Liquid chromatography is a method of separating the components of a mixture. In chemical and biochemical processing, the technique is used to purify a desired product, which may be mixed with by-products, starting materials, and reagents as a result of some previous processing step. In the field of chemical analysis, liquid chromatography is widely employed as a means of identifying and quantifying the components in a sample.
Separation occurs as the mixture is swept by a mobile fluidic phase through a column containing an immobile substrate referred to as the stationary phase. The rate at which any one component passes through the column depends on the nature of the interactions with the stationary phase. In general, the basis of these interactions may be adsorption, chemical bonding, polarity, solubility, or molecular filtration. The transit through a column of a component that interacts strongly with the stationary phase is retarded compared with a component that interacts weakly. Consequently, the components of a mixture emerge from the column at different times, allowing them to be collected or analysed separately.
It is normal practice to pass the fluidic stream through an ultraviolet (UV) absorption cell when it emerges from the column. The fluid within the cell is illuminated with UV light from a lamp, and the absorbance is measured with a photometer. This method of detection is non-destructive, and all of the flow may be passed through the cell without the components of the mixture undergoing any transformation. However, while good detection sensitivity can be achieved using UV absorption, even multiple wavelength or dispersive instruments are rarely capable of determining the chemical identities of the components with any confidence.
Alternatively, a mass spectrometer can be used to detect and identify the separated components. As the technique is destructive, only a small fraction of the fluidic stream should be diverted to the mass spectrometer if the intention is to make further use of the separated components. Those of skill in the art are aware that the molecular weight of a component can be deduced from its mass spectrum, and that this is often sufficient to identify the component with a high level of confidence.
Analysis by mass spectrometer requires that the biomolecules gain an electric charge and are released from solution. The resulting ions may then be separated according to their mass-to-charge ratio (m/z) by electric fields, magnetic fields, or combinations thereof and subsequently detected by an ion detector. Mass analysers are operated in vacuum to ensure that the trajectories of the ions are dominated by the applied fields rather than by collisions with neutral gas molecules. However, widely used methods of soft ionisation (techniques that generate ions without also causing fragmentation) operate at atmospheric pressure. Electrospray ionisation, atmospheric pressure chemical ionisation, and atmospheric pressure photo-ionisation are common examples. When one of these ionisation techniques is used, ions and ambient neutral gas molecules are drawn into the vacuum system housing the mass analyser through a small aperture. This aperture can become contaminated or blocked if high concentrations of involatile solutes are also present in the fluidic stream. While only electrospray ionisation will be considered below, it will be understood that many of the benefits of systems provided in accordance with the present teaching are also applicable when other forms of atmospheric pressure ionisation are employed.
In electrospray ionisation, the fluidic stream is passed through a capillary tube with a sharp tip to which a high voltage is applied. A partial separation of the positively and negatively charged ions in solution occurs in response to the electric field. Ions with the same polarity as the applied voltage become concentrated in an extruded volume at the tip of the emitter known as a Taylor cone. Droplets with an excess charge (an imbalance between the numbers of positive and negative ions) are ejected from the Taylor cone when the applied electric field is sufficient to overcome the surface tension of the fluid. Wholly aqueous solutions do not spray well, as the surface tension of water is high. Typically, an organic solvent that is miscible with water, such as acetonitrile or methanol, is added to the solvent system to reduce the surface tension. The charge density of a free droplet increases as solvent evaporates and its diameter decreases. This continues until such time as the electrostatic repulsion between like charges exceeds the surface tension, and the droplet undergoes fission. The fissioning of successive generations of progeny droplets eventually leads to the excess charge being carried by single ions surrounded by weakly bound shells of neutral solvent molecules.
Mass spectral peak intensities generally increase linearly with analyte concentration up to 10−5 to 10−3 M. At higher concentrations, the response levels-off or saturates. This is a reflection of the fact that the number of free ions produced cannot exceed the available excess charge. The chemical composition of the fluidic stream (the chemical identities of the solutes and their concentrations) is of great importance. The mass spectral peak intensity of an analyte of interest will normally decrease when the concentration of other solute ions is increased, as these compete for the finite amount of excess charge available. This phenomenon is known as ion suppression.
Those of skill in the art are aware that it is important to consider the mechanisms by which ions may be generated in solution prior to the electrospray process. Ionic compounds such as salts yield solvated positive and negative ions on dissolution and often give a strong response. Covalent compounds typically become charged through the addition of an acidic or a basic modifier to the solution. If the analyte has a basic moiety such as an amine, the addition of an acid to the solvent system results in protonation and the generation of positively charged ions. Formic acid at a concentration of 0.1% (v/v) is often used. If the analyte has an acidic moiety such as a carboxylic acid group, the addition of a base results in deprotonation and the generation of negatively charged ions. Biomolecules typically have numerous basic or acidic centres and can consequently become multiply charged. Covalent compounds may also become charged by forming adducts. For example, if NaCl is present, proteins in solution can acquire a positive charge by forming adducts with the Na+ ions. This somewhat complicates the interpretation of mass spectra as peaks due to protonation/deprotonation and adduct formation may be present, depending on the composition of the solution.
In view of the forgoing discussion, characteristics of electrospray ionisation mass spectrometry that are relevant to the analysis of components dissolved in a fluidic stream may be summarised as:
High concentrations of involatile solutes result in contamination of the vacuum interface.
The signal level associated with a component of interest can become suppressed by the presence of other solutes.
The response saturates when the overall concentration of solutes is in excess of 10−5 to 10−3 M.
Wholly aqueous solutions do not spray well as the surface tension is too high.
The ionisation of covalent compounds is promoted by acidic or basic modifiers.
It is therefore clear, and well understood by those of skill in the art, that while the information that can be derived from mass spectrometry is useful, its application in all fields is not trivial and indeed in certain instances, it may not be a suitable analysis tool at all.
In this context it is useful to appreciate that biomolecules require careful handling during chromatographic separations and other processing steps, as they are prone to chemical transformations and modifications that render them biologically inactive, a process known as denaturation. Proteins, peptides, polypeptides, antibodies, enzymes, hormones, oligosaccharides, lipids, nucleic acids, and oligonucleotides are examples of biomolecules. They can be derived from natural sources, or synthesised. While the molecular backbone of a biomolecule may remain intact unless subjected to harsh conditions, changes to the native secondary, tertiary, and quaternary conformations can result in a loss of bioactivity, reduced solubility, and a tendency to aggregate. Elevated temperatures, extremes of pH, organic solvents, and non-physiological concentrations of salt are known to cause denaturation. Consequently, biomolecules are typically stored and processed in solutions that mimic natural physiological environments, for example, tris-buffered saline (TBS), an aqueous solution of 50 mM tris(hydroxymethyl)aminomethane and 150 mM NaCl adjusted to pH 7.6 with hydrochloric acid. Higher NaCl concentrations are encountered during certain chromatographic techniques, including, for example, affinity chromatography.
Unfortunately, TBS and other similar buffers used in biochemical processing are incompatible with electrospray ionisation mass spectrometry. The high concentrations of buffering agent and NaCl result in strong ion suppression, complex mass spectra dominated by clusters and adducts, and contamination of the vacuum interface with involatile material. Furthermore, the solution is slightly alkaline, resulting in very limited protonation of basic centres. Any attempt to directly acidify the solution in order to promote protonation is initially counteracted by the buffering capacity of the buffer system. It will therefore be apparent that the analysis of biomolecules using mass spectrometry is not necessarily appropriate or feasible, as the carefully tailored aqueous buffers used to preserve the fragile native conformations of biomolecules result in low sensitivity, data analysis difficulties, and a need for frequent servicing of the vacuum interface.
It is known to address these problems using dialysis. Dialysis is a technique used in chemical processing to change the composition of solutions, and is ideally suited to the task of desalting biomolecule samples prior to analysis by mass spectrometry. It is an example of a mass transfer operation, a group of processes that can be employed to effect changes in composition without chemical reaction. In dialysis, two solutions with different compositions are separated by a semi-permeable membrane that allows the passage of molecules through narrow pores. In order to attain thermodynamic equilibrium, molecules diffuse from one side of the membrane to the other until the concentrations in the two solutions are equal. Usually, the aim is to substantially remove one or more unwanted low molecular weight components from a mixture while retaining desired high molecular weight components. This can be achieved if the pores in the membrane are wide enough to allow passage of the smaller molecules, but too narrow to transmit larger molecules.
Manual batch dialysis involves filling an envelope fabricated from semi-permeable membrane (often a section of tubing tied off at each end) with the solution to be dialysed. This is immersed in a bath containing a large volume of the second solution, or dialysate. Over the course of hours or even days, unwanted low molecular weight components diffuse out of the membrane envelope and into the dialysate, leaving behind a purified solution of the high molecular weight components. It is known to prepare biomolecule samples for mass spectrometric analysis using this method.
Flow processing by dialysis can be used for the in-line purification of fluidic streams, a familiar example being the clinical treatment of whole blood by haemodialysis machines. The technique involves pumping the fluidic stream to be purified through a first channel of a dialysis cell, and the dialysate through a second channel of the dialysis cell, the two channels being separated by a semi-permeable membrane. It is known to prepare biomolecule samples for mass spectrometric analysis using flow dialysis. Liu and co-workers have described in Analytical Chemistry vol. 68, 3295-3299 (1996) the clean-up and analysis of a very dilute solution of an oligonuleotide, which was pumped continuously through a flow dialysis cell and thereafter to an electrospray emitter by a syringe pump. The dialysate flow rate was at least sixty-fold greater than the flow rate of the oligonucleotide solution.
While this described process did achieve an analysis of biomolecules by mass spectrometry, there is a continued need for a means of repetitively sampling the effluent from a chromatography column, diluting the sampled material in a solvent system that aids the electrospray process, and efficiently performing dialysis on the resulting solution to remove components that would otherwise suppress the detection sensitivity, complicate the analysis, and contaminate the mass spectrometer. The process is required to be completed promptly so that the biomolecules emerging from the chromatography column can be identified without delay. Minimal dialysate consumption and a low-cost, compact, fluidic pumping arrangement are also desirable.
Liu and Verma have described an apparatus comprising a fluidic pump, a chromatography column, a needle valve splitter, a microdialysis assembly, and an electrospray ionisation mass spectrometer in the Journal of Chromatography A, vol. 835, 93-104 (1999). The needle valve splitter was set such that a minor fraction of the fluidic stream leaving the chromatography column was diverted to the dialysis assembly and thereafter the electrospray emitter. The dead volume of the dialysis assembly, including associated tubing was measured to be approximately 15-20 μL. Consequently, the flow rate through the dialysis system was chosen to be 10-20 μL/min so as to limit the delay to approximately 1 min, i.e. 1 min elapsed before material diverted from the main stream reached the electrospray emitter. A 2% solution of acetic acid in methanol was added to the flow at the electrospray emitter using a syringe pump in order to enhance the signal levels. Earlier introduction was avoided as the semi-permeable membrane used was intolerant of acidic conditions (pH<4), and because the authors believed that the efficiency of the dialysis process would be low in the presence of an organic solvent. The dialysate was separately supplied by gravity feed from a reservoir. Apart from the need for two additional fluidic streams (one to supply the dialysate and one to provide the acetic acid solution), a disadvantage of this configuration is that the flow rate through the dialysis cell is not independent of the flow rate through the chromatography column. If the latter is increased without also altering the needle valve setting, the residence time of the fluid in the dialysis cell decreases, leading to less efficient desalting. A further disadvantage of this configuration is that it is unsuitable for electrospray sources designed to operate at reduced flow. As the flow rate that may be accepted by the electrospray emitter also determines the flow rate through the entire dialysis assembly and associated tubing, the time taken for material diverted from the main stream to reach the electrospray emitter becomes unacceptably long when the operating regime is in the nanoliter to low microliter per minute range.
There therefore remain a number of problems associated with monitoring of biomolecule separations by mass spectrometry when the mobile phase is incompatible with common ionisation techniques and reliable operation of the vacuum interface.
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