviii) 270-280 mm; (xxix) 280-290 mm; (xxx) 290-300 mm; and ( xxxi) preferably has a length selected from the group consisting of> 300 mm.

  The second column preferably contains a C4, C8 or C18 stationary phase.

  (I) <1 μm; (ii) 1-2 μm; (iii) 2-3 μm; (iv) 3-4 μm; (v) 4-5 μm; (vi) 5- 6 μm; (vii) 6-7 μm; (viii) 7-8 μm; (ix) 8-9 μm; (x) 9-10 μm; (xi) 10-15 μm; (xii) 15-20 μm (xiii) 20-25 μm; (xiv) 25-30 μm; (xv) 30-35 μm; (xvi) 35-40 μm; (xvii) 40-45 μm; (xviii) 45-50 μm; ( xix) preferably comprising particles having a size selected from the group consisting of> 50 μm.

  The second column comprises (i) <100 angstroms; (ii) 100-200 angstroms; (iii) 200-300 angstroms; (iv) 300-400 angstroms; (v) 400-500 angstroms; (vi) 500- (Vii) 600-700 angstrom; (viii) 700-800 angstrom; (ix) 800-900 angstrom; (x) 900-1000 angstrom; and (xi)> 1000 angstrom It is preferable that the particle | grains which have are included.

  In the third (preloading) mode of operation, salt and / or other contaminants are at least partially or substantially removed from the sample mixture and exit the second column (ie, precolumn). preferable.

  In the third mode of operation, the relative concentration of the analyte in the sample mixture is stored in the second column, stored by the second column, or otherwise retained by the second column. Preferably increased substantially while being retained in the second column.

  Preferably, the liquid chromatography system switches to the first mode of operation after the third mode of operation.

  According to another aspect of the present invention, an analytical instrument including the above-described liquid chromatography system is provided.

  The analytical instrument comprises: (i) an ultraviolet (UV) detector; (ii) an ultraviolet (UV) array detector; (iii) an infrared (IR) detector; (iv) an ion mobility separator; (v) an ion mobility. (Vi) Visible ultraviolet (UV) detector; (vii) Nuclear Magnetic Resonance (NMR) detector; (viii) Electrospray Light Scattering Detector (ELSD); (ix ) Further liquid chromatography system (LC-LC); (x) Refractive index (RI) detector; (xi) Visible light detector; (xii) Chemiluminescence detector; and (xiii) Fluorescence detector Preferably it is selected.

  According to another aspect of the present invention, a mass spectrometer including the above-described liquid chromatography system is provided.

  Preferably, the mass spectrometer further includes an ion source connected to the first column.

  The ion source includes: (i) Electrospray (ESI) ion source; (ii) Atmospheric Pressure Chemical Ionisation (“APCI”) ion source; (iii) Atmospheric Pressure ionization (Atmospheric Pressure ionization) Photo ionization ("APPI") ion source; (iv) Laser desorption ionization ("LDI") ion source; (v) Inductively Coupled Plasma ("ICP") ion source; (vi ) Electron Impact (“EI”) ion source; (vii) Chemical Ionisation (“CI”) ion source; (viii) Field Ionisation (“FI”) ion source; (ix) Fast Atomic collision (Fast Atom Bombardment, "FAB") ion source; (x) Liquid Secondary Ion Mass Spectrometry ("LSIMS") ion source; (xi) Atmospheric Pressure Ionisation, " API ") ion source; (xii) Field Desorption," FD " (Xiii) Matrix Assisted Laser Desorption Ionisation ("MALDI") ion source; (xiv) Desorption / Ionisation on Silicon ("DIOS") ion source preferably selected from the group consisting of: (xv) a desorption electrospray ionization ("DESI") ion source; and (xvi) a nickel-63 radioactive ion source.

  The ion source preferably includes a continuous ion source or a pulsed ion source.

  The liquid chromatography system transitions from the first mode of operation to the second mode of operation in determining whether important analyte ions have been eluted from or released from the ion source. It is preferable to switch.

  The mass spectrometer preferably further includes a mass analyzer. The mass analyzer comprises: (i) an orthogonal acceleration time-of-flight mass analyzer; (ii) an axial acceleration time-of-flight mass analyzer; (iii) a quadrupole mass analyzer; (iv) a Penning mass analyzer; v) Fourier Transform Ion Cyclotron Resonance ("FTICR") mass spectrometer; (vi) 2D or linear quadrupole ion trap; (vii) Paul or 3D quadrupole ion trap; and ( viii) Preferably selected from the group consisting of a magnetic sector mass analyzer.

According to another aspect of the invention,
Providing a first column, a first fluid transport system for transporting a first fluid to the first column, and a second fluid transport system for transporting a second different fluid to the first column;
Passing the fluid through the first column at a first flow rate by the first fluid transport system; and subsequently
Substantially diverting the first fluid from the first column and passing the second different fluid through the first column at a second different flow rate by the second fluid transport system;
A liquid chromatography method is provided.

  According to another aspect of the present invention, a mass spectrometry method including the above-described liquid chromatography method is provided.

  Said preferred embodiment advantageously allows for an extended time on important analytes or analytes eluting from the liquid chromatography column, thus increasing the observed number of ions and increasing the signal to noise ratio. Allows (or allows further experiments to be performed when multiple components co-elute).

  The preferred chromatography system includes a liquid chromatography pump having three pumping trays A, B and C. Of the pumping trays, two of A and B are preferably used for solvent gradient formation, and the third C is preferably used for loading and elution of the sample at a relatively low flow rate. An advantage of the preferred chromatography system is that the preferred system does not require any restrictor for flow rate control. Furthermore, a post column valve is not required, and it is preferred not to use a post column valve because the preferred system is not adversely affected by chromatographic performance caused by the introduction of dead volume.

  The preferred embodiment enables peak parking to be performed in an advanced manner compared to other known approaches to peak parking. In particular, when a mass analyzer, mass spectrometer or other analytical instrument identifies the presence of an important species or analyte, it is preferable to send a pulse, signal or other indicator to the liquid chromatography pumps A, B. The solvent gradient depending on the solvent channels A, B is then preferably stopped substantially instantaneously and the flow from the pumps A, B is both reduced or substantially stopped. It is preferable. The valve is preferably switched so that it has the effect of substantially removing (or less preferably, but significantly reducing) the column head pressure. The valve also allows the sample flow from the auxiliary pump C to be operated at a lower flow rate towards the input to the column. Subsequently, it is preferred that a rise at low pressure occurs, whereby the sample or analyte passes through or elution from the analytical column occurs at a relatively low pressure, and therefore a peak parking effect occurs effectively. It is preferable.

  The system may switch from a peak parking mode of operation when an additional pulse, signal or other indication is received. The further pulse, signal or indicator may be sent from, for example, a mass analyzer, mass spectrometer or other analytical instrument. However, according to another embodiment, the system automatically activates from the peak parking mode of operation after setting or after a predetermined time, or according to or in response to one or more other predetermined criteria. May be switched automatically. When the system switches from peak parking mode of operation, it is preferably subsequently switched to return the valve to its original position. Subsequently, it is preferable to restart the set flow rate, and subsequently, it is preferable to continue the solvent gradient depending on the solvent channels A and B from where it was previously stopped.

  A particularly advantageous property of the preferred embodiment is that the preferred liquid chromatography system does not suffer from pressure relaxation problems because the head pressure is preferably turned off almost or substantially instantaneously. . The column pressure is then preferably allowed to start up depending on the flow rate of the auxiliary isocratic pump C operating at a relatively low flow rate. A further advantage of the preferred system is that no post-column valve is required and therefore chromatographic separation is maintained in the system.

  Various embodiments of the invention will now be described with reference to the accompanying drawings, which are merely exemplary.

  FIG. 1A shows a liquid chromatography split flow system according to a preferred embodiment during the pre-column loading mode of operation. FIG. 1B shows a liquid chromatography split flow system according to a preferred embodiment during the standard flow rate elution mode of operation. And FIG. 1C shows a liquid chromatography split flow system according to a preferred embodiment during the reduced flow rate elution mode of operation.

  FIG. 2A shows a liquid chromatography direct flow system according to a preferred embodiment during the pre-column loading mode of operation. FIG. 2B shows a liquid chromatography direct flow system according to a preferred embodiment during the standard flow rate elution mode of operation. And FIG. 2C shows a liquid chromatography direct flow system according to a preferred embodiment during the reduced flow rate elution mode of operation.

FIG. 3 shows example data obtained using the split flow liquid chromatography system shown in FIGS.
FIG. 4 shows example data obtained using the direct flow liquid chromatography system shown in FIGS.

  A preferred embodiment for performing variable flow chromatography in a split flow chromatography system will now be described with reference to FIGS. 1A, 1B and 1C. The preferred chromatography system preferably includes 10-port switching valves V1, V2. Different sized tubes and capillaries may be used to complete the system. In each figure, the position of the valve rotor is indicated by a bold line. For example, for the valve V1 shown in FIG. 1A, port 1 is connected to port 2, port 3 is connected to port 4, port 5 is connected to port 6, port 7 is connected to port 8, 9 is connected to the port 10.

  The split flow chromatography system shown in FIGS. 1A, 1B and 1C is used with an analytical column 21 having, for example, an inner or inner diameter of 180 μm or less. The split ratio is preferably dependent on the back pressure of the restrictor compared to the back pressure of the precolumn 6 plus the analytical column 21.

  FIG. 1A shows the position of the valve rotor in the pre-column loading mode of operation. The sample is preferably injected (injected) into the system via an auxiliary pump and autosampler 1, preferably at a flow rate of several tens of microliters per minute. The sample then preferably passes through tube 2, filter 3 and tube 4 and goes to port V1 (4) in the first 10-port switching valve V1. The sample then passes towards port V1 (3) and leaves the valve via port V1 (3), then passes through tube 5 and is further connected to port V1 (6) Trapped in the pre-column 6 formed. Preferably, the sample or analyte is trapped in the precolumn 6 while fluid passes through the precolumn 6 to the port V1 (5). The fluid then leaves the valve via port V1 (5). The fluid then passes through the tube 7 to the port V2 (1) in the second 10-port switching valve V2. The fluid then passes towards port V2 (10) and is then discarded via tube 8.

  In the pre-column loading mode of operation shown in FIG. 1A and described above, the solvent flow continues during this time through the analytical column 21 connected to the ion source of the mass spectrometer. The solvent flow is maintained by two pump trays 9, 10 forming part of solvent channels A, B. It is preferable that the liquid or solvent from the two pump trays 9 and 10 is moved to the mixing T section 13 through the tubes 11 and 12. Preferably, the two solvents are subsequently mixed in the mixing T section, and the resulting mixed solvent subsequently passes through the tube 14 to the valve port V2 (4) and to the port V2 (5) It is preferable to go inside. Subsequently, the mixed solvent passes through the tube 15 and travels toward the T section 16 for splitting. The restrictor arm of the dividing T section 16 goes to the port V2 (2) via the pipe 17. Fluid from port V2 (2) flows towards port V2 (3) and then passes through restrictor 18 and is eventually discarded. However, the flow for analysis (flow) passes through the dividing T section 16 and then passes through the pipe 19 to the port V1 (7) in the first 10-port switching valve V1. The analytical flow then passes through port V1 (7) and from there to port V1 (8), then through tube 20 and into port V1 (1). The analytical flow then passes through port V1 (1), from there to V1 (2) and then into analysis column 21.

  The analytical column 21 is preferably connected to a nanoflow spray device, such as an electrospray ionization ion source or other ion source, which is optimal for operation at such relatively low flow rates. Are preferably arranged. At least some of the analyte ions generated by the spray device or ion source are subsequently subjected to mass spectrometry (or more generally general analysis) mass spectrometer body (or It is preferable to pass toward another type of analytical instrument).

  In the pre-column loading mode of operation described above in connection with FIG. 1A, the plug 22 connected to port V2 (6) is not used for the specific mode of operation. However, the plug 22 is used in a reduced flow rate elution mode of operation, which will be discussed in more detail later in connection with FIG. 1C.

  After salt or other contaminants have been removed from the sample stored in the pre-column 6 and a loading / desalting period has occurred, valve V1 subsequently continues from the pre-column loading mode of operation shown in FIG. 1A. It is preferred to switch to the standard flow rate elution mode of operation shown in FIG. 1B and discussed in more detail below.

  FIG. 1B shows a preferred split flow chromatography system in standard flow rate elution mode of operation. The fluid is arranged to flow from the auxiliary pump and autosampler 1, for example at a flow rate of 0.4 microliters per minute. The fluid passes through the tube 2 and goes to the filter 3. After passing through the filter 3, the fluid subsequently passes through the pipe 4 and goes to the port V1 (4) in the first 10-port switching valve V1. The fluid then passes through port V1 (4) and from there to port V1 (5), and then passes through tube 7 to port V2 (1). The fluid then passes through port V2 (1) and from there to port V2 (10) and then passes through tube 8 and is discarded. In this mode, the back pressure in the aforementioned flow path (flow path) is extremely low.

  The solvent gradient of the liquid chromatography is preferably performed or maintained during the standard flow rate elution mode of operation, and the following method is used to flow through the precolumn 6 before flowing through the analytical column 21. It is preferable to arrange by. The liquid or solvent from the two pump trays 9 and 10 in the solvent channels A and B is preferably moved to the mixing T section 13 through the tubes 11 and 12. The resulting mixed solvent then passes through the tube 14 to the valve port V2 (4) and then passes into the port V2 (5). It is preferable that the mixed solvent subsequently passes through the port V2 (5), exits from the port V2 (5), passes through the pipe 15, and goes to the T section 16 for division. The restrictor arm of the part for splitting (split) goes to the port V2 (2) via the pipe 17. The fluid then passes through port V2 (2) and from there to port V2 (3), and then passes through restrictor 18 and is discarded. However, the analysis flow (flow) passes through the pipe 19 and goes to the port V1 (7). The mixed solvent then passes toward port V1 (6). The analytical flow or mixed solvent then passes through port V1 (6) and from there through precolumn 6 and then through tube 5 and into port V1 (3). The analytical flow comprising the mixed solvent and any analyte released from the pre-column subsequently passes through port V1 (3) to port V1 (2) and then to analytical column 21 Head inside. The analytical column 21 is preferably coupled to a nanoflow spray device such as an electrospray ionization ion source or other ion source, which is optimally arranged for such relatively low flow rate operation. It is preferable that At least some of the resulting analyte ions are then preferably passed through a mass spectrometer for subsequent mass analysis.

  In the standard flow rate elution mode of operation described above, the tube 20 interconnecting the valve ports V1 (1) and V1 (8) is not used, and similarly, the plug 22 connected to the port V2 (6). Also do not use.

  When important species or important analytes are detected by a mass spectrometer, mass analyzer or other analytical instrument, it is preferable to send a pulse, signal or other indicator to pumps A, B. The system then preferably switches to the reduced flow rate elution mode of operation described in more detail below with reference to FIG. 1C.

  FIG. 1C shows a preferred split flow chromatography system in reduced flow rate elution mode of operation. In the reduced flow rate elution mode of operation, valve V2 switches from the position it was in the standard flow rate elution mode of operation described above with reference to FIG. 1B. The switching of the valve V2 has an effect of effectively removing the back pressure to the pre-column 6 and the analysis column 21. The flow rate from the two pumps 9, 10 in the solvent channels A, B may be reduced according to a programmable split ratio, or the solvent gradient is stopped or stopped at a certain solvent concentration. May be. The effective solvent flow rate is therefore effectively reduced. It is preferable that the solvent from the second solvent channels A and B pass through the pipes 11 and 12 and go to the T section 13 for mixing. It is preferable that the mixed solvent subsequently passes through the pipe 14 and goes to the port V2 (4) in the second 10-port switching valve V2. The mixed solvent then preferably passes through port V2 (4) to port V2 (3) and then to restrictor 18 and is preferably discarded.

  In this mode of operation, the flow from the auxiliary pump and autosampler 1 preferably serves to generate a reduced flow rate through the pre-column 6 and the analytical column 21, as described below. The fluid flow passes through the tube 2 toward the filter 3. The fluid then passes through the tube 4 and goes to the port V1 (4). The fluid then flows from port V1 (4) to port V1 (5). Preferably, the fluid subsequently passes through the tube 7 and flows towards V2 (1). Preferably, the fluid subsequently passes from port V2 (1) to port V2 (2) and then to the dividing T section 16 via the tube 17. The arm including the tube 15 is preferably connected to the port V2 (5) and is preferably dead-ended by the plug 22 of the port V2 (6). As a result, a gentle rise in pressure occurs. The fluid then passes through the tube 19 to the port V1 (7). The fluid then passes towards port V1 (6) and into precolumn 6. Any analyte that elutes from the precolumn 6 continues to elute and preferably passes through tube 5 to port V1 (3) by the flow of solvent. The analyte and solvent then pass through port V1 (2) and into analysis column 21. This causes an effective elution time extension for any eluting species.

  In the reduced flow rate elution mode of operation, the fluid provided by the third pump and introduced into the tube 2 preferably comprises an aqueous solution or solvent (preferably containing 1% formic acid). If the aqueous solution or solvent is not the same as the aqueous solution or solvent that is preferably dispensed from solvent channel A, it is preferably substantially the same. Therefore, in this mode of operation, the system is effectively switched temporarily so that the solvent passing through the precolumn 6 is approximately equivalent to the solvent used at the beginning of the solvent gradient process. Therefore, in this mode of operation, it is preferred that the progress of the liquid chromatographic separation is temporarily stopped or otherwise stopped.

  In the reduced flow rate elution mode of operation described above, tube 8 and tube 20 are preferably not used.

  After a predetermined, preferably programmable time, the chromatography system may switch back from the reduced flow rate elution mode of operation to the standard flow rate elution mode of operation described above with reference to FIG. 1B. preferable.

  Hereinafter, a direct flow as another embodiment will be described with reference to FIGS. 2A, 2B, and 2C. The direct flow mode can be typically performed using an analytical column 21 having an inner or inner diameter of 320 μm or more.

  FIG. 2A shows the preferred valve rotor position in the pre-column loading mode of operation of the direct flow chromatography system. The sample is preferably injected into the system via an auxiliary pump and autosampler 1, preferably at a flow rate of several tens of microliters per minute. The sample passes through the tube 2 and goes to the filter 3. The sample then goes through the tube 4 to the port V1 (4) in the first 10-port switching valve. The sample then passes from port V1 (4) towards port V1 (3) and then passes through tube 5. The sample is subsequently trapped in the precolumn 6. The fluid continues to pass through the precolumn 6 and goes to port V1 (6). The fluid then passes through port V1 (5). The fluid then travels through the tube 23 toward the port V2 (6). The fluid then passes towards port V2 (7) and is then preferably discarded via tube 24.

  In this mode of operation, the solvent flow preferably continues to pass through the analytical column 21 by the following method. The liquid from the two pump trays 9, 10 in the solvent channels A, B is preferably moved to the mixing T section 13 through the tubes 11, 12. The solvent is preferably mixed in the T section 13 and the mixed solvent then continues through the tube 14 and toward the valve port V2 (4). It is preferable that the mixed solvent subsequently passes toward the port V2 (5), and then passes through the pipe 25 toward the port V1 (7). It is preferable that the mixed solvent subsequently passes toward the port V1 (8) and then passes through the pipe 20 and toward the port V1 (1). Subsequently, it is preferable that the mixed solvent finally passes from the port V1 (1) toward the port V1 (2) and then toward the analysis column 21. The analytical column 21 may be coupled to a nanoflow spray device such as an electrospray ionization ion source or other ion source, the nanoflow spray device being arranged to operate at a relatively high flow rate. Also good. At least some of the analyte ions generated by the spray device or ion source are subsequently subjected to mass spectrometry (or more generally general analysis) mass spectrometer body (or It is preferable to pass toward another type of analytical instrument).

  In the pre-column loading mode of operation described above, it is preferable not to use the plug 26.

  After the salt or other contaminant is removed from the sample stored in the pre-column 6 and a loading / desalting period has occurred, the valve V1 is subsequently followed by the pre-column of operation shown in FIG. 2A. It is preferred to switch from loading mode to the standard flow rate elution mode of operation shown in FIG. 2B and discussed in more detail below.

  FIG. 2B shows a preferred direct flow chromatography system in standard flow rate elution mode of operation. The fluid is arranged to flow from the auxiliary pump and autosampler 1, for example at a flow rate of 0.4 microliters per minute. The fluid preferably passes through the tube 2 and travels to the filter 3. It is preferable that the fluid subsequently passes through the pipe 4 and goes to the port V1 (4) in the first 10-port switching valve V1. Preferably, the fluid then passes towards port V1 (5) and then passes through tube 23 and towards port V2 (6). Preferably, the fluid then passes through port V2 (6) and then from there to port V2 (7), and then passes through tube 24 and is discarded. In this mode, the back pressure in the aforementioned flow path (flow path) is extremely low.

  The solvent gradient of the liquid chromatography is preferably performed and maintained during the standard flow rate elution mode of operation, and the following method is used to flow through the precolumn 6 before flowing through the analytical column 21. It is preferable to arrange by. The liquid or solvent from the two pump trays 9 and 10 in the solvent channels A and B is preferably moved to the mixing T section 13 through the tubes 11 and 12. It is preferable that the solvent is subsequently mixed in the mixing T section 13, and the mixed solvent subsequently passes through the tube 14 to the valve port V2 (4), and then the port V2 (5) It is preferable to pass inward. It is preferable that the mixed solvent subsequently passes through the port V2 (5) and then travels through the pipe 25 to the port V1 (7). It is preferable that the mixed solvent subsequently passes from the port V1 (7) toward the port V1 (6). It is preferable that the mixed solvent subsequently passes through the precolumn 6. Any analyte eluted from the pre-column 6 preferably flows through the tube 5 with the solvent and travels to port V1 (3). The solvent and the released analyte are then preferably passed through valve V1 (2) and then preferably passed through analytical column 21.

  The analytical column 21 is preferably connected to a nanoflow spray device such as an electrospray ionization ion source or other ion source, which is optimally arranged for operation at relatively high flow rates. It is preferable. At least some of the analyte ions generated by the spray device or ion source are subsequently subjected to mass spectrometry (or more generally general analysis) mass spectrometer body (or It is preferable to pass toward another type of analytical instrument). In the standard elution mode of operation described above with respect to FIG. 2B, tube 20 and plug 26 are not used.

  When important species or important analytes are detected by a mass spectrometer, mass analyzer or other analytical instrument, it is preferable to send pulses, signals or other indicators to pumps 9, 10 in solvent channels A, B . The system then preferably switches to the reduced flow rate elution mode of operation described in more detail below with reference to FIG. 2C.

  FIG. 2C shows a preferred direct flow chromatography system in reduced flow rate elution mode of operation. In the reduced flow rate elution mode of operation, valve V2 is preferably switched from the position it was in the standard flow rate elution mode of operation described above with reference to FIG. 2B. This switching of the valve V2 effectively removes the back pressure to the pre-column 6 and the analytical column 21. The flow rate from the two pumps 9, 10 in the solvent channels A, B is effectively stopped and the liquid chromatography gradient is effectively stopped or stopped at the current composition or solvent gradient. The solvent from the pumps 9 and 10 passes through the pipes 11 and 12 and travels to the mixing T section 13. The solvent is mixed in the mixing T section 13, and the mixed solvent subsequently passes through the pipe 14 and goes to the port V 2 (4). The mixed solvent then goes to port V2 (3). Port V2 (3) is coupled to plug 26 in this mode of operation. This flow rate is then preferably stopped to maintain the pump head pressure.

  Here, the flow from the auxiliary pump connected to the pipe 2 preferably serves to cause a decrease in the solvent flow rate through the pre-column 6 and the analytical column 21, as described below. The aqueous solvent preferably flows through the tube 2 toward the filter 3. Preferably, the fluid then passes through the tube 4 and goes to the port V1 (4). Preferably, the fluid then flows from port V1 (4) to port V1 (5). The fluid then passes through the tube 23 and goes to the port V2 (6). The fluid then flows towards port V2 (5). The fluid then flows through the tube 25 and goes to the port V1 (7). The fluid then passes towards port V1 (6) and into precolumn 6. Any analyte that elutes from the precolumn 6 continues to elute and preferably passes through tube 5 to port V1 (3) by the flow of solvent. It is preferred that the solvent and any eluted analytes subsequently pass into port V1 (2) and analysis column 21. This causes an effective elution time extension for any eluting species.

  In the reduced flow rate elution mode of operation, the fluid provided in the tube 2 by the third pump preferably comprises an aqueous solution or solvent (preferably containing 1% formic acid). The aqueous solution or solvent is preferably substantially the same if it is not the same as the aqueous solution or solvent dispensed by or from solvent channel A. Therefore, in this mode of operation, the system is temporarily switched to use a solvent that is approximately equivalent to the solvent used at the beginning of the solvent gradient process. Thus, in this mode of operation, it is preferred that the progress of the liquid chromatography separation is effectively temporarily stopped or otherwise stopped.

  In the reduced flow rate elution mode of operation described above, tube 20 and tube 24 are preferably not used.

  After a pre-determined, preferably programmable time, the chromatography system may switch back from the reduced flow rate elution mode of operation to the standard flow rate elution mode of operation described above with reference to FIG. 2B. preferable.

  Figure 3 shows the chromatogram obtained by injecting 200 fmol of BSA digest into a 75 μm internal diameter column formed as part of a Waters CapLC (RTM) HPLC system operating in split flow mode. Indicates gram.

  Figure 4 is also obtained by injecting 500 fmol of BSA digest into a 180 µm internal diameter column formed as part of a Waters CapLC (RTM) HPLC system operating in direct flow mode. The obtained chromatogram is shown.

In both cases, Data Dependent Acquisition (DDA) experiments were set up such that 4 ions were selected from MS / MS. Trace 1 in FIGS. 3 and 4 shows the TIC and is the base peak of MS. Trace 2 in FIGS. 3 and 4 shows the TIC and is the base peak of MS / MS.
The collision energy in the MS / MS mode was kept relatively low so as to preserve the parent ion in the MS / MS mode. Traces 3 and 4 in FIGS. 3 and 4 show ions in MS mode that were not selected for MS / MS.

  This data shows that a significant peak parking effect was achieved in the MS / MS mode of operation and that chromatographic separation was well maintained for ions that were not chosen for MS / MS.

  Further embodiments are also contemplated (not shown) that use a different connection arrangement for valve V1, allowing the flow from auxiliary pump C to be diverted through a restrictor in standard flow rate elution mode. This has the effect of creating or increasing back pressure, which reduces the pressure shock seen by the pump when entering the reduced flow rate elution mode of operation.

  The flow selection valves V1, V2 may include valves having other numbers of ports, according to other less preferred embodiments. For example, valves V1, V2 may include 6, 7, 8, 9, or more than 10 ports.

  The effective isocratic pump flow with pumps 9, 10 in the standard flow rate elution mode of operation and the auxiliary pump in the reduced flow rate elution mode of operation may be varied to vary the peak elution profile in different experiments. It is also contemplated.

  Although the preferred and less preferred embodiments have been described with reference to liquid chromatography systems, it is also contemplated that the disclosed chromatography system can be used as part of a gas chromatography system.

  While the invention has been described in terms of preferred embodiments, those skilled in the art will recognize that various changes in form and detail are possible without departing from the scope of the invention as set forth in the appended claims. is there.

Fig. 3 shows a liquid chromatography split flow system according to a preferred embodiment during the pre-column loading mode of operation. Fig. 3 shows a liquid chromatography split flow system according to a preferred embodiment during the standard flow rate elution mode of operation. Fig. 3 shows a liquid chromatography split flow system according to a preferred embodiment during a reduced flow rate elution mode of operation. Fig. 3 shows a liquid chromatography direct flow system according to a preferred embodiment during the pre-column loading mode of operation. Figure 2 shows a liquid chromatography direct flow system according to a preferred embodiment during the standard flow rate elution mode of operation. Fig. 3 shows a liquid chromatography direct flow system according to a preferred embodiment during a reduced flow rate elution mode of operation. Figure 2 shows example data obtained using the split flow liquid chromatography system shown in Figures 1A-1C. FIG. 2 shows example data obtained using the direct flow liquid chromatography system shown in FIGS.

Claims (62)

The first column,
A first fluid transport system for transporting a first fluid to the first column; and
A liquid chromatography system comprising a second fluid transport system for transporting a second different fluid to the first column;
In a first mode of operation, the first fluid transport system passes the first fluid through the first column at a first flow rate; and
In a second mode of operation, the first fluid is substantially diverted from the first column and the second fluid transport system diverts the second different fluid to a second different flow rate. Pass through the first column,
Liquid chromatography system. The liquid chromatography system according to claim 1, wherein the first column includes a high performance liquid chromatography (HPLC) column. (I) <50 μm; (ii) 50-100 μm; (iii) 100-200 μm; (iv) 200-300 μm; (v) 300-400 μm; (vi) 400- 500 μm; (vii) 500-600 μm; (viii) 600-700 μm; (ix) 700-800 μm; (x) 800-900 μm; (xi) 900-1000 μm; (xii) 1.0-1.5 mm (xiii) 1.5-2.0 mm; (xiv) 2.0-2.5 mm; (xv) 2.5-3.0 mm; (xvi) 3.0-3.5 mm; (xvii) 3.5-4.0 mm; (xviii) 4.0-4.5 mm; ( xix) 4.5-5.0 mm; (xx) 5.0-5.5 mm; (xxi) 5.5-6.0 mm; (xxii) 6.0-6.5 mm; (xxiii) 6.5-7.0 mm; (xxiv) 7.0-7.5 mm; (xxv) 7.5-8.0 mm; (xxvi) 8.0-8.5 mm; (xxvii) 8.5-9.0 mm; (xxviii) 9.0-9.5 mm; (xxix) 9.5-10.0 mm; and (xxx)> 10.0 mm The liquid chromatography system according to claim 1, wherein the liquid chromatography system has an internal diameter. (I) <10 mm; (ii) 10-20 mm; (iii) 20-30 mm; (iv) 30-40 mm; (v) 40-50 mm; (vi) 50- 60 mm; (vii) 60-70 mm; (viii) 70-80 mm; (ix) 80-90 mm; (x) 90-100 mm; (xi) 100-110 mm; (xii) 110-120 mm (xiii) 120-130 mm; (xiv) 130-140 mm; (xv) 140-150 mm; (xvi) 150-160 mm; (xvii) 160-170 mm; (xviii) 170-180 mm; ( (xxix) 180-190 mm; (xx) 190-200 mm; (xxi) 200-210 mm; (xxii) 210-220 mm; (xxiii) 220-230 mm; (xxiv) 230-240 mm; (xxv) 240-250 mm; (xxvi) 250-260 mm; (xxvii) 260-270 mm; (xxviii) 270-280 mm; (xxix) 280-290 mm; (xxx) 290-300 mm; and (xxxi)> The liquid chromatography system according to claim 1, which has a length selected from the group consisting of 300 mm. The liquid chromatography system according to any one of claims 1 to 4, wherein the first column contains a C4, C8 or C18 stationary phase. (I) <1 μm; (ii) 1-2 μm; (iii) 2-3 μm; (iv) 3-4 μm; (v) 4-5 μm; (vi) 5- 6 μm; (vii) 6-7 μm; (viii) 7-8 μm; (ix) 8-9 μm; (x) 9-10 μm; (xi) 10-15 μm; (xii) 15-20 μm (xiii) 20-25 μm; (xiv) 25-30 μm; (xv) 30-35 μm; (xvi) 35-40 μm; (xvii) 40-45 μm; (xviii) 45-50 μm; ( 6. A liquid chromatography system according to any of claims 1 to 5, comprising particles having a size selected from the group consisting of xix)> 50 [mu] m. (I) <100 angstroms; (ii) 100-200 angstroms; (iii) 200-300 angstroms; (iv) 300-400 angstroms; (v) 400-500 angstroms; (vi) 500- (Vii) 600-700 angstrom; (viii) 700-800 angstrom; (ix) 800-900 angstrom; (x) 900-1000 angstrom; and (xi)> 1000 angstrom The liquid chromatography system according to claim 1, comprising particles having The liquid chromatography system according to any of claims 1 to 7, wherein the first fluid transport system comprises one, two, or more than two pumps. The liquid chromatography system according to any of claims 1 to 8, wherein the first fluid transport system comprises one or more piston pumps, syringe pumps or peristaltic pumps. The liquid chromatography system according to claim 1, wherein the first fluid transport system comprises an aqueous solvent or solution transport device. The liquid chromatography system of claim 10, wherein the aqueous solvent or solution transport device dispenses an aqueous solvent or solution in use. The liquid chromatography system according to claim 1, wherein the first fluid transport system includes an organic solvent transport device. The liquid chromatography system of claim 12, wherein the organic solvent transport device distributes the organic solvent in use. 14. The liquid of claim 13, wherein the organic solvent is selected from the group consisting of (i) acetonitrile; (ii) methanol; (iii) propanol; (iv) one alcohol; and (v) tetrahydrofuran (THF). Chromatography system. 13. A liquid chromatography system according to claim 10 and 12, wherein streams from the aqueous solvent or solution transport device and the organic solvent transport device are mixed in use to provide an isocratic fluid stream to the first column. . 16. A liquid chromatography system according to any of claims 1 to 15, wherein the second fluid transport system comprises one, two or more than two pumps. The liquid chromatography system according to any of claims 1 to 16, wherein the second fluid transport system comprises one or more piston pumps, syringe pumps or peristaltic pumps. 18. A liquid chromatography system according to any of claims 1 to 17, wherein the second fluid transport system comprises a sample transport device. 19. A liquid chromatography system according to any of claims 1 to 18, wherein the second fluid transport system provides an isocratic fluid flow to the first column in use. (I) <10 nl / min; (ii) 10-20 nl / min; (iii) 20-30 nl / min; (iv) 30-40 nl / min; (v) 40- (Vi) 50-60 nl / min; (vii) 60-70 nl / min; (viii) 70-80 nl / min; (ix) 80-90 nl / min; (x) 90- (Xi) 100-200 nl / min; (xii) 200-300 nl / min; (xiii) 300-400 nl / min; (xiv) 400-500 nl / min; (xv) 500- 600 nl / min; (xvi) 600-700 nl / min; (xvii) 700-800 nl / min; (xviii) 800-900 nl / min; (xix) 900-1000 nl / min; (xx) 1- 100 μl / min; (xxi) 100-200 μl / min; (xxii) 200-300 μl / min; (xxiii) 300-400 μl / min; (xxiv) 400-500 μl / min; (xxv) 500- 600 μl / min; (xxvi) 600-700 μl / min; (xxvii) 700-800 μl / min; (xxviii) 800-900 μl / min; (xxix) 900-1000 μl / min; (xxx) 1.0- 2.0 ml / min; (xxxi) 2.0-3.0 ml / min; (xxxii) 3.0-4.0 ml / min; (xxxiii) 4.0-5.0 ml / min; (xxxiv) 5.0-6.0 ml / min; (xxxv) 6.0- 7.0 ml / min; (xxxvi) 7.0-8.0 ml / min; (xxxvii) 8.0-9.0 ml / min; (xxxviii) 9.0-10.0 ml / min; and (xxxix)> 10.0 ml / min The liquid cup according to claim 1. Romatography system. (I) <10 nl / min; (ii) 10-20 nl / min; (iii) 20-30 nl / min; (iv) 30-40 nl / min; (v) 40- (Vi) 50-60 nl / min; (vii) 60-70 nl / min; (viii) 70-80 nl / min; (ix) 80-90 nl / min; (x) 90- (Xi) 100-200 nl / min; (xii) 200-300 nl / min; (xiii) 300-400 nl / min; (xiv) 400-500 nl / min; (xv) 500- 600 nl / min; (xvi) 600-700 nl / min; (xvii) 700-800 nl / min; (xviii) 800-900 nl / min; (xix) 900-1000 nl / min; (xx) 1- 100 μl / min; (xxi) 100-200 μl / min; (xxii) 200-300 μl / min; (xxiii) 300-400 μl / min; (xxiv) 400-500 μl / min; (xxv) 500- 600 μl / min; (xxvi) 600-700 μl / min; (xxvii) 700-800 μl / min; (xxviii) 800-900 μl / min; (xxix) 900-1000 μl / min; (xxx) 1.0- 2.0 ml / min; (xxxi) 2.0-3.0 ml / min; (xxxii) 3.0-4.0 ml / min; (xxxiii) 4.0-5.0 ml / min; (xxxiv) 5.0-6.0 ml / min; (xxxv) 6.0- 7.0 ml / min; (xxxvi) 7.0-8.0 ml / min; (xxxvii) 8.0-9.0 ml / min; (xxxviii) 9.0-10.0 ml / min; and (xxxix)> 10.0 ml / min The liquid cup according to claim 1. Romatography system. The liquid chromatography system according to claim 1, wherein the second flow rate is substantially lower than the first flow rate. The ratio of the second flow rate to the first flow rate is: (i) <1; (ii) 0.1-1; (iii) 0.01-0.1; (iv) 0.001-0.01; (v) 0.0001-0.001; (vi) The liquid chromatography system according to claim 1, which is selected from the group consisting of 0.00001-0.0001; (vii) 0.000001-0.00001; (viii) 0.0000001-0.000001; (ix) <0.0000001. 24. A liquid chromatography system according to any of claims 1 to 23, wherein the second flow rate in the second mode of operation is not zero. The liquid chromatography system determines, analyzes, measures, detects, and predicts that, in use, one or more important analytes or one or more components have emerged, eluted or transmitted from the first column. 25. A liquid chromatography system according to any of claims 1 to 24, wherein upon evaluation, the first mode of operation is switched to the second mode of operation. 26. A liquid chromatography system according to any of claims 1 to 25, wherein the liquid chromatography system switches from the second mode of operation to the first mode of operation after a predetermined time in use. The predetermined time is: (i) <1 second; (ii) 1-10 seconds; (iii) 10-20 seconds; (iv) 20-30 seconds; (v) 30-40 seconds; (vi) (Vii) 50-60 seconds; (viii) 60-70 seconds; (ix) 70-80 seconds; (x) 80-90 seconds; (xi) 90-100 seconds; (xii) 100- 110 seconds; (xiii) 110-120 seconds; (xiv) 120-130 seconds; (xv) 130-140 seconds; (xvi) 140-150 seconds; (xvii) 150-160 seconds; (xviii) 160-170 seconds ; (xix) 170-180 seconds; (xx) 180-190 seconds; (xxi) 190-200 seconds; (xxii) 200-210 seconds; (xxiii) 210-220 seconds; (xxiv) 220-230 seconds; ( (xxv) 230-240 seconds; (xxvi) 240-250 seconds; (xxvii) 250-260 seconds; (xxviii) 260-270 seconds; (xxix) 270-280 seconds; (xxx) 280-290 seconds; (xxxi) 27. The liquid chromatography system of claim 26, selected from the group consisting of: 290-300 seconds; and (xxxii)> 300 seconds. The liquid chromatography system, in use, switches from the first mode of operation to the second mode of operation at time t ₁ , wherein t ₁ is (i) ≦ 10 seconds; (ii) ≦ 9 seconds; (iii ) ≤ 8 seconds; (iv) ≤ 7 seconds; (v) ≤ 6 seconds; (vi) ≤ 5 seconds; (vii) ≤ 4 seconds; (viii) ≤ 3 seconds; (ix) ≤ 2 seconds; (x) ≦ 1 second; (xi) ≦ 0.75 second; (xii) ≦ 0.5 second; (xiii) ≦ 0.25 second; (xiv) ≦ 0.1 second; and (xv) substantially instantaneous, claim Item 28. The liquid chromatography system according to any one of Items 1 to 27. 29. A liquid chromatography system according to any of claims 1 to 28, wherein in the second mode of operation, fluid dispensed from the first fluid transport system is substantially diverted away from the first column and discarded. . In the second mode of operation, at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.9% of the first fluid dispensed from the first fluid transport system is 30. A liquid chromatography system according to any of claims 1 to 29, substantially not transmitted to the first column. 6. In the second mode of operation, substantially 100% of the fluid dispensed from the first fluid transport device is diverted from the first column or substantially not transmitted to the first column. Item 31. The liquid chromatography system according to any one of Items 1 to 30. When the liquid chromatography system switches from the first mode of operation to the second mode of operation, the column head pressure for the first column is substantially reduced or removed at time t ₂ , t ₂ is (i) ≤ 10 seconds; (ii) ≤ 9 seconds; (iii) ≤ 8 seconds; (iv) ≤ 7 seconds; (v) ≤ 6 seconds; (vi) ≤ 5 seconds; (vii) ≤ 4 Seconds; (viii) ≤ 3 seconds; (ix) ≤ 2 seconds; (x) ≤ 1 second; (xi) ≤ 0.75 seconds; (xii) ≤ 0.5 seconds; (xiii) ≤ 0.25 seconds; (xiv) ≤ 0.1 seconds 32. The liquid chromatography system according to any of claims 1-31, selected from the group consisting of: and (xv) substantially instantaneous. 35. The fluid according to any of claims 1-32, wherein in the first mode of operation, the fluid dispensed by the first fluid transport system causes an analyte to pass from the second column to the first column. Liquid chromatography system. 34. The method of any of claims 1-33, wherein, in the first mode of operation, the first fluid transport system serves to keep the flow of fluid through the first column substantially constant or regular. Liquid chromatography system. In the first mode of operation, the first fluid passes through the first column at a flow rate of x ml / min, and in the second mode of operation, the first fluid is y ml / min. The liquid chromatography system according to claim 1, wherein the liquid chromatography system passes through the first column at a flow rate. y is (i) ≤ 0.2 x; (ii) 0.15-0.20 x; (iii) 0.10-0.15 x; (iv) 0.05-0.10 x; (v) 0.01-0.05 x; (vi) ≤ 0.01 x; ( 36. The liquid chromatography system of claim 35, selected from the group consisting of vii) substantially zero; and (viii) 0. An analyte with a specific mass to charge ratio is in said first mode of operation: (i) <1 second; (ii) 1-2 seconds; (iii) 2-3 seconds; (iv) 3-4 seconds (v) 4-5 seconds; (vi) 5-6 seconds; (vii) 6-7 seconds; (viii) 7-8 seconds; (ix) 8-9 seconds; (x) 9-10 seconds; ( xi) 10-15 seconds; (xii) 15-20 seconds; (xiii) 20-25 seconds; (xiv) 25-30 seconds; (xv) 30-35 seconds; (xvi) 35-40 seconds; (xvii) 37. The liquid chromatography system of any of claims 1-36, having a peak elution time selected from the group consisting of: 40-45 seconds; (xviii) 45-50 seconds; and (xix)> 50 seconds. An analyte with a specific mass to charge ratio is in said second mode of operation: (i) <1 second; (ii) 1-10 seconds; (iii) 10-20 seconds; (iv) 20-30 seconds (v) 30-40 seconds; (vi) 40-50 seconds; (vii) 50-60 seconds; (viii) 60-70 seconds; (ix) 70-80 seconds; (x) 80-90 seconds; ( xi) 90-100 seconds; (xii) 100-110 seconds; (xiii) 110-120 seconds; (xiv) 120-130 seconds; (xv) 130-140 seconds; (xvi) 140-150 seconds; (xvii) 150-160 seconds; (xviii) 160-170 seconds; (xix) 170-180 seconds; (xx) 180-190 seconds; (xxi) 190-200 seconds; (xxii) 200-210 seconds; (xxiii) 210- 220 seconds; (xxiv) 220-230 seconds; (xxv) 230-240 seconds; (xxvi) 240-250 seconds; (xxvii) 250-260 seconds; (xxviii) 260-270 seconds; (xxix) 270-280 seconds 38. A liquid chromatograph according to any of claims 1 to 37, having a peak elution time selected from the group consisting of: (xxx) 280-290 seconds; (xxxi) 290-300 seconds; and (xxxii)> 300 seconds. Graphy system. The analyte having the specific mass to charge ratio is: (i) <100; (ii) 100-200; (iii) 200-300; (iv) 300-400; (v) 400-500; (vi ) 500-600; (vii) 600-700; (viii) 700-800; (ix) 800-900; (x) 900-1000; (xi) 1000-1100; (xii) 1100-1200; (xiii) 1200-1300; (xiv) 1300-1400; (xv) 1400-1500; (xvi) 1500-1600; (xvii) 1600-1700; (xviii) 1700-1800; (xix) 1800-1900; (xx) 1900 39. The liquid chromatography system of claims 37 and 38, having a mass to charge ratio selected from the group consisting of: -2000; and (xxi)> 2000. 40. The liquid chromatography system according to any of claims 1-39, wherein in a third mode of operation, a sample mixture comprising an analyte is dispensed from the second fluid transport device. In the third mode of operation, the analyte is stored in the second column, stored by the second column, or otherwise retained by the second column, or in the second column. 41. The liquid chromatography system of claim 40, wherein the liquid chromatography system is retained. 42. The liquid chromatography system according to claim 41, wherein the second column comprises a High Performance Liquid Chromatography (HPLC) column. (I) <50 μm; (ii) 50-100 μm; (iii) 100-200 μm; (iv) 200-300 μm; (v) 300-400 μm; (vi) 400- 500 μm; (vii) 500-600 μm; (viii) 600-700 μm; (ix) 700-800 μm; (x) 800-900 μm; (xi) 900-1000 μm; (xii) 1.0-1.5 mm (xiii) 1.5-2.0 mm; (xiv) 2.0-2.5 mm; (xv) 2.5-3.0 mm; (xvi) 3.0-3.5 mm; (xvii) 3.5-4.0 mm; (xviii) 4.0-4.5 mm; ( xix) 4.5-5.0 mm; (xx) 5.0-5.5 mm; (xxi) 5.5-6.0 mm; (xxii) 6.0-6.5 mm; (xxiii) 6.5-7.0 mm; (xxiv) 7.0-7.5 mm; (xxv) 7.5-8.0 mm; (xxvi) 8.0-8.5 mm; (xxvii) 8.5-9.0 mm; (xxviii) 9.0-9.5 mm; (xxix) 9.5-10.0 mm; and (xxx)> 10.0 mm 43. A liquid chromatography system according to claim 41 or 42 having an internal diameter of (I) <10 mm; (ii) 10-20 mm; (iii) 20-30 mm; (iv) 30-40 mm; (v) 40-50 mm; (vi) 50- 60 mm; (vii) 60-70 mm; (viii) 70-80 mm; (ix) 80-90 mm; (x) 90-100 mm; (xi) 100-110 mm; (xii) 110-120 mm (xiii) 120-130 mm; (xiv) 130-140 mm; (xv) 140-150 mm; (xvi) 150-160 mm; (xvii) 160-170 mm; (xviii) 170-180 mm; ( (xxix) 180-190 mm; (xx) 190-200 mm; (xxi) 200-210 mm; (xxii) 210-220 mm; (xxiii) 220-230 mm; (xxiv) 230-240 mm; (xxv) 240-250 mm; (xxvi) 250-260 mm; (xxvii) 260-270 mm; (xxviii) 270-280 mm; (xxix) 280-290 mm; (xxx) 290-300 mm; and (xxxi)> 44. A liquid chromatography system according to claim 41, 42 or 43, having a length selected from the group consisting of 300 mm. 45. A liquid chromatography system according to any of claims 41 to 44, wherein the second column comprises a C4, C8 or C18 stationary phase. (I) <1 μm; (ii) 1-2 μm; (iii) 2-3 μm; (iv) 3-4 μm; (v) 4-5 μm; (vi) 5- 6 μm; (vii) 6-7 μm; (viii) 7-8 μm; (ix) 8-9 μm; (x) 9-10 μm; (xi) 10-15 μm; (xii) 15-20 μm (xiii) 20-25 μm; (xiv) 25-30 μm; (xv) 30-35 μm; (xvi) 35-40 μm; (xvii) 40-45 μm; (xviii) 45-50 μm; ( 46. A liquid chromatography system according to any of claims 41 to 45, comprising particles having a size selected from the group consisting of xix)> 50 [mu] m. (I) <100 angstroms; (ii) 100-200 angstroms; (iii) 200-300 angstroms; (iv) 300-400 angstroms; (v) 400-500 angstroms; (vi) 500- (Vii) 600-700 angstrom; (viii) 700-800 angstrom; (ix) 800-900 angstrom; (x) 900-1000 angstrom; and (xi)> 1000 angstrom 47. The liquid chromatography system according to any of claims 41 to 46, comprising particles having 48. In any one of claims 41 to 47, in the third mode of operation, salt and / or other contaminants are at least partially or substantially removed from the sample mixture and exit the second column. Liquid chromatography system. In the third mode of operation, the relative concentration of the analyte in the sample mixture is stored in the second column, stored by the second column, or otherwise retained by the second column. 49. A liquid chromatography system according to any of claims 41 to 48, wherein the liquid chromatography system increases substantially while being retained or retained in the second column. 50. A liquid chromatography system according to any of claims 41 to 49, wherein the liquid chromatography system switches to the first mode of operation after the third mode of operation. The analytical instrument containing the liquid chromatography system in any one of Claims 1-50. The analytical instrument comprises: (i) an ultraviolet (UV) detector; (ii) an ultraviolet (UV) array detector; (iii) an infrared (IR) detector; (iv) an ion mobility separator; (v) an ion mobility. (Vi) Visible ultraviolet (UV) detector; (vii) Nuclear Magnetic Resonance (NMR) detector; (viii) Electrospray Light Scattering Detector (ELSD); (ix ) Further liquid chromatography system (LC-LC); (x) Refractive index (RI) detector; (xi) Visible light detector; (xii) Chemiluminescence detector; and (xiii) Fluorescence detector 52. The analytical instrument of claim 51, which is selected. A mass spectrometer comprising the liquid chromatography system according to claim 1. 54. The mass spectrometer of claim 53, further comprising an ion source coupled to the first column. The ion source is (i) an Electrospray ("ESI") ion source; (ii) an atmospheric pressure chemical ionization ("APCI") ion source; (iii) an atmospheric pressure photoionization (Atmospheric Pressure ionization) Photo ionization ("APPI") ion source; (iv) Laser desorption ionization ("LDI") ion source; (v) Inductively Coupled Plasma ("ICP") ion source; (vi ) Electron Impact (“EI”) ion source; (vii) Chemical Ionisation (“CI”) ion source; (viii) Field Ionisation (“FI”) ion source; (ix) Fast Atomic collision (Fast Atom Bombardment, "FAB") ion source; (x) Liquid Secondary Ion Mass Spectrometry ("LSIMS") ion source; (xi) Atmospheric Pressure Ionisation, " API ") ion source; (xii) Field Desorption," FD " (Xiii) Matrix Assisted Laser Desorption Ionisation ("MALDI") ion source; (xiv) Desorption / Ionisation on Silicon ("DIOS") ion source 55. A mass spectrometer according to claim 54, selected from the group consisting of: (xv) a desorption electrospray ionization ("DESI") ion source; and (xvi) a nickel-63 radioactive ion source. 56. A mass spectrometer as claimed in claim 54 or 55, wherein the ion source comprises a continuous ion source. 56. A mass spectrometer as claimed in claim 54 or 55, wherein the ion source comprises a pulsed ion source. In determining whether important analyte ions have eluted from or released from the ion source, the liquid chromatography system moves from the first mode of operation to the second mode of operation. The mass spectrometer according to any one of claims 54 to 57, which is switched. 59. A mass spectrometer according to any of claims 53 to 58, further comprising a mass analyzer. The mass analyzer comprises: (i) an orthogonal acceleration time-of-flight mass analyzer; (ii) an axial acceleration time-of-flight mass analyzer; (iii) a quadrupole mass analyzer; (iv) a Penning mass analyzer; v) Fourier Transform Ion Cyclotron Resonance ("FTICR") mass spectrometer; (vi) 2D or linear quadrupole ion trap; (vii) Paul or 3D quadrupole ion trap; and ( 60. The mass spectrometer of claim 59, selected from the group consisting of viii) magnetic sector mass analyzer. Providing a first column, a first fluid transport system for transporting a first fluid to the first column, and a second fluid transport system for transporting a second different fluid to the first column;
Passing the fluid through the first column at a first flow rate by the first fluid transport system; and subsequently
Substantially diverting the first fluid from the first column and passing the second different fluid through the first column at a second different flow rate by the second fluid transport system;
Liquid chromatography method. 62. A mass spectrometry method comprising the liquid chromatography method according to claim 61.

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