JPH07198570A - Preparation of electric spray ion source, device for preparing eluate mixture and analyzer - Google Patents

Abstract

PURPOSE: To provide an electric spray performance improving technique under the existance of specific high velocity liquid chromatography additive such as trifluoroacetic acid which behaves as ion species in chromatographed effluent. CONSTITUTION: A liquid chromatographing device 60 to select effluent, mixture including strong acid anion, a preparing means (to add additive fluid to elusion liquid mixture) 62 to accept sample (elusion liquid mixture) from the liquid chromatographing device 60 and remove the restraining effect of chromatography additive fluid 28 including weak acid, an electric spray device 64 connected to the preparing device to accept sample from the preparing device and a mass analyzing device 66 connected to an electric spray chamber are provided.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention generally relates to the presence of certain high performance liquid chromatography (HPLC) additives such as trifluoroacetic acid (TFA), which behaves as ionic species in chromatographic eluates. TECHNICAL FIELD The present invention relates to a technique for improving electrospray performance, and specifically to a method for preparing an electrospray ion source, a device for preparing an eluate mixture, and an analyzer.

[0002]

BACKGROUND OF THE INVENTION Electrospray ionization mass spectrometry has become very popular recently as a technique for linking high performance liquid chromatography (HPLC) and mass spectrometry. The electrospray phenomenon (also known as electrohydrodynamic spray) is the process of transforming a liquid surface into a spray consisting of fine, highly charged droplets in the presence of a strong electric field. In electrospray mass spectrometry, a solution of non-volatile organic molecules and biopolymers (such as proteins and DNA) is electrosprayed to produce a large number of small, highly charged droplets containing the component of interest. . The solvent is rapidly evaporated from the droplets and the residual biopolymer ions are transferred to the mass spectrometer through an orifice or capillary, where the mass-charge ratio (m
/ Z) is accurately measured. As the boundary between liquid chromatography and mass spectrometry, electrospray's widespread popularity derives not least from its sensitivity, but also multi-loading and mathematical deconvolution.
n) based on standard mass spectrometry measurements,
It also derives from its ability to analyze large molecular weight compounds. Among other things, these features make electrospray mass spectrometry (ESMS) useful in biochemistry and protein science.

A substantial obstacle to obtaining the full benefit of electrospray mass spectrometry, and most particularly to a significant extent of ESMS sensitivity, is the presence of signal suppressing solvent in the chromatographic eluate. Certain solvents or mobile phase additives that are widely used in high performance liquid chromatography significantly reduce the sensitivity of subsequent ESMS analysis. For example, trifluoroacetic acid (TFA) has excellent chromatographic ion-pairing properties, low UV in the range (200-220 nm) where proteins and peptides absorb.
It is widely used in high performance liquid chromatography due to its special suitability for protein and peptide related chromatography due to its light absorption and its volatility. However, one drawback for TFA is
It is this compound that suppresses the signal in the ESMS analysis of the HPLC eluate. That is, the presence of TFA in a sample analyzed by mass spectrometry interferes with accurate detection of the type and amount of ions present in the sample.

To remove TFA from the eluate, high performance liquid chromatography (HPLC) eluate can be processed by intermediate purification techniques or other attempts to obtain either HPLC or ES.
TFA because it sacrifices sensitivity of MS or both
The presence of signal suppressors such as is associated with major limitations. Therefore, sample preparation methods for electrospray mass spectrometry should be used immediately after removal from the chromatographic column and without modification of the chromatographic separation or purification method.
Most preferably, the eluate can be supplied. It is extremely important to overcome the signal suppressing effect of TFA and similar solutions and to achieve the maximum qualitative and quantitative sensitivity possible with ESMS.

Attempts to overcome the signal suppressing effects of TFA and other solvents have focused on the high electrical conductivity imparted to solutions by the presence of such ionic species. Conductivity is
Interferes with electrospray methods that require a voltage drop across the electrospray needle. Mechanical modification of electrospray needles is limited in overcoming signal suppression but has some effectiveness. Another substantial mechanical solution is the implementation of enhanced atomization by ultrasound or other means. Such mechanical solutions show only a slight improvement in sensitivity, and the results show only a small part of the sensitivity of mass spectrometry in the absence of signal suppressing solvent.

The surface tension of the eluate also interferes with the electrospray process. US Pat. No. 5,122,670 (1992)
, Et al., A sheath solvent such as 2-methoxymethanol taught by Mychleest.
The use of a solvent results in limited effectiveness in overcoming signal suppression. However, according to either of these solutions, the improvement in sensitivity is only on the order of 2 or 5 times and does not approach the level of sensitivity obtained in the absence of signal suppressing ionic species. .

[0007] None of the approaches to date has mentioned the fact that the ionic species present in the chromatographic solution specifically interact with the peptide or protein in question, so that there is a problem of signal suppression. Are still left. No single technique to improve sensitivity to date has described this interaction as an impediment to the restoration of signal sensitivity. Mass spectrometry is one of the most powerful analytical tools available, and protein scientists and biochemists have found that in protein characterization and peptide separation,
With the increasing focus on mass spectrometry, the need for sensitivity and uncompromising qualitative information becomes even more compelling.

[0008]

SUMMARY OF THE INVENTION The present invention is directed to a method (electrospray ion source preparation method) and apparatus (eluate mixture preparation apparatus and analyzer) for improving the sensitivity of electrospray mass spectrometry in the presence of a signal suppressing solvent. And to provide. The present invention provides a more sensitive technique of electrospray ionization mass spectrometry, especially when the chromatographic eluate contains TFA and other similar ionic species.

[0009]

SUMMARY OF THE INVENTION In summary, the present invention provides for the formation of a weak acid in high concentration relative to the amount of strong acid or anion of another ionic species present in the chromatographic eluate prior to or during electrospray droplet formation. Teaches to add. TF
A By adding a weak acid at a concentration higher than that of the other eluent ionic species, a competitive equilibrium between the weak acid and the chromatographic eluent ionic species is established. Weak acids can react with the components of interest (in the sample) in the chromatographic eluate.
The FA interaction is disrupted and sensitivity to electrospray mass spectrometry is restored to substantially 100%.

The present invention allows for direct sample transfer from a chromatographic column to an electrospray mass spectrometer without the need for intermediate purification or suppression separation, previously obtained only in samples where ionic species were not present. Up to the standard
Restores mass spectrometry sensitivity. The method according to the invention for preparing an electrospray ion source comprises (1) selecting a chromatographic eluent mixture, (2) adding a liquid or gaseous additive stream to the eluent to form a mixture. The steps of: (3) controlling mixing in the product mixture; and (4) introducing the product mixture into the electrospray chamber. Alternatively, when the additive fluid is a gas, it is added directly to the electrospray chamber. In this configuration, the mixing of the eluate and the added fluid is performed immediately before being introduced into the mass spectrometer. In most cases, the eluent is selected by inclusion of an ion-pair acid, and other eluent additives falling into this category are known or obvious to chromatographic technicians, Trifluoroacetic acid (TFA), heptfluorobutyric acid (HFB
It is one selected from the group consisting of A) and hydrochloric acid (HCl), but is not limited thereto.

The additive fluid contains a weak acid at a concentration such that the concentration of the weak acid in the product mixture is at least 5 times the concentration of ion-pair acid. Optimal concentrations will vary with the nature of the acid selected. The weak acid has a dissociation constant (pK _a ) of 3 or greater and is often selected from the group consisting of acetic acid, formic acid, propionic acid, butyric acid, valeric acid, and malonic acid. Other weak acids with similar properties are also suitable.

The additive fluid may include a volatile carrier solvent selected on the basis of its low surface tension and its miscibility with the eluate. The carrier solvent is often
It is selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, 2-methoxyethanol, n-butanol / isopropanol, n-butanol / acetonitrile, and n-butanol / 2-methoxyethanol. A carrier solvent is necessary to overcome surface tension when nebulization is not facilitated. Other carrier solvents with similar properties can be selected to complete this step.

In one embodiment, the volume of liquid introduced into the electrospray chamber is significantly reduced by utilizing a flow divider to direct a portion of the effluent or product mixture to a collector. . If the flow divider splits a portion of the product mixture (ie, after the additive fluid has been pressed into the eluent mixture or mixed with the eluent mixture), the eluent mixture and additional fluid may be further combined. Delay coils or other means that can be mixed are available.

[0014]

EXAMPLES The method and apparatus according to the present invention restore signal sensitivity to electrospray mass spectrometry in the presence of signal suppressing solvent. Electrospray mass spectrometry (E) in which the chromatographic eluate in question contains ionic species such as trifluoroacetic acid
The reduced sensitivity of SMS) has significantly impeded the efforts of biochemists and protein scientists. The exact process of electrospray ionization has not been clearly characterized, but the generally accepted theory is depicted in FIG. 1 as a four-step process: 1) introduced from needle 12. A stream of liquid 10 is atomized and the weighted droplets produced by the atomization form water fumes (not shown) 2)
A smaller droplet having a larger surface charge-volume ratio (sometimes described as a "charge / area" ratio) that results in the loading droplet being heated and the solvent evaporating from the droplet upon heating. 3) Coulomb explosion occurs, which is produced by (not shown), and smaller droplets 14 are produced with a reduction in the surface-charge ratio for each droplet 14.
And 4) "ion evaporation" occurs, and the load sample ion 1
6 are sprayed from smaller droplets with high surface-charge density. This ion generation process is important for electrospray because it creates desolventized ions from solution without evaporation of the analyte. This process ionizes non-volatile, heat labile compounds by electrospray. Ionic species such as TFA interfere with the ionization process in steps 1 and 4 and this interference manifests as signal suppression.

To date, all efforts to overcome the signal suppression effect of ionic species have focused on the disturbance in step 1 which is a result of the high conductivity due to the presence of ionic species. However, the "ion evaporation" of step 4 is also suppressed by the interaction of the ionic species and the analyte. The mere description of a Step 1 type of interference did not succeed in restoring sensitivity by more than a 2-5 fold substantially smaller recovery effect.
The methods and apparatus described herein are the first to address the problem of ionic interactions, overcome both types of interference, and practically restore any signal sensitivity.

FIG. 2 is a schematic diagram of an apparatus incorporating the present invention. The apparatus includes an electrospray chamber 18; an eluent mixture 22 containing a signal suppressor from an eluate input device 21, and an introduction device 20 having a second end 24 in the electrospray chamber 18; an addition fluid 28. At a point between the flow input device 26, such as a conduit for guiding to the introduction device 20, and the two ends of the introduction device 20, before entering the electrospray chamber 18, , A mixing chamber 30 for mixing the eluent mixture 22 with an addition fluid 28 to produce a product mixture 32.
Or tee (T-shaped tube);

The eluent mixture 22 contains a strong ion-pairing acid, typically trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA), or hydrochloric acid (HCl), which exhibits chromatographically useful characteristics. These strong acids are 3
It has a dissociation constant with the following values. Other ion pair chromatographic additives are self-evident for routine expert analysis.

Additive fluid 28 contains a weak acid concentration such that the concentration of weak acid in product mixture 32 is on the order of five times the ion-to-acid concentration of eluent mixture 22. The optimum relative concentrations of weak and strong acids are the relative dissociation constants (pK _a ).
Determined by The weak acid in the additional vulcanizate 28 is selected from the group of acids having a dissociation constant of 3 or more. Usually, the weak acid is
It is selected from the group consisting of acetic acid, formic acid, propionic acid, butyric acid, benzoic acid, and malonic acid. Other suitable acids will be apparent to the ordinary analyst.

The introduction of the additive fluid 28 must be carried out so that the additive fluid 28 and the eluent mixture 22 are sufficiently mixed in order to establish a competitive equilibrium between the ionic species and the weak acid for the analyte molecule. I have to. However, it is generally desirable to broaden the chromatographic band and not induce excessive lengthwise mixing that reduces chromatographic resolution.

A mixing device 34, such as a delay coil or packed column, is arranged in the path of the product mixture 32 in the introduction device 20 after the mixing chamber 30 and before the electrospray chamber 18. The details of the mixing parameters associated with introducing the sample into the electrospray mass spectrometer are well known to those skilled in the relevant art. The individual operating parameters are given in the examples provided at the end of this section.

The majority of the stream of product mixture 32 is the collector 3.
8 and a small portion in the introduction device electrospray chamber 1
A flow divider 36 for dividing the flow of the product mixture 32 in the introduction device 20 so that it continues to flow toward 8.
Other means are provided.

Addition fluid 28 is added to form a product mixture 32.
The best results have been obtained with the arrangement depicted in FIG. This arrangement minimizes band spreading and increases mixing, but at the expense of flow input device 26.
Requires a high pressure pump as part of it, and consumes relatively more additive fluid 28.

FIG. 3 shows that the flow divider 36 has the eluent mixture 2
2 shows another embodiment arranged to split the two streams before they enter the mixing chamber 30. Such an arrangement consumes less added fluid 28 and can be operated by a low cost infusion pump (not shown), but results in a large amount of band spreading and poor mixing efficiency. As described above with reference to FIG. 1, a delay coil 34 or other delay device such as a packed column may be added after the mixing chamber 30 to facilitate mixing and equilibration.

Based on the effectiveness of unenhanced droplet formation (pure electrostatic spraying), the addition fluid 28 further contains methanol, ethanol, isopropanol, acetonitrile,
2-methoxyethanol, n-butanol / isopropanol, n-butanol / acetonitrile, and n-
It may contain an organic carrier solvent selected from the group consisting of butanol / 2-methoxyethanol. Other suitable carrier solvents also apply to the average analysis.

FIG. 4 shows another embodiment of the invention in which the spray facilitating means 42 is connected to the second end of the path of the introduction device 20 for introducing the product mixture into the spray chamber. Higher flow rates can be used if the spray is facilitated by various means (ultrasonic, heat, or air),
And a flow divider is needed. Furthermore, because the assisted spray overcomes the interference of ionic species with the droplet generation step,
It is possible to exclude the organic carrier solvent (which promotes droplet formation) from the added fluid 28, leading to a more concentrated solution of the selected weak acid. Higher concentrations allow introduction at appropriate lower flow rates.

FIG. 5 is a schematic diagram showing another embodiment in which the additive fluid 28 is introduced directly into the electrospray chamber 18.

FIG. 6 is a schematic diagram of another embodiment in which additive fluid 28 is directed via a sheath flow arrangement. The sheath flow arrangement requires precise alignment of the electrospray needle tips 44,46. As noted above, it is necessary to mix the additive fluid 28 (introduced as a sheath liquid) with the eluent mixture 22 to allow competitive equilibrium, thus interfering with the mixing of large analytes, etc. in the eluate mixture. , The sheath arrangement is not ideally suitable. However, 100 μl / min. At the following flow rates, the sheath flow arrangement is preferred because it requires a small, inexpensive infusion pump 50 and consumes only a small amount of added fluid 28.

FIGS. 7 and 8 show a comparison between the signal suppression effect of TFA, which is one chromatographic solvent, and the recovery effect regarding the signal sensitivity when the present invention is carried out.
The results were obtained with the arrangement as shown in FIG. 4, i.e. with additive fluid containing weak acid but no carrier, and with assisted spray. 52 of FIG. 7 shows the mass spectrometry result of the peptide sample containing 0.1% TFA in the eluate. In FIG. 8, 54 indicates that the fluid consisting of propionic acid is 100 μl / min. 3 shows mass spectrometry results of the same peptide sample added at different flow rates. By comparing the peak height of 52 in FIG. 7 with the peak height of 54 in FIG. 8, a significant increase in signal sensitivity is observed.

9 and 10 show that the present invention substantially includes the arrangement shown in FIG. 2, ie, a mixing tee tube in the inlet path before the flow divider and a carrier in the addition fluid, Figure 3 shows the data obtained from test samples for pure electrostatic (non-assisted) electrospray. 56 in FIG. 9 is 0.1%
2 shows the results of mass spectrometry of an untreated eluate containing TFA of. Figure 58, 58 shows the increase in peak height obtained when a solvent comprising propionic acid in isopropanol was added prior to electrospraying according to the present invention.

FIG. 11 shows a system according to the present invention.
FIG. 3 shows a system according to the invention for receiving an eluate and directing an additive fluid such that the signal suppressing effect of ion-pair acid in the chromatographic effluent is overcome by the high performance liquid chromatographic device 60, the method described above. Includes a preparation device 62, an electrospray device 64, and a mass spectrometer 66 described in. The system may include a device (not shown) for assisted spraying.

In summary, the figures and data provided herein demonstrate that the signal-suppressing effect of chromatographic additives such as TFA is substantially eliminated by the present invention.

[Theory] Studies are currently underway on the exact nature of the chemical interactions that occur during electrospray. The reasons for the success of the present invention are not yet completely clear, but nonetheless provide one theory useful for discussion.

For the specimen M, the product ions are mainly in the form of (M + nH) ^{n +} , where n ≧ 1. These ions are generated in solution, and the charge properties facilitate extraction of ions from the solution.

In the aqueous solution, the protonated trifluoroacetic acid CF ₃ COOH is present in the acid / base equilibrium state.

[0035]

[Chemical formula 1] 1. ^{_{CF 3 COOH = CF 3 COO -}} + H 3 O +
K _a = 0.5

CF ₃ C which is the trifluoroacetic acid anion
OO ⁻ can form an ion pair with a positively charged analyte ion.

[0037]

[Chemical formula 2] 2. _{CF 3 COOH + (M + nH} ) n + = _{^{[(M + nH) n + ·}} nCF 3 COO - ]

The uncharged ion pair produced does not undergo ion evaporation. In this way, TFA effectively prevented the production of analyte ions.

If other acids such as acetic acid are added to the solution, an acid / base equilibrium similar to Equation 1 exists.

[0040]

[Chemical Formula 3] 3. _{H 2 O + CF 3 COOH =} CF 3 COO -
^{_{+ H 3 O + K a =}} 1.8 × 10 -5

If acetic acid is present in a sufficiently high concentration, there may be two competitive equilibria. _CF 3 COO acetate anion ^- presence of has the potential to compete with TFA ions into the ion pair of the analyte cations. However, because the acetate ion pair is relatively unstable, the analyte cations do not continue to undergo ionic evaporation.

[0042]

[Chemical formula 4] CF ₃ COO ⁻ + (M + nH) ^{n +} = [(M + nH) ^{n +} · nCF ₃ COO ⁻ ] = CF ₃ COO ⁻ + (M + nH) ^{n +}

The relatively high concentration of H ₃ O ⁺ from excess acetic acid then shifts the equilibrium of equation 1 to the left, reducing the amount of ion pair CF ₃ COO ⁻ present and consequently the analyte ions. Do not evaporate.

Due to the relatively large equilibrium coefficients of equations 1 and 3, the concentration of acetic acid and other acids significantly exceeds that of TFA. This idea holds for ion pair acids other than TFA (eg HFBA, HCl) and organic acids other than acetic acid (eg formic acid, propionic acid).
The relative efficiency of other combinations will depend on several factors known to those skilled in the art, such as equilibrium constants and volatility.

Given the conceptual structure described above, there are several variables that can be optimized for various operating characteristics. Problem solving parameters can be divided into three main groups: fixed properties (defined by the user's application), chemical parameters, and operating parameters.

A critical advantage of the present invention is that it directly links chromatographic techniques such as high performance liquid chromatography with electrospray mass spectrometry without sacrificing either performance. . Analysts can utilize the method of the present invention without modifying the chromatographic conditions. To this end, the following conditions should be considered as a standard starting point for HPLC performance in the situation where the eluent in question is obtained from a high performance liquid chromatographic column.

[0047]

Table 1 HPLC flow rate (10 to 1500 μl / min.) (Eluent flow rate) HPLC mobile phase organic solvent% (0 to 100%) (eluent carrier) HPLC mobile phase organic solvent (methanol, acetonitrile, isopropanol) (elution) Liquid carrier) HPLC mobile phase buffer / additive (TFA, etc.) (eluent additive) HPLC column size and stationary phase (irrelevant) HPLC column temperature (irrelevant)

For standard conditions as described above, a range of chemical solutions (additional fluids) can be used in accordance with the present invention. Some acids can be added or "teeed" in the eluent to counteract the potency of ion pair acids (eg TFA). Those tested include formic acid, acetic acid, propionic acid, butyric acid, valeric acid, and malonic acid. At present, propionic acid gives the best results with virtually 100% signal sensitivity.
The result will be recovered. The concentration of acid in the addition fluid depends on the nature of the acid selected.

The acid is usually prepared as a solution in a carrier solvent. The two components make the added fluid. In the case of unassisted electrospray, the carrier solvent is a highly conductive solution of TFA in the course of the droplet formation step.
Play an important role. In assisted electrospray, the carrier solvent is less important and, under certain conditions, is omitted altogether because the formation of droplets is mechanically assisted.

The additive fluid must be introduced at a constant flow rate that optimally maintains the chromatographic separation in the eluate. In the preferred embodiment, where the additive fluid is added to the eluent and mixing is done in the mixing chamber prior to encountering the dividing means or flow divider, the optimum flow rate is approximately 400.
μl / min. Met. Other embodiments have different optimal flow rates.

To reiterate, the mixing of the eluate mixture with the optimum of addition fluid is important for best results.
The optimum time is empirically determined as the amount of mixing time required for the competitive equilibrium established between ionic species, be it a strong acid, a salt of a strong acid, or an acid introduced into the additive fluid. . For some analytes, such as large globular proteins, it is also necessary to allow extra contact time for equilibration. Generally, the required equilibration time can be provided by a delay coil or packed column, neither of which can be mixed without interfering with the chromatographic separation.

As described above, the present invention relates to [1] a method for preparing an electrospray ion source of the type using an ionization chamber, wherein _a ) a strong acid anion having _a dissociation constant (pK _a ) of 3 or less is used. Selecting an eluent mixture containing; b) adding an additive fluid containing a weak acid having a dissociation constant of 3 or more to the eluate mixture; c) producing a mixture in which the weak acid is present at a higher concentration than the strong acid; d) controlling the mixing of the resulting mixture; and e) introducing the resulting mixture into the ionization chamber;
And has the following preferred embodiments.

[2] In the method of [1], the step of selecting an eluent mixture in which the strong acid is selected from the group consisting of trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA) and hydrochloric acid (HCl). It is characterized by including.

[3] In the method of [2], the weak acid further comprises the step of adding an additive fluid selected from the group consisting of acetic acid, formic acid, propionic acid, butyric acid, benzoic acid and malonic acid. To do.

[4] The method of [3] is characterized by further including the step of dividing the flow of the eluate mixture and directing a part of the flow toward the collector.

[5] The method of [3] is characterized by further including a step of dividing the produced mixture and directing a part of the mixture to a collector.

[6] The method of [3] is characterized in that it further comprises the step of adding an addition fluid containing a volatile carrier solvent which is miscible with the aqueous solvent.

[7] In the method of [6], the carrier solvent is a mixture selected from the group consisting of n-butanol / isopropanol, n-butanol / acetonitrile, and n-butanol / 2-methoxyethanol. Characterized in that it includes the step of adding the added fluid that has been added.

[8] The method of [6] is characterized in that the carrier solvent further comprises the step of adding an additive fluid selected from the group consisting of methanol, ethanol, isopropanol, methoxyethanol, and acetonitrile. .

[0060]

[9] The method of [6] is characterized by further including a step of dividing the flow of the eluate mixture and directing a part of the flow toward the collector.

[10] In the method of [6],
Characterized in that it comprises the step of splitting the stream of the produced mixture and directing a part of it towards the collector.

[11] In the method of [3],
Characterized in that it includes the step of facilitating droplet formation of the product mixture in the electrospray chamber.

The present invention also relates to [12] an apparatus for preparing an eluent mixture containing a signal suppressor for introduction into an electrospray ionization chamber, which is a) 1) opened to receive the eluent mixture. An introduction device having a first end; 2) a second end opening to the electrospray ionization chamber; 3) an introduction path for communication between the first end and the second end; b) an input means connected to a selected portion of the introduction device for introducing the addition fluid into the eluent mixture: and c) an introduction device for mixing the eluate mixture with the addition fluid to form a mixture. And a mixing means connected to the selected portion, which has the following preferred embodiment.

[13] The apparatus of [12] is characterized in that the product mixture is produced at a point before the product mixture is introduced into the electrospray chamber.

[14] In the apparatus of [12], a pressurizing means connected to a selected portion of the introducing device is further provided to pressurize the liquid in the introducing device before introducing into the electrospray chamber. It is characterized by including.

[15] The apparatus of [14] further comprising splitting means connected to selected portions of the introduction device for splitting the product mixture stream and directing a portion to the collector. Is characterized by.

[16] The apparatus of [13] further includes a dividing means connected to a selected portion of the introduction device for dividing the flow of the eluate mixture and directing a part of the flow toward the collector. It is characterized by

[17] The apparatus of [15] is characterized in that the mixing means includes a delay device.

Furthermore, the present invention is [18] an analytical apparatus, comprising: a) a liquid chromatograph device; b) a preparation means for receiving a sample from the liquid chromatograph device and removing the inhibitory effect of the chromatographic additive; c) an electrospray device connected to the preparation device and receiving the sample from the preparation device; and d) a mass spectrometric device connected to the electrospray chamber, which has the following preferred embodiments.

[19] In the analyzer of [18], the preparation means comprises: a) 1) a first end opened to receive the eluate mixture; 2) a second end to the electrospray ionization chamber. An introduction device having an opening; 3) an introduction passage providing communication between the first end and the second end; b) a selection of an introduction device for introducing an addition fluid into the eluate. An input means connected to the part; and c) a mixing means connected to a selected part of the introduction device for mixing the eluate mixture with the addition fluid and producing a mixture. .

[20] In the analyzer of [19], the input means guides the added fluid containing a weak acid having _a dissociation constant (pK _a ) of 3 or more.

[21] In the analyzer of [20], the input means guides a weak acid selected from the group consisting of acetic acid, formic acid, propionic acid, butyric acid, benzoic acid and malonic acid.

[22] In the analyzer of [20], the input means further introduces an addition fluid containing a carrier solvent which is volatile and miscible with the aqueous solvent.

[23] In the analyzer of [22], the input means introduces a carrier solvent selected from the group consisting of methanol, ethanol, isopropanol, methoxyethanol, and acetonitrile.

[24] In the analyzer of [22], the input means is a mixture, and n-butanol / isopropanol, n-butanol / acetonitrile, and n
-Deriving a carrier solvent selected from the group consisting of -butanol / 2-methoxyethanol.

[25] The analyzer of [20] is characterized by further including a spray promoting device connected to a selected portion of the introducing device.

[26] The analyzer of [19] is characterized in that the added fluid is gaseous and the input means is directly connected to the electrospray chamber.

[27] In the analytical device of [22], further, the flow splitting means may be provided at selected portions of the device prior to directing the additive fluid into the eluent mixture so as to reduce the volume of the eluent mixture. It is characterized in that it comprises connected flow dividing means.

[28] In the analytical device of [22], further, the flow splitting means reduces the volume of the product mixture before the product mixture is introduced into the electrospray chamber in a selected portion of the device. And a flow dividing means connected to the.

[29] The analyzer of [22] is characterized in that the mixing means includes a delay device.

[0081]

EFFECTS OF THE INVENTION The present invention allows for direct sample transfer from a chromatographic column to an electrospray mass spectrometer without the need for intermediate purification or suppression separations, while the sample was previously free of ionic species. The sensitivity of mass spectrometry can be restored to the level obtained only in. In short, according to the present invention, the signal suppressing effect of the chromatographic additive such as TFA can be substantially eliminated by 100%.

[Brief description of drawings]

FIG. 1 is a schematic diagram of an electrospray device.

FIG. 2 is a schematic diagram showing a preferred embodiment of the device of the present invention.

FIG. 3 is a schematic diagram showing another embodiment of the device of the present invention.

FIG. 4 is a schematic diagram showing another embodiment of the device of the present invention.

FIG. 5 is a schematic diagram showing another embodiment of the device of the present invention.

FIG. 6 is a schematic diagram showing another embodiment of the device of the present invention.

FIG. 7: Acid (T according to the invention for solvent induced signal suppression)
It is a figure which shows the effect of FA) additive.

FIG. 8 shows the effect of the acid (propionic acid) additive according to the present invention on solvent induced signal suppression.

FIG. 9 shows the effect of an additive with carrier (TFA) according to the invention on solvent-induced signal suppression.

FIG. 10 shows the effect of an additive with a carrier according to the invention (solvent containing propionic acid in isopropanol) on solvent-induced signal suppression.

FIG. 11 is a schematic diagram showing a system arrangement according to the present invention.

[Explanation of symbols]

 12 needles 14 small droplets 16 loaded sample ions 18 electrospray chamber 20 introduction device 21 eluent input device 22 eluent mixture 26 flow input device 28 additive fluid 30 mixing chamber 32 product mixture 34 delay coil 36 flow divider 38 collector 44 , 46 Electrospray needle tip 60 High-performance liquid chromatograph device 62 Preparation device 64 Electrospray device 66 Mass spectrometer

Claims (3)

[Claims] 1. A method of preparing an electrospray ion source of the type using an ionization chamber, comprising the steps of: _a ) selecting an eluent mixture containing a strong acid anion having _a dissociation constant (pK _a ) of 3 or less; b. ) Adding an additive fluid containing a weak acid having a dissociation constant of 3 or more to the eluent mixture; c) producing a mixture in which the weak acid is present in higher concentration than the strong acid; d) controlling the mixture of the produced mixture And e) introducing the resulting mixture into the ionization chamber;
A method for preparing an electrospray ion source, comprising: 2. An apparatus for preparing an eluent mixture containing a signal suppressor for introduction into an electrospray ionization chamber, comprising: a) 1) a first end open for receiving the eluent mixture; 2) a second end opening to the electrospray ionization chamber; 3) an introduction device having an introduction path for communication between the first end and the second end: b) addition to the eluate mixture. Input means connected to a selected portion of the introduction device for introducing a fluid: and c) connected to a selected portion of the introduction device to mix the eluent mixture with the addition fluid to produce a mixture. An apparatus for preparing an eluate mixture, which comprises: 3. A liquid chromatographic device; b) a preparation means for receiving a sample from the liquid chromatographic device and removing the inhibitory effect of the chromatographic additive; c) a preparation device connected to the preparation device. An analyzer comprising a receiving electrospray device; and d) a mass spectrometric device connected to the electrospray chamber.