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(11) | EP 0 655 769 A1 |
| (12) | EUROPEAN PATENT APPLICATION |
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| (54) | Method and apparatus for preparing an electrospray ion source sample |
| (57) A method, apparatus and system for preparing ions for electrospray and restoring
signal sensitivity to mass spectrometric analysis of chromatographic effluents containing
ion pairing species, in particular, strong acids or the salts of strong acids. Through
the addition of high concentrations of weak acid before or during droplet formation
stage, the suppressive effects of TFA and like chromatographic additives are eliminated
and the signal sensitivity of spectrometric analysis restored. |
[0001] This invention relates to a method and apparatus for preparing an electrospray ion source, for example to improved electrospray performance in the presence of certain high-performance liquid chromatography (HPLC) additives, such as trifluoroacetic acid (TFA), which behave as ionic species in chromatographic effluent.
[0002] Electrospray ionization mass spectrometry has recently gained enormous popularity as a technique bridging high-performance liquid chromatography (HPLC) and mass spectrometric analysis. The electrospray phenomenon (also known as electrohydrodynamic atomization) is a process of disintegration of a liquid surface in the presence of a strong electric field into a spray of fine, highly charged droplets. In electrospray mass spectrometry, solutions of involatile organic molecules and biopolymers (such as proteins and DNA) are electrosprayed to produce a large number of small, highly charged droplets containing the components of interest. The solvents are rapidly evaporated from the droplets and the residual biopolymer ions are transported through orifices or capillaries into a mass spectrometer where mass-to-charge ratios (m/z) are accurately determined. As an interface between liquid chromatography and mass spectrometry, electrospray's popularity is due not in small part to its sensitivity, but to its ability to analyze compounds of large molecular weight on standard mass spectrometry instrumentation through the techniques of multiple charging and mathematical deconvolution as well. These characteristics, among others, make electrospray mass spectrometry (ESMS) particularly useful in biochemistry and protein science.
[0003] A substantial impediment to reaping the full benefits of electrospray mass spectrometry, most particularly to the remarkable degree of ESMS sensitivity, is the presence of signal-suppressing solvents in the chromatographic effluents. Certain solvents or mobile phase additives widely used in high-performance liquid chromatography severely curtail the sensitivity of subsequent analysis through ESMS. For example,trifluoroacetic acid (TFA) is widely used in high-performance liquid chromatography because it is uniquely suited to protein and peptide related chromatography owing to its excellent chromatographic/ion pairing characteristics, its low UV absorbance in the range in which proteins and peptides absorb (200-220 nm), and its volatility. One drawback to TFA, however, is that it suppresses signal in ESMS analysis of HPLC effluent. That is to say, the presence of TFA in a sample being analyzed by mass spectroscopy will prevent the accurate detection of type and quantity of ions present in the sample.
[0004] The presence of a signal suppressor such as TFA is all the more limiting since subjecting high-performance liquid chromatography (HPLC) effluent to intermediate purification techniques or other attempts to remove TFA from the effluent compromises the sensitivity of either the HPLC, the ESMS, or both. Therefore, it is most desirable that any sample preparation method for electrospray mass spectrometry be able to accommodate effluent directly after removal from the chromatographic column and without modification of the chromatographic separation or purification method. It is critically important to overcome the signal suppressive effects of TFA and similar solutions and to realize the maximum qualitative and quantitative sensitivity of which ESMS is capable.
[0005] Attempts to overcome the signal-suppressive effects of TFA and other solvents have focused on the high conductivity imparted to a solution by the presence of such ionic species. Conductivity interferes with the electrospray process, which requires a voltage drop relative to the electrospray needle. Mechanically altering the electrospray needle is of some, albeit limited, effectiveness in overcoming signal suppression. Another essentially mechanical solution is the implementation of assisted nebulization, through ultrasound or other means. These mechanical approaches show only a slight improvement in sensitivity, with results demonstrating only a small fraction of the sensitivity of mass spectrometric analysis in the absence of signal suppressing solvents.
[0006] Surface tension of the effluent also interferes with the electrospray process. The use of a sheath solvent such as 2 methoxyethanol as taught by Mylchreest in United States Patent No. 5,122,670 (1992) results in limited effectiveness in overcoming signal suppression. However, with any of these approaches, the improvement in sensitivity is only on the order of two to five-fold and does not begin to approach the level of sensitivity attained in the absence of signal suppressing ionic species.
[0007] The problem of signal suppression remains because none of the approaches to date has addressed the fact that the ionic species present in the chromatographic solution specifically interact with the peptides or proteins of interest. None of the techniques to improve sensitivity to date has addressed this interaction as an impediment to restoration of signal sensitivity. Since mass spectrometry is one of the most powerful analytical tools available, and as protein scientists and biochemists focus increasingly on mass spectrometric analysis in the characterization of protein and peptide separations, the need for sensitivity and uncompromised qualitative information grows ever more acute.
[0009] According to an aspect of the present invention, there is provided a method of preparing an electrospray ion source as specified in claim 1.
[0010] According to another aspect of the present invention, there is provided apparatus for preparation of an effluent mixture as specified in claim 6.
[0011] It is possible to provide a more sensitive method of electrospray ionization mass spectrometric analysis, especially where the chromatographic effluent contains TFA or other similar ionic species. Preferably, a weak acid is added before or during the electrospray droplet formation stage in high concentrations relative to the amount of the anions of a strong acid or other ionic species present in chromatographic effluent. By adding a concentration of a weak acid that is greater than the concentration of TFA or other effluent ionic species, a competitive equilibrium between the weak acid and the chromatographic effluent ionic species can be established. The weak acid disrupts the TFA interaction with the components of interest (analytes) in the chromatographic effluent and restores virtually 100% sensitivity to the electrospray mass spectrometry analysis.
[0012] There can be allowed direct transfer of samples from the chromatographic column to the electrospray mass spectrometer, without the need for intermediate purification or suppression separation, and restores mass spectrometry sensitivity to levels attainable heretofore only in samples from which ionic species were absent. The method for preparing an electrospray ion source preferably includes the steps of (1) selecting the chromatographic effluent mixture; (2) adding an additional flow, either in liquid or gas state, to the effluent mixture to form a resultant mixture; (3) controlling mixing within the resultant mixture; and (4) introducing the resultant mixture into the electrospray chamber. Alternatively, when the additional flow is a gas, it may be added directly to the electrospray chamber. In that configuration, the mixing of the effluent and the additional flow fluid takes place just prior to introduction into a mass spectrometer. In most instances, the effluent will be selected because it contains an ion pairing acid, and frequently, but not exclusively, is one of a group primarily consisting of: trifuoroacetic acid (TFA), heptofluorobutyric acid (HFBA), and hydrochloric acid (HCl), although other effluent additives which fall into this category are well known or will be otherwise apparent to chromatographers.
[0013] The additional flow fluid may contain a weak acid in a concentration such that in the resultant mixture, the weak acid concentration may be at least five times that of the ion-pairing acid. Optimal concentration varies according to the nature of the chosen acid. The weak acid will have a dissociation constant (pKa) of 3 or greater, and may be chosen from the group consisting of the following acids: acetic, formic, propionic, butyric, valeric, and malonic. Other weak acids with similar properties may be suitable.
[0014] The additional flow fluid may also contain a volatile carrier solvent that is selected on the basis of its low surface tension and miscibility with the effluent. The carrier solvent may be chosen from the group consisting of methanol, ethanol, isopropanol, acetonitrile, 2-methoxyethanol, n-butanol/isopropanol, n-butanol/acetonitrile, and n-butanol/2-methoxyethanol. The carrier solvent may be necessary to overcome surface tension where nebulization is not otherwise assisted. Other carrier solvents with similar properties may be selected to accomplish this step.
[0015] In one embodiment, the volume of liquid introduced into the electrospray chamber is significantly reduced by using a flow splitter and diverting some of either the effluent or the resultant mixture into a collector. In the case where the flow splitter diverts part of the resultant mixture (i.e., after the additional flow fluid has been pumped into or otherwise mixed with the effluent mixture), a delay coil or other means for permitting further mixing of the effluent mixture and the additional flow fluid may be used.
[0016] An embodiment of the present invention is described below, by way of example only, with reference to the accompanying drawings, in which:
Fig. 1 is a simplified schematic representation of an electrospray apparatus;
Fig. 2 is a schematic of a preferred embodiment of electrospray apparatus;
Fig. 3 is a schematic of an alternate embodiment of electrospray apparatus;
Fig. 4 is a schematic of another embodiment of electrospray apparatus;
Fig. 5 is a schematic of a further embodiment of electrospray apparatus;
Fig. 6 is a schematic of another embodiment of electrospray apparatus;
Figs. 7A and 7B are graphs showing the effects of an acid additive on solvent-induced signal suppression;
Figs. 8A and 8B are graphs showing the effect of an additive with a carrier on solvent-induced signal suppression; and
Fig. 9 is a schematic of a preferred embodiment of system configuration.
[0017] The method and apparatus described below restore signal sensitivity to electrospray mass spectrometry in the presence of signal-suppressing solvents. The decreased sensitivity of electrospray mass spectrometry (ESMS) where the chromatographic effluent of interest contains ionic species such as trifluoroacetic acid has severely hampered the efforts of biochemists and protein scientists. Although the precise process of electrospray ionization has not been definitely characterized, the generally accepted theory is sketched in Fig 1 as a four step process whereby: 1) a stream of liquid 10 introduced through a needle 12 is nebulized, the charged droplets formed via nebulization forming a plume (not shown); 2) the charged droplets are heated and the heat causes vaporization of solvent from the droplets which, in turn, results in smaller droplets with greater surface charge to volume ratio (sometimes described as "charge to area" ratio) (not shown); 3) coulombic explosions occur, whereby even smaller droplets 14 are produced, with a decrease in the surface-to-charge ratio per droplet; and 4) "ion evaporation" occurs, wherein charged analyte ions 16 are ejected from the smaller droplets with high surface-to-charge density. This ion generation process is the key to electrospray because it creates a desolvated ion from a solution without going through a vaporization of the analyte. This allows non-volatile and thermally unstable compounds to be ionized by electrospray. Ionic species such as TFA interfere with the ionization process at step one and at step four, and the interference reveals itself in signal suppression.
[0018] To date, all efforts to overcome the signal suppressive effects of ionic species have focused on the interference at step 1, which is the result of high conductivity due to the presence of ionic species. However, step four "ion evaporation" is also inhibited due to the interaction of the ionic species and the analyte. By only addressing step one type interference, no method has succeeded in restoring the sensitivity more than two to five-fold - essentially a small restorative effect. The method and apparatus described are the first to address the problem of ionic interaction and to overcome both types of interference and, thereby, to restore virtually all signal sensitivity.
[0019] Fig. 2 is a schematic of a preferred embodiment of apparatus which includes an electrospray chamber 18; a conductive device 20 that receives from the effluent input device 21, the effluent mixture 22 containing signal suppressors and that has a second end 24 within the electrospray chamber 18; flow input device 26 such as tubing, for introducing additional flow fluid 28 to the conductive device 20; and at some point between the two ends of the conductive device, a mixing chamber 30 or tee for mixing the effluent mixture 22 with the additional flow fluid 28 to form a resultant mixture 32 at a point in the conductive device 20 before the resultant mixture 32 enters the electrospray chamber 18.
[0020] The effluent mixture 22 contains a strong ion-pairing acid which demonstrates characteristics useful in chromatography, commonly trifluoroacetic acid (TFA), heptafluorobutyric acid (HFBA), or hydrochloric acid (HCl). These strong acids have dissociation constants with values of less than 3. Other ion pairing chromatographic additives will be evident to analysts of ordinary skill.
[0021] The additional flow fluid 28 contains a weak acid in a concentration such that the concentration of the weak acid in the resultant mixture 32 is on the order of five times that of the effluent mixture's 22 ion pairing acid. The optimal relative concentrations of the weak and strong acid depend on the relative dissociation constants (pKa). The weak acid in the additional flow fluid 28 is chosen from the group of weak acids with a dissociation constant of greater than 3. Commonly, the weak acid is chosen from the group consisting of: acetic, formic, propionic, butyric, benzoic, and malonic acid. Other suitable acids will be apparent to the average analyst.
[0022] The introduction of the additional flow fluid 28 must be accomplished in such a way that there is sufficient mixing between the additional flow fluid 28 and the effluent mixture 22 to allow competitive equilibrium between the ionic species and the weak acid with respect to analyte molecules. However, it is generally advisable not to induce excessive longitudinal mixing which may result in chromatographic band broadening and loss of chromatographic resolution.
[0023] A mixing device 34, such as a delay coil or packed column, may be positioned in the path of the resultant mixture 32 in the conductive device 20 after the mixing chamber 30 and before the electrospray chamber 18. The particulars of mixing parameters as related to the introduction of samples into an electrospray mass spectrometric analyzer are familiar to those skilled in the relevant art. Specific procedural parameters appear in the examples provided at the end of this section.
[0024] A flow splitter 36 or other means for splitting the resultant mixture 32 flow in the conductive device 20 so that the bulk of the resultant mixture 32 flow is diverted into a collector 38 while a small portion continues flowing in the conductive device towards the electrospray chamber 18.
[0025] The best results have been obtained with an arrangement depicted in Fig 2 where the additional flow fluid 28 is added, and the resultant mixture 32 flow is then split. This arrangement minimizes bandbroadening and increases mixing but requires an extra high-pressure pump as part of the flow input device 26 and consumes relatively more additional flow fluid 28.
[0026] Fig 3 illustrates an alternative embodiment where the flow splitter 36 is positioned to split the effluent mixture 22 flow before it enters the mixing chamber 30. Such an arrangement consumes less additional flow fluid 28 and can be operated with a low cost syringe pump (not shown), but introduces large amounts ofbandbroadening and mixes less efficiently. As is noted above in reference to Fig 1, a delay coil 34, or other delay device such as a packed column, can be added after the mixing chamber 30 to assist mixing and equilibration.
[0027] Depending on the effectiveness of unaided droplet formation (purely electrostatic nebulization), the additional flow fluid 28 may further contain an organic carrier solvent chosen from the group consisting of methanol, ethanol, isopropanol, acetonitrile, 2-methoxyethanol, n-butanol/isopropanol, n-butanol/acetonitrile, and n-butanol/2-methoxyethanol. Other suitable carrier solvents will be apparent to the average analyst.
[0028] Fig. 4 shows an alternate embodiment in which a nebulization assist 42 is coupled to the second end of the conductive device 20 passage which introduces the resultant mixture into the electrospray chamber. In cases where nebulization is assisted through any of the various means (ultrasound, thermal or pneumatic), higher flow rates can be used and a flow splitter may be unnecessary. Moreover, since assisted nebulization overcomes the interference of ionic species with the droplet formation step, it is possible to omit the organic carrier solvent (which assists droplet formation) from the additional flow fluid 28 and to introduce a more concentrated solution of the selected weak acid. Higher concentration allow introduction at proportionately lower flow rates.
[0029] Fig.5 is a schematic of an alternate embodiment in which the additional flow fluid 28 is introduced directly into the electrospray chamber 18.
[0030] Fig. 6 is a schematic of an alternate embodiment in which the additional flow fluid 28 is introduced via a sheath flow configuration. A sheath flow configuration requires precise positioning of electrospray needle tips 44, 46. Because mixing of the additional flow fluid 28 (which would be introduced in the manner of a sheath liquid) with the effluent mixture 22 is necessary to allow competitive equilibration as discussed above, the sheath configuration is not ideally suited where impediments to mixing exist, such as a bulky analyte in the effluent mixture. However, with flow rates below 100 microliters per minute, the sheath flow configuration may be preferred since it requires only a small, inexpensive syringe pump 50 and consumes only a small quantity of additional flow fluid 28.
[0031] Figs. 7A and 7B show a comparison of the signal suppression effects of one chromatographic solvent, TFA, and the restorative effect on signal sensitivity when the current invention is practiced. The results were obtained with a configuration such as that illustrated in Fig. 4, i. e. additional flow fluid with weak acid but no carrier, and assisted nebulization. Graph 52 of Fig. 7A shows the mass spectrometric analysis of a peptide sample with 0.1% TFA in the effluent. Graph 54 of Fig. 7B show the results of the same peptide sample but with the additional flow fluid of propionic acid added at a rate of 100 microliters per minute. The radical increase in signal sensitivity can be see by comparing the height of the peaks from graph 52 with those in graph 54.
[0032] Figs. 8A and 8B show data obtained from testing samples with the system of Fig. 2, i. e. a mixing tee in the conductive passage prior to the flow splitter, with carrier in the additional flow fluid and purely electrostatic (unassisted) electrospray. Graph 56 of Fig. 8A shows mass spectrometric results of an untreated effluent containing 0.1% TFA. Graph 58 of Fig. 8B shows the increased peak height obtained when propionic acid in isopropanol is added prior to electrospray.
[0033] Fig. 9 shows a system which comprises a high performance liquid chromatographic device 60, the preparation apparatus 62 as described in the present application which receives effluent and introduces an additional flow fluid 28 so that through the method herein described, the signal-suppressing effects of ion pairing acids in the chromatographic effluent are overcome, an electrospray apparatus 64, and a mass spectrometer 66. The system may also include a device for assisted nebulization (not shown).
[0034] In sum, as the drawings and data presented herein demonstrate, the signal suppressive effects of chromatographic additives such as TFA are virtually 100% eliminated.
[0035] There is ongoing investigation concerning the exact nature of the chemical interactions taking place in the course of electrospray. While the reason for the success of the described embodiments is not yet absolutely certain, nonetheless, one theory useful for discussion purposes is presented below.
[0036] For an analyte, M, the resulting ions are typically of the form (M + nH)n+ where n ³ 1. It is generally accepted that these ions are formed in solution and the charged character is the impetus for lifting the ion out of solution.
[0037] In aqueous solutions, protonated trifluoroacetic acid CF₃COOH exists in an acid/base equilibrium
1. CF₃COOH = CF₃COO⁻ + H₃O⁺ Ka =0.5
[0038] The trifluoroacetate anion, CF₃COO- can form ion pairs with positively charged analyte ions:
2. CF₃COOH + (M + nH)n+ = [(M + nH)n+ * nCF₃COO⁻]
[0039] The resulting uncharged ion pair is not subject to ion evaporation. In this way, the TFA has effectively prevented the analyte ion formation.
[0040] If another acid, such as acetic acid, is added to the solution, a similar acid/base equilibrium to equation 1 exists:
3. H₂O + CH₃COOH = CH₃COO⁻ + H₃0⁺ Ka= 1.8 x 10⁻⁵
[0041] If the acetic acid is present in high enough concentration, two possible competitive equilibriums can exist. The presence of the acetate anion, CH₃COO-, can compete with the TFA ion for ion pairing of the analyte cations. However, since the acetate ion pair is relatively less stable, the analyte cation can subsequently be freed for ion evaporation.
4. CH₃COO⁻ + (M + nH)n+= [(M + nH)n+ * nCH₃COO⁻] = CH₃COO⁻ + (M + nH)n+
[0042] Additionally, the relatively high concentration of H₃O⁺ from the excess of acetic acid will force the equilibrium in Equation 1 toward the left, reducing the amount of ion pairing CF₃COO⁻ present and consequently freeing analyte ions for ion evaporation.
[0043] Note that because of the relative magnitudes of the equilibrium coefficients in equations 1 and 3, the concentration of acetic or other acid must greatly exceed the concentration of TFA. This concept holds true for ion-pairing acids other than TFA (e.g. HFBA, HCl) and organic acids other than acetic acid (e.g. formic acid, propionic acid). The relative efficiency of other combinations will depend on a number of factors familiar to those skilled in the art, such as equilibrium constants and volatility.
[0044] Given the foregoing conceptual framework, there are a number of variables that can be optimized for different operating characteristics. Solution parameters can be broken into three main groups: fixed characteristics (which are defined by the user application), chemical parameters and operating parameters.
[0045] A key benefit of the described examples is a direct connection between the chromatographic techniques, such as high performance liquid chromatography, and electrospray mass spectrometry, without compromising the performance of either. The analyst may use the method without modifying the chromatographic conditions. To that end, in the circumstance where the effluent in question is derived from a high performance liquid chromatography column, the following conditions should be considered as a standard starting point based on practice in HPLC:
HPLC Flow rate (10µl/min- 1500µl/min) (effluent flow rate)
HPLC Mobile Phase % Organic (0-100%)(effluent carrier)
HPLC Mobile Phase Organic (Methanol, Acetonitrile, Isopropanol)(effluent carrier)
HPLC Mobile Phase Buffers/Additives(e.g. TFA)(effluent additive)
HPLC Column Dimensions and Stationary Phase (irrelevant)
HPLC Column Temperature (irrelevant)
[0046] With standard conditions like those stated above, a range of chemical solutions (additional flow fluids) can be used. A number of acids can be added or " teed" into the effluent to counteract the ion pairing acid (e.g. TFA). Those tested include formic, acetic, propionic, butyric, valeric, and malonic. To date, the best results have been achieved with propionic acid, which results in virtually 100% restoration of signal sensitivity. Concentration of the acid in the additional flow fluid depends on the nature of the acid selected.
[0047] The acid is typically prepared in a solution of carrier solvent. These two components make up the bulk of the additional flow fluid. In cases of unassisted electrospray, the carrier solvent plays an important role in allowing high conductivity solutions of TFA to undergo the droplet formation step. In assisted electrospray, droplet formation is mechanically assisted and, consequently, the carrier solvent is less important and, under some conditions, may be omitted entirely.
[0048] The additional flow fluid should be introduced at a fixed flow rate which optimally preserves the chromatographic separation in the effluent. In the preferred embodiment where the additional flow fluid is added to the effluent and mixing occurs in the mixing chamber prior to encountering the splitting means or flow splitter, an optimal flow rate has been approximately 400ml/min. Alternate embodiments may have different optimal flow rates.
[0049] To reiterate, optimal mixing of the effluent mixture and the additional flow fluid provides the best results. Optimal mixing is empirically defined as that amount of mixing time necessary for a competitive equilibrium to be established between the ionic species whether a strong acid or the salt of a strong acid, and the acid introduced in the additional flow fluid. For some analytes, such as large, globular proteins, it may also be necessary to allow additional contact time for equilibration. Generally, the needed equilibration time can be supplied by a delay coil or a packed column, either of which allow mixing without disturbing the chromatographic separation.
1. A method of preparing an electrospray ion source of the type which includes an ionization
chamber (18) comprising the steps of:
a) selecting an effluent mixture (22) which contains the anions of a strong acid that has a dissociation constant (pKa) of three or less;
b) adding to the effluent mixture an additional flow fluid (28) that contains a weak acid that has dissociation constant of three or greater;
c) forming a resultant mixture (32) wherein the weak acid is present in greater concentration than the strong acid;
d) controlling mixing of the resultant mixture (32); and
e) introducing the resultant mixture (32) into the ionization chamber (18).
2. A method as in claim 1, including the step of selecting the strong acid from the group
consisting of trifluoroacetic acid(TFA), heptafluorobutyric acid (HFBA), hydrochloric
acid (HCl).
3. A method as in claim 1 or 2, including the steps of selecting for the additional flow
fluid (28) a weak acid from the group consisting of acetic, formic, propionic, butyric,
benzoic, and malonic acid; and adding to the additional flow fluid a carrier solvent
that is volatile and miscible with aqueous solvents and selected from the group consisting
of methanol, ethanol, isopropanol, methoxy, ethanol, and acetonitrile.
4. A method as in any preceeding claim, including the step of splitting either the flow
of the effluent mixture (22), or the flow of the resultant mixture (32), and thereby
to divert a portion thereof into a collector (38).
5. A method as in any preceeding claim, including the step of assisting droplet formation
of the resultant mixture (32) in the electrospray chamber (18).
6. Apparatus for preparation of an effluent mixture containing signal suppressors for
introduction into an electrospray ionization chamber (18) comprising:
a) a conductive device (20), including a first open end operative to receive the effluent mixture (22); a second end that opens into the electrospray ionization chamber (18); a conductive passage communicating between the first and second ends; and
b) input arrangement connected to a selected portion of the conductive device for introducing additional flow fluid (28) to the effluent mixture; and
c) mixing arrangement (30) connected to a selected portion of the conductive device for mixing the effluent mixture (22) with the additional flow fluid (28) to form a resultant mixture (32).
7. Apparatus as in claim 6, where the resultant mixture (32) is formed at a point prior
to introduction of the resultant mixture in which the mixing arrangement is located
upstream of the electrospray chamber into the electrospray chamber (18).
8. Apparatus as in claim 6 or 7, including a pressurizing device connected to a selected
portion of the conductive device (20) for pressurizing liquids in the conductive device
before their introduction into the electrospray chamber (18).
9. Apparatus as in claim 6, 7 or 8, including a flow splitter (36) connected to a selected
portion of the conductive device (26) for splitting either the resultant mixture flow
or the effluent mixture (22) flow so as to divert a split portion into a collector
(36).